*Review* **Cell Wall Integrity and Its Industrial Applications in Filamentous Fungi**

**Akira Yoshimi 1,2,† , Ken Miyazawa 2,3,† , Moriyuki Kawauchi <sup>1</sup> and Keietsu Abe 2,4,\***


**Abstract:** Signal transduction pathways regulating cell wall integrity (CWI) in filamentous fungi have been studied taking into account findings in budding yeast, and much knowledge has been accumulated in recent years. Given that the cell wall is essential for viability in fungi, its architecture has been analyzed in relation to virulence, especially in filamentous fungal pathogens of plants and humans. Although research on CWI signaling in individual fungal species has progressed, an integrated understanding of CWI signaling in diverse fungi has not yet been achieved. For example, the variety of sensor proteins and their functional differences among different fungal species have been described, but the understanding of their general and species-specific biological functions is limited. Our long-term research interest is CWI signaling in filamentous fungi. Here, we outline CWI signaling in these fungi, from sensor proteins required for the recognition of environmental changes to the regulation of cell wall polysaccharide synthesis genes. We discuss the similarities and differences between the functions of CWI signaling factors in filamentous fungi and in budding yeast. We also describe the latest findings on industrial applications, including those derived from studies on CWI signaling: the development of antifungal agents and the development of highly productive strains of filamentous fungi with modified cell surface characteristics by controlling cell wall biogenesis.

**Keywords:** filamentous fungi; cell wall integrity; signaling pathway; surface sensor; protein kinase C; mitogen-activated protein kinase; plant pathogen; application; fungicide; drug target; culture; productivity

## **1. Introduction**

Many microorganisms, especially fungi, have evolved as decomposers of terrestrial plants, which are primary producers. Fungi are considered to be among the most successful taxa in terrestrial ecosystems. The success of fungi is thought to be due to their ability to form filamentous cells, called hyphae, which form a network called mycelium; this ability allows fungi to invade solid substrates and acquire nutrients efficiently from the inside of the substrates that are difficult to penetrate for unicellular microorganisms [1–3]. The invasion of a solid substrate by filamentous fungi begins with contact between substrate surface and the fungi. When filamentous fungi invade solid substrates, their cells are exposed to oxidative stress; changes in osmolality, temperature, and pH; and chemical compounds, including pheromones [4]. Therefore, biochemical reactions at the cell surface affect fungal growth, and fungal cell surface structures, which form the interface between

**Citation:** Yoshimi, A.; Miyazawa, K.; Kawauchi, M.; Abe, K. Cell Wall Integrity and Its Industrial Applications in Filamentous Fungi. *J. Fungi* **2022**, *8*, 435. https://doi.org/ 10.3390/jof8050435

Academic Editors: María Molina and Humberto Martín

Received: 8 February 2022 Accepted: 20 April 2022 Published: 23 April 2022

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**Copyright:** © 2022 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/).

substrates and fungi, play an important role. Understanding the structure of the fungal cell wall and the regulation of its construction may lead to applications in controlling fungal pathogens and the effective utilization of filamentous fungi.

In this review, we focus on the cell wall integrity (CWI) signaling that regulates cell wall construction and remodeling. The cell wall, the outermost layer of the fungal cell, maintains cell morphology, protects the cells, and transmits the external stimuli inside the cell. Fungal CWI signaling has been studied in detail in the budding yeast *Saccharomyces cerevisiae* (reviewed by Levin [5,6], Gustin et al. [7], and Chen and Thomer [8]). In the CWI pathway of *S. cerevisiae*, perturbations of the cell wall are detected by the Wsc-type and Mid-type cell surface sensors. The signal is then consecutively transmitted through the following components: the GDP/GTP exchange factor Rom, the small GTPase Rho1, protein kinase C (PKC), the mitogen-activated protein (MAP) kinase cascade (MAP kinase kinase kinase Bck1; a pair of MAP kinase kinases Mkk1/Mkk2, and the MAP kinase Mpk1/Slt2), and the transcription factors (TFs) Rlm1 and Swi4, a subunit of the Swi4–Swi6 TF complex. The other signaling pathways in *S. cerevisiae* are the high osmolarity glycerol (HOG) pathway (MAP kinase: Hog1 kinase), filamentous and invasive growth (FG) pathway (Kss1 kinase), and pheromone pathway (Fus3 kinase) [9,10]. Extensive crosstalk between these pathways in *S. cerevisiae* has been documented [11,12]. In this review, we refer to the central pathway involved in CWI via PKC–Mpk1/Slt2 or their orthologs as the CWI PKC pathway. When describing the entire system that contributes to the maintenance of CWI, including not only the CWI PKC pathway but also other signaling pathways, we refer to it as CWI signaling.

Our research in this area has resulted in some industrial applications. Here, we discuss the similarities and differences between the functions of CWI signaling factors in filamentous fungi and in yeast, including cell surface sensors in Section 2 and downstream components in Section 3. We describe the development of antifungal agents based on the analysis of CWI signaling in Section 4, and the development of fungal culture technology using strains with modified cell surface structures in Section 5.

#### **2. Cell Surface Sensors of Cell Wall Integrity Signaling Pathway**

#### *2.1. Wsc- and Mid-Type Sensors*

Filamentous fungi grow by invading and decomposing solid substrates [3], and these features are used for solid-state fermentation in industrial applications [13,14]. These processes are initiated by a contact between the substrate surface and the fungal cell surface. Fungi perceive information at the contact surface and transmit it into the cells. Cell surface sensors embedded in the cell wall are important in this process, and in sensing and responding appropriately to environmental stresses. Perturbation of the cell wall may affect fungal survival, so changes in cell wall structure as such must also be sensed. Cell wall sensors in fungi were first studied in *S. cerevisiae* (for detailed reviews, see [15,16]). We provide an overview of sensor proteins in fungi in Table 1. The membrane-spanning sensors of the *S. cerevisiae* CWI PKC pathway consist of two sub-families: Wsc-type sensors (Wsc1–3) and Mid-type sensors (Mid2 and Mtl1). All of them have a transmembrane region and an extracellular region; the latter is rich in serine and threonine residues and is highly *O*-mannosylated. At the N-terminus, only the Wsc type has the Wsc domain (also referred to as the cysteine-rich domain, CRD), but only the Mid type has an *N*-glycosylated asparagine residue. The glycan chains of the extracellular region of the sensor proteins are interact with cell wall polysaccharides. These proteins function as mechanosensors. Stimuli in the cell wall and the resulting distortion of the plasma membrane are sensed as force that tilts and stretches the serine/threonine-rich region, which acts like a nanospring [15,16]. This structural change results in a conformational change in the cytoplasmic tail, which triggers downstream signal transmission.

The dimorphic fungus *Candida albicans* forms so-called invasive filaments during host invasion. Strains lacking Wsc-type sensors show little change in susceptibility to cell wall stresses, and the formation of invasive filaments does not differ from that of the wild-type strain [17]. These data suggest that the Wsc-type sensors are not crucial for CWI in this fungus.

In filamentous fungi, homologs of the *S. cerevisiae* Wsc1–3 cell wall sensors were identified in silico in a model filamentous fungus *Aspergillus nidulans* [18], and their function was analyzed [19,20]. WscA has a Wsc-domain, a serine- and threonine-rich region, a transmembrane region, and a C-terminal intracellular domain. WscA was considered to be a substrate for *O*-D-mannosyltransferase Pmt because Wsc1 and Mid2 are mannosylated by Pmt; this was confirmed using an HA-tagged WscA-expressing strain [19]. Futagami et al. [20] showed that WscA and WscB, both Wsc1 orthologs in *A. nidulans*, are *N*- and *O*-glycosylated and are localized in the cell wall. Disruption of *wscA* results in abnormal growth and reduced conidiation. The conidial formation is also reduced in the *wscB* deletion strain, but to a lesser extent. The *wscAwscB* double-disruption strain is viable, but its growth retardation is more severe than that caused by *wscA* single deletion [20]. Whereas yeast Wsc1 is involved in stress response under alkaline conditions [21], Wsc-type sensors of *A. nidulans* are thought to sense cell wall changes under acidic conditions [20]. Loss of WscA alters the transcript levels of genes for cell wall α-1,3-glucan synthases (*agsA* and *agsB*), resulting in an increase in the content of alkali-soluble glucan [20]. Loss of Wsc-type sensors also enhances the phosphorylation of a mitogen-activated protein (MAP) kinase, MpkA [20]. These results and the absence of α-1,3-glucan in yeast suggest that *A. nidulans* Wsc-type sensors have a somewhat different sensing spectrum and downstream signaling pathway from those of *S. cerevisiae* [20]. Futagami et al. [22] showed that a Mid-type sensor protein, MtlA, in *A. nidulans* is highly *O*-glycosylated and localized to the cell surface. Loss of MtlA decreases conidial formation, increases sensitivity to cell wall inhibitors, such as calcofluor white (CFW), congo red (CR), and micafungin, an echinocandin antifungal, and decreases cell wall glucan and chitin content [22]. Thus, the CWI sensor MtlA is important for cell wall stress tolerance and cell wall maintenance in this fungus [22].

The function of Wsc1–3 and the Mid-type sensor MidA has been reported in the human pathogenic fungus *Aspergillus fumigatus* [23]. The disruption of *A. fumigatus wsc1*, a gene for a Wsc1 homolog of *S. cerevisiae*, increases sensitivity to caspofungin, an echinocandin antifungal, and additional disruption of *wsc3* reduces colony growth and conidial formation. Disruption of *midA* alone does not affect colony growth, but disruption of *midA* in the *wsc1wsc3* double-disruption strain results in severe growth retardation and severe reduction of conidial formation [23]. Disruption of *wsc2* does not affect colony growth or conidiation. MidA, but not Wsc1–3, is essential for the tolerance to CFW, CR, and high-temperature stress [23]. The functions of Wsc1, Wsc3, and MidA partly overlap, and they are involved in vegetative growth and conidiation [23].

In *Neurospora crassa*, loss of WSC-1, a homolog of *S. cerevisiae* Wsc1, increases sensitivity to caspofungin and CFW and strongly reduces the formation of aerial hyphae and conidia [24]. The *wsc-2* gene encodes another Wsc-type sensor; the *wsc-2* disruption strain has a phenotype similar to that of the wild type, but with a slightly reduced growth rate and conidial formation [24]. Disruption of *wsc-1* also reduces the basal level of phosphorylation and stress-induced activity of MAK-1, a MAP kinase in the CWI PKC pathway in *N. crassa*. Disruption of *wsc-2* has a negligible effect on MAK-1 activation by cell wall stress. The authors of [24] concluded that WSC-1 and WSC-2 are required for MAK-1 activation in *N*. *crassa* and that both function as cell wall sensors.

The entomopathogen *Beauveria bassiana* has at least nine proteins with a single Wscdomain [25,26]. Among them, Wsc1A–E are localized in the hyphal cell wall or membrane, and the deletion of each of them increases sensitivity to cell wall perturbation, osmotic stress, oxidative stress, and metal ions, and also delays germination and reduces resistance to UV-B and/or heat stress [25]. None of the deletions have a significant effect on vegetative growth, conidial formation, or virulence [25]. The ninth Wsc sensor, Wsc1I, which contains not only a Wsc domain but also an N-terminal DUF1996 domain (domain of unknown function 1996), is localized to the vacuoles and cell wall/membrane and is involved in sensitivity to osmotic stress, oxidative stress, and cell wall stress compounds [26]. In a *wsc1I* deletion strain, the phosphorylation level of the MAP kinase Hog1 is greatly reduced under osmotic, oxidative, and cell wall stresses, suggesting that Wsc1I senses a variety of cell stresses upstream of the Hog1 pathway [26]. Overall, the data suggest some variations of Wsc- and Mid-type sensors among fungal species.

*J. Fungi* **2022**, *8*, 435


**Table 1.** Major fungal surface sensors whose functions have been analyzed.




*J. Fungi* **2022**, *8*, 435

**Table 1.** *Cont*.


\* Abbreviations: CWI, cell wall integrity; CFG, caspofungin; CAF, caffeine; SDS, sodium dodecyl sulfate; CW, cell wall; CR, congo red; CFW, calcofluor white.

#### *2.2. Other Types of Cell Surface Sensors*

In addition to the Wsc- and Mid-type sensors, several other types of sensor proteins function at the cell surface in filamentous fungi (Table 1). In *N. crassa*, HAM-7 was identified as a factor associated with anastomosis and sexual development [49]. It has a typical signal peptide at the N-terminus and a glycosylphosphatidylinositol (GPI) anchor signal at the C-terminus [24,49]; it is GPI-anchored to the plasma membrane, and the N-terminal extracellular domain is thought to be localized in the cell wall space. The loss of HAM-7 affects vegetative growth, hyphal branching pattern, and the formation of protoperithecia, but not the sensitivity to cell wall stress compounds [24]. Similar to WSC-1, HAM-7 is required for the activation of MAK-1 MAP kinase, and a strain deficient in both WSC-1 and HAM-7 shows severe phenotypic alterations such as compact colonies, poor formation of aerial hyphae, almost no conidiation, defective cell fusion, and no formation of protoperithecia [24]. Since these alterations are the same as those caused by the deficiency of the MAK-1 pathway, WSC-1 and HAM-7 are considered to be the major sensors upstream of the MAK-1 pathway, although their functions might differ [24].

Signaling mucins are anchored to the plasma membrane, are localized in the cell wall space, and function upstream of MAP kinases [50]. Signaling mucins have a typical signal peptide, a highly glycosylated extracellular inhibitory mucin domain, a single transmembrane domain, and a short intracellular tail. The signaling mucin Msb2 of *S. cerevisiae* is an upstream sensor of the FG and HOG pathways and is activated by nutrient starvation and by cleavage of the extracellular domain [51]. In addition to Msb2, the mucinlike protein Hkr1 is present in *S. cerevisiae* [34] and is involved in sensing cell wall damage by zymolyase [52], which degrades the β-1,3-glucan network [52,53]. Although Hkr1 orthologs have been found in *Ashbya gossypii*, which is closely related to *S. cerevisiae*, they have not been found in other filamentous fungi examined, such as *A. fumigatus*, *Fusarium graminearum*, *Magnaporthe grisea* (currently *Magnaporthe oryzae*, synonymous to *Pyricularia oryzae*), and *N. crassa* [54]. In *C. albicans*, Msb2 deficiency leads to increased sensitivity to cell wall stresses and loss of invasive phenotypes [17,55]. Together with the data on the Wsc-type sensor-defective strains [17], this finding suggests that the sensing of cell wall changes in this fungus is dependent more on the Msb2 sensor than on the Wsc-type sensors.

In some plant pathogenic fungi, the function of Msb2 homologs has been analyzed in relation to their virulence [43,46,56]. In the soil-borne vascular wilt fungus *Fusarium oxysporum*, loss of Msb2 leads to phenotypic alterations that overlap with those caused by the deficiency of the Fmk1 MAP kinase pathway (ortholog of the FG pathway in *S. cerevisiae*), including defects in penetration of cellophane membranes, adhesion to host plant roots, and virulence to the host plant [43]. Unlike Fmk1 deficiency, *msb2* deletion confers sensitivity to cell wall stress compounds, and this sensitivity is enhanced by a double knock-out of *msb2* and *fmk1* [43]. These observations indicate that Msb2 is involved in invasive growth and infection upstream of Fmk1, and also in the cell wall stress response through a pathway distinct from the CWI PKC pathway [43]. In the rice blast fungus *M. oryzae*, MoMsb2 functions upstream of the Pmk1 MAP kinase pathway as a sensor for hydrophobicity and cutin monomers on the plant surface; MoMsb2 is involved in appressorium formation in cooperation with MoSho1, which is thought to be an osmo-sensor [45]. Appressorium formation and host invasion via Pmk1 activation involve the interaction of MoMsb2 with the small GTPase Ras2, and MoMsb2 function partially overlaps with that of the mucinlike protein Cbp1 [56], which was originally identified as a chitin-binding protein [57] and lacks the mucin and transmembrane domains. As in other fungi, Msb2 of *Botrytis cinerea* functions as a surface sensor upstream of Bmk1 MAP kinase (ortholog of the Kss1 in *S. cerevisiae*) but seems to have little relevance to the CWI PKC pathway [46]. In *A. nidulans*, Msb2 is involved in adhesion and biofilm formation, cell wall stress tolerance, vegetative growth, and conidiation under nutrient deficiency via both the CWI PKC and FG pathways [40]. In *A. fumigatus*, MsbA has a similar function, with a particularly strong effect on the CWI PKC pathway [41]. The deficiency of MsbA in *A. fumigatus* alters host immune responses and increases virulence, which has been attributed to changes in the cell

wall structure [41]. Generally, other types of sensors in filamentous fungi are associated with the CWI PKC pathway, but their contribution varies among fungal species.

#### **3. Signal Transduction Downstream of Cell Surface Sensors**

#### *3.1. Rom2 and Rho1*

In *S. cerevisiae*, the cytoplasmic tails of Wsc- and Mid-type sensors interact with the guanine nucleotide exchange factor (GEF) Rom2 [15,16]. This interaction activates the small GTPase Rho1 by converting it into the GTP-bound state. Rho1-GTP activates PKC [15,16], and is also required for the activity of β-1,3-glucan synthase Fks1 (reviewed by Wagener et al. [58]).

The function of Rom2 has been reported in pathogenic fungi *A. fumigatus* [59] and *Candida* species [60]. Since the deletion of *rom2* was suggested to be lethal in *A. fumigatus*, a conditional strain was used for analysis [59]. Under *rom2*-suppressive conditions, this strain has a severe growth defect, a complete loss of conidiation, and an increased sensitivity to cell wall inhibitors [59]. In *A. fumigatus*, Rom2 is localized to the hyphal tip and septa, and *rom2* suppression increases basal levels of phosphorylation of MpkA MAP kinase [59]. Co-immunoprecipitation of HA-tagged Rom2 with Rho1 confirmed their interaction [59]. These results suggest that Rom2 is involved in the activation of the CWI PKC pathway by acting between Wsc- and Mid-type sensors and Rho1 [59].

In a human pathogen, *Candida glabrata*, a temperature-sensitive (ts) mutation in the *rom2* gene has been identified during the analysis of essential genes in ts mutant strains [60]. In *C. albicans*, a strain carrying the same ts mutation was generated; it had colony defects because of the lysis phenotype at a temperature shift without osmostabilizer, as in the ts mutant of *C. glabrata* [60]. These data and the fact that a heterozygous mutant (*Rom2*/*rom2*) but not null mutant (*rom2*/*rom2*) was obtained suggest that the *rom2* gene is essential for viability in these *Candida* species and that *Candida* Rom2 is involved in the CWI PKC pathway, in line with its functional similarities with the yeast Rom2 [60].

The function of Rho1 has been analyzed in *Aspergillus* species [61–64]. In *A. fumigatus*, AfRho1 forms a complex with β-1,3-glucan synthase Fks1 [61] and, together with Rho3, is localized to the hyphal tip under normal growth conditions and seems to control the CWI PKC pathway and the cytoskeleton [62]. Among five subfamilies of small GTPases, the Rho subfamily is most extensively characterized [65]. The industrial fungus *Aspergillus niger* has six Rho GTPases, and RhoA plays a central role in polarity establishment and survival, RhoB and RhoD are important for the CWI PKC pathway, and RhoD is important for septum formation, while RhoC has a minor function [63]. RacA and CftA (Cdc42) also maintain polarity, but RacA seems to contribute more than Cdc42 in *A. niger* [63]. In *A. nidulans*, RhoA is involved in polar growth, branching, and cell wall biogenesis [64]. In *F. oxysporum*, the loss of the *rho1* gene is not lethal but results in severe growth defects with abnormal cell walls; the cell wall alteration is thought to activate immune responses in host plants [66].

The basidiomycete *Ustilago maydis* causes corn smut disease; an Rho1 homolog of *U. maydis* is required for vegetative growth and is associated with cell polarity and cytokinesis, and Rho1 loss results in abnormalities in budding and chitin deposition [67]. In another basidiomycete, the edible mushroom *Grifola frondosa*, loss of Rho1 results in reduced mycelial growth, decreased amount of cell wall polysaccharides, and increased sensitivity to cell wall stress [68].

#### *3.2. Protein Kinase C*

At the N-terminus, PKC has C1 and C2 cysteine-rich domains, and Rho1 is thought to interact with the C1 domain to regulate the activity of the CWI PKC pathway [69]. Rho1 also binds the N-terminal HR1A domain of PKC, but this binding seems to be involved in a pathway independent of the CWI PKC pathway [70,71]. At the C-terminus, PKC has a serine/threonine kinase domain and a hydrophobic tail with an NFD (Asn-Phe-Asp) motif, the phenylalanine residue of which coordinates the substrate ATP to activate PKC [69].

Comparative genomics has been applied to factors associated with the CWI PKC pathway in human pathogenic fungi such as *C. albicans* and *A. fumigatus* and in plant pathogens such as *M. grisea* and *U. maydis* vs. those in *S. cerevisiae* [54]. The CWI PKC pathway seems to be conserved in most fungal species [54,72]. In *C. albicans*, PKC deficiency leads to cell lysis in both the budding and hyphal growth forms that can be ameliorated by osmotic stabilization [73].

The function of PKC of filamentous fungi has been extensively studied in aspergilli, in particular in *A. nidulans* (see our detailed review [74]). Loss of *pkcA* in *A. nidulans* is lethal [75,76]. Repression of this gene increases sensitivity to cell wall stress agents such as caspofungin and CFW and leads to abnormal cell wall structure [77,78]. PkcA has pleiotropic effects and regulates mitosis, germination, secondary metabolism, and farnesol-induced cell death [75–78]. PkcA inhibits apoptosis induction via the MpkA [79]. Expression of a constitutively active PkcA mutant in *A. nidulans* increases transcript levels of several chitin synthase genes (*chsB*, *chsC*, *chsD*, *csmA*, and *csmB*) and the α-1,3-glucan synthase gene *agsB* [80]. These findings indicate that PkcA in *A. nidulans* regulates the transcription of cell wall-related genes, and at least in this fungus, PkcA seems to play a central role in the CWI PKC pathway. In *A. fumigatus*, *pkcA* is thought to be an essential gene, and the analysis using a non-essential mutant of *pkcA* suggests that PkcA functions upstream of the MAP kinase MpkA [81]. In *N. crassa*, PKC is associated with the lightresponse signaling pathway and is essential for growth [82,83].

The loss of PKC is lethal in many filamentous fungi, so there are not many examples of functional analysis of PKC in plant pathogens. In *M. oryzae*, RNAi-based analysis has shown that the repression of *pkc1* causes severe growth retardation and considerably affects the transcription of genes involved in cell wall remodeling, autophagy, signal transduction, and secondary metabolism [84]. Sugahara et al. [85] found that a filamentous fungus-specific PKC inhibitor suppresses hyphal melanization in *M. grisea* by suppressing the expression of melanin synthesis-related genes, which are required for pathogenicity of some plant pathogenic fungi [86,87]. Overall, PKC is important in survival and pathogenesis and the development of drugs targeting fungal PKC may be an effective strategy.

#### *3.3. MAP Kinase Cascades Involved in Cell Wall Integrity and Their Targets*

In *S. cerevisiae*, the MAP kinase kinase kinase Bck1 activates a pair of redundant MAP kinase kinases Mkk1/Mkk2, and they activate the MAP kinase Mpk1/Slt2 [8]. Scaffold protein Spa2 mediates the interaction between the MAP kinase kinases and MAP kinase [8]. Mpk1/Slt2 phosphorylates the transcription factors (TFs) Rlm1 and Swi4, a subunit of the Swi4–Swi6 TF complex. At least 25 CW-related genes, including genes for β-1,3-glucan synthase and chitin synthases, are regulated by Mpk1/Slt2 [88]. Cell wall stress compounds such as CFW, CR, and zymolyase lead to Mpk1/Slt2 activation [89–91]. The transcriptional response to CR is almost exclusively dependent on Mpk1/Slt2 and Rlm1 [92], but the response to cell damage caused by zymolyase requires both CWI and HOG pathways [91]. In response to CWI damage, a complex transcriptional response program associated with altering metabolism and remodeling the cell wall is elaborately implemented [92,93]. Activation of the Mpk1/Slt2 pathway also required for stimulation of calcium influx through the plasma membrane Ca2+ channels Cch1–Mid1, resulting in calcineurin activation, TF Crz1 dephosphorylation, its nuclear translocation and transcriptional regulation of genes related to adaptation to cell wall and cytoplasmic stresses [8]. Mpk1/Slt2 is also activated by hyperosmotic stress, which is dependent on the activation of the Mid2 sensor and Hog1 MAP kinase [8]. The regulation of cell wall biogenesis in fungi is linked to various aspects of morphological control and stress responses through active crosstalk with other signaling pathways [8,94].

Although not all of the MAPK orthologs of fungal pathogens such as *C. albicans* have been functionally analyzed fully, their position in the pathway seems to reflect the *S. cerevisiae* paradigm [72]. In *C. albicans*, the response of the CaSko1 transcription factor to caspofungin depends on the Psk1 PAK kinase but not on the Hog1 MAP kinase [72]. In contrast to *S. cerevisiae* Ste11, a MAP kinase kinase kinase of the Kss1 pathway, there is no evidence that *C. albicans* Ste11 activates Hog1 [72]. The Cas5 transcription factor also contributes to the transcriptional response to caspofungin and has no ortholog in *S. cerevisiae* [72].

Among MAP kinases of filamentous fungi, the orthologs of the genes of the CWI PKC pathway have been analyzed in *A. nidulans*, and the differences in their functions between *A. nidulans* and *S. cerevisiae* have been discussed [18]. In *A. nidulans*, the loss of the Mpk1/Slt2 ortholog MpkA modulates conidial germination and polar growth and increases sensitivity to cell wall stress compounds such as micafungin and CFW [18]. The most distinctive difference between *A. nidulans* MpkA and *S. cerevisiae* Mpk1/Slt2 is in their target genes. The transcription of most cell wall-related genes is MpkA-independent, whereas transcription of synthase genes for α-1,3-glucan, which is absent in the cell wall of *S. cerevisiae*, depends on the TF RlmA via MpkA [18]. Transcription of *fksA* for β-1,3 glucan synthase and *chsB* for chitin synthase is MpkA-dependent under some cell wall stresses [22,80], but factors involved in the transcriptional regulation of many other cell wall-related genes are largely unknown.

In *A. nidulans*, MpkB, an ortholog of Kss1 and Fus3 MAP kinases of the FG and pheromone pathways of *S. cerevisiae* and MpkA have the same phosphorylation motif, and MpkB deletion increases sensitivity to micafungin [95]. Similarly, an MpkB-deficient strain of *A. fumigatus* has an increased sensitivity to caspofungin [96], but at least in *A. nidulans*, MpkB is not involved in the transcriptional regulation of cell wall-related genes [95], suggesting that MpkB may be involved in CWI in a different way. The Kss1/Fus3 orthologous pathway has been extensively studied as a virulence-related factor (see Jiang et al. [97] for a concise and systematic review on this and Mpk1/Slt2 pathways). This pathway is important for appressorium formation and invasive growth of the rice blast fungus *M. oryzae*, and orthologous pathways function similarly in many other appressorium-forming plant pathogens. An MpkB ortholog Chk1 in the southern corn leaf blight fungus *Cochliobolus heterostrophus* regulates not only sexual-asexual development and pathogenicity, but also adaptation to oxidative and heavy-metal stresses [98].

The function of the orthologous Mpk1/Slt2 pathway, especially its involvement in virulence, varies among species in plant pathogenic fungi [97]. Mps1 MAP kinase is dispensable for appressorium formation in *M. oryzae*, but the Mpk1/Slt2 orthologs play a pivotal role in the early stages of appressorium formation in *Colletotrichum lagenarium* and *Colletotrichum gloeosporioides*. They are also involved in various growth processes and pathogenicity-related functions in plant pathogenic fungi: the loss of the Mpk1/Slt2 ortholog leads to severe defects in aerial hyphal formation and sporulation in *M. oryzae* and increases formation of aerial hyphae and decreases that of sclerotium in *Sclerotinia sclerotiorum*.

Among basidiomycetes, the Mpk1/Slt2 orthologous pathway has been analyzed in the pathogenic yeast *Cryptococcus neoformans*. Most of the components of the CWI PKC pathway are conserved except for the sensor proteins in comparison with ascomycete fungal pathogens, and the Mpk1 orthologs seem to share common functions related to cell wall biogenesis, heat stress response, and virulence [99,100]. In *C. neoformans*, Pkc1 activity is important in dynamic morphological changes during infection, especially in changes to the large (so-called Titan) cells and capsule formation [100]. In the edible mushroom *Ganoderma lucidum*, the target of rapamycin (TOR) pathway, which plays a central role in cell growth, regulates cell wall synthesis via an Mpk1/Slt2 ortholog, indicating the potential relationship between the TOR and CWI PKC pathway [101]. In this fungus, the Swi6 ortholog appears to function downstream of the Mpk1/Slt2 pathway; Swi6 has two splice variants, and the variant Swi6B appears to be associated with regulation of the CWI PKC pathway [102]. The development of various CWI regulatory mechanisms in different fungi is due to the complex evolution of the CWI pathway to suit their survival strategies. The development of antifungal agents targeting these unique systems may be effective and is described in Section 4. In addition, the unique technology to increase production by controlling the cell surface structure, which has been developed on the basis of the analysis of CWI signaling, is discussed in Section 5.

#### **4. Cell Wall Integrity as a Drug Target for Antifungal Agents**

#### *4.1. Compounds That Inhibit the Synthesis of Cell Wall Polysaccharides*

The development of antifungal drugs is important in medicine for fungal disease treatment and in agriculture for crop protection. Because the fungal cell wall is essential for survival and its architecture is fungus-specific, factors involved in its construction may be effective targets for antifungal agents. The echinocandin class compounds, such as caspofungin, micafungin, and anidulafungin are semisynthetic lipopeptides; they have been widely used for more than 30 years since their development [103]. Echinocandins have superior antifungal activity against *Candida* spp. and *Aspergillus* spp. and are therapeutic agents in particular for esophageal candidiasis, invasive candidiasis, and invasive aspergillosis [103]. They are also active against some other ascomycetes, including *Alternaria* spp. and *Bipolaris* spp., but not against basidiomycete *C. neoformans* or any zygomycetes [103,104]. Echinocandins inhibit the synthesis of β-1,3-glucan, an essential cell wall component in many fungi; for example, they impair the activity of glucan synthases encoded by the *FKS1* and *FKS2* genes in *S. cerevisiae* [103,104]. The emergence of resistant strains has been reported in some *Candida* spp., and an amino acid substitution in Fks1 seems to contribute to the resistance [104]. Because *Aspergillus* spp. growth is not completely suppressed by echinocandins, it is rather difficult to distinguish whether they are resistant or not, but resistant mutants have been generated in the laboratory [105]. Resistant strains have been also isolated in clinical situations, raising concerns about the increase in the incidence of such strains [105].

The nucleoside antibiotics, blasticidin S and polyoxins, are known as forerunners for antibiotics used for agriculture [106]. Blasticidin S inhibits protein synthesis, whereas polyoxins inhibit cell wall synthesis in target fungi [106–108]. Polyoxin A was isolated from *Streptomyces cacaoi* in 1965 [109,110] as a new nucleoside compound and was marketed in 1967 [106]. Polyoxins are structurally similar to the substrate for biosynthesis of chitin (UDP-*N*-acetylglucosamine) [107,108,111], which is an essential cell wall component of plant pathogenic fungi [106–108]. Nikkomycins are structurally related to polyoxins [111]. Polyoxins and nikkomycins are taken up by the fungi and mimic the substrate of chitin synthase, antagonistically inhibiting cell wall chitin synthesis [106–108,111,112].

Some dyes such as CFW and CR are used for laboratory experiments. CFW and CR bind to the fungal cell wall components chitin and glucans [113] and inhibit cell wall synthesis [114]. Their mechanism of action is well summarized by Ram and Klis [114]. Both compounds have two sulfonic acid groups, which are negatively charged under slightly acidic to basic conditions; this makes the compounds soluble and active against fungi [114]. Under these conditions, they cannot pass through the plasma membrane because they each carry two negative charges and are thought to target compounds on the outside of the cell wall [114]. CFW and CR bind to β-linked-glucans in vitro, but CFW preferentially stains chitin in fungal cell wall [114]. Among the cell wall polysaccharides, β-1,3-glucan interacts strongly with CR but not as strongly with CFW in vitro [113]. In *A. nidulans*, the strain with increased cell surface exposure of β-1,3-glucan due to the loss of α-1,3-glucan from the cell wall has increased CR adsorption [115]. In addition, CR adsorbs more on purified β-1,3 glucan or chitin and less on mutan (bacterial α-1,3-glucan) in vitro [115]. Overall, CFW and CR are thought to act by binding to chitin and β-linked-glucan chains, thereby inhibiting the assembly of chitin and β-linked-glucans and weakening the cell wall [114]. Recently, several transcription factors involved in CR sensitivity and CR dynamics in fungal cells have been analyzed [116]. The amorphous cell surface polysaccharide, galactosaminogalactan (GAG),

interferes with the uptake of CR into the fungal cell [116]. CR-resistant strains form larger abnormal swollen ("Quasimodo") cells than the wild-type or CR-sensitive strains [116]. Those cells adsorb more CR, leading to CR removal from the culture media and resulting in the acquisition of CR resistance [116]. CR affects the transcription of the genes related to primary and secondary metabolism and toxin efflux systems, suggesting that damage to the fungal cell wall can cause serious adverse effects for fungal growth [116].

#### *4.2. Compounds That Act on the CWI Signaling*

Factors involved in fungal signaling systems are often unique to fungi and so may be effective targets for antifungal drugs. For example, dicarboximide fungicides such as iprodione and procymidone, and phenylpyrrole fungicides such as fludioxonil have been used for many years to control crop diseases [117–120]. These fungicides convert type III histidine kinases to phosphatases, which deactivate the histidine-containing phosphotransfer intermediator Ypd1, resulting in abnormal activation of the downstream HOG pathway, and disturb the fungal osmotic response signaling system [117–119].

Recently, Beattie and Krysan [121] developed a high-throughput screening system for antifungal agents based on the adenylate kinase (AK) assay. AK released during fungal cell lysis phosphorylates an ADP-containing reagent, and the generated ATP is detected by luciferase. Using this method, the authors found that compound PIK-75 inhibits growth of not only *A. fumigatus* but also *C. neoformans*, and that PIK-75 activity at least in part is due to the loss of CWI. This assay allows cell-wall active compounds to be identified even if their action on the cell wall is indirect [121].

As described in *3.1*, PKC is an important signaling factor associated with cell wall biogenesis. Therefore, PKC inhibitors, such as staurosporine, enzastaurin, and ruboxistaurin, can be expected to have excellent antifungal activity. For example, staurosporine strongly inhibits PKC in filamentous fungi [85]. However, these inhibitors cannot be used as antifungal agents because they also inhibit human PKC. To screen for specific inhibitors of fungal cell wall biogenesis, Sugahara et al. [85] conducted an in silico screening to target PKC of *M. oryzae*. The overall concept of the study is depicted in Figure 1. A threedimensional MgPkc1 structure was modeled to screen for compounds that might inhibit its kinase domain, and the candidate compounds were tested for antifungal activity against *M. grisea.* Among them, Z-705 had the highest inhibitory effect. Chimeric *PKCs* encoding the regulator domain from *S. cerevisiae* and the kinase domain from *S. cerevisiae* (control), *M. grisea* or *A. nidulans* were integrated in the *S. cerevisiae* genome, and Z-705 specifically inhibited chimeric PKCs with the kinase domain from filamentous fungi, but not with that from *S. cerevisiae* [85]. The inhibitory effect was comparable to that of staurosporine, a well-known PKC inhibitor. This compound also inhibited hyphal melanization induced by cell wall stress in *M. grisea*, which is necessary for the infection process [86,87] (Figure 1). In *M. grisea*, Mps1 acts downstream of Pkc1 and regulates gene expression for synthase of α-1,3-glucan that confers the ability to evade the host plant immune system [122,123]. From this perspective, Pkc1 inhibition is also important for reducing pathogenicity. We believe that the efficacy of drugs targeting the cell wall of filamentous fungi is increasing, and the development of such drugs should be further promoted in the future.

**Figure 1.** Diagram of drug development to target the CWI signaling pathway in filamentous fungi. In *M. oryzae*, Pkc1 is a protein kinase C, Mps1 is a MAP kinase, and Pig1 and Rlm1 are transcription factors. *SCD* (encoding scytalone dehydratase), *3HNR* (trihydroxy-naphthalene reductase), and *4HNR* (1,3,6,8-tetrahydroxy-naphthalene reductase) are involved in the biosynthesis of 1,8-dihydroxynaphthalene (DHN) melanin in several plant pathogens, including *M. oryzae. AGS1* encodes α-1,3-glucan synthase. Abbreviations: AP, appressorium; GT, germ tube; CO, conidium; IH, invasive hyphae; AG, α-1,3-glucan. **5. Improvement of Productivity by Modification of Macromorphology in Filamentous Figure 1.** Diagram of drug development to target the CWI signaling pathway in filamentous fungi. In *M. oryzae*, Pkc1 is a protein kinase C, Mps1 is a MAP kinase, and Pig1 and Rlm1 are transcription factors. *SCD* (encoding scytalone dehydratase), *3HNR* (trihydroxy-naphthalene reductase), and *4HNR* (1,3,6,8-tetrahydroxy-naphthalene reductase) are involved in the biosynthesis of 1,8 dihydroxynaphthalene (DHN) melanin in several plant pathogens, including *M. oryzae. AGS1* encodes α-1,3-glucan synthase. Abbreviations: AP, appressorium; GT, germ tube; CO, conidium; IH, invasive hyphae; AG, α-1,3-glucan.

#### **Fungi**  Another applied approach derived from studies on cell wall biogenesis in filamen-**5. Improvement of Productivity by Modification of Macromorphology in Filamentous Fungi**

tous fungi is the improvement of culture characteristics to increase productivity by controlling the surface properties of fungal cells and fungal morphology. Here, we describe some examples of this approach. *5.1. Phenotypes of α-1,3-Glucan-Deficient Mutants*  Another applied approach derived from studies on cell wall biogenesis in filamentous fungi is the improvement of culture characteristics to increase productivity by controlling the surface properties of fungal cells and fungal morphology. Here, we describe some examples of this approach.

#### As described above, expression of α-1,3-glucan synthase genes (*agsA*, *agsB*) is controlled by MAP kinase MpkA in *A. nidulans* [18]. Single or double disruption of the two *5.1. Phenotypes of α-1,3-Glucan-Deficient Mutants*

α-1,3-glucan synthase genes of *A. nidulans* has revealed that the single disruption of *agsB* and the double disruption of *agsA* and *agsB* cause complete loss of cell wall α-1,3-glucan but are not lethal [115]. *Aspergillus fumigatus* has three α-1,3-glucan synthase genes, and disruption of all the three genes (Δ*ags*) is not lethal [124,125]. The germinating conidia of *A*. *fumigatus* Δ*ags* do not aggregate [124]. The hyphae of α-1,3-glucan-deficient mutants of *A*. *nidulans* such as Δ*agsB* and Δ*agsA*Δ*agsB* are dispersed in shake-flask cultures, whereas those of the parental strain form tightly aggregated pellets [18]. In *A*. *nidulans*, the *agsB* gene is clustered with the α-amylase-encoding genes *amyD* and *amyG* [126]. An intracellular α-amylase AmyG hypothetically contributes to synthesis of the primer molecule for α-1,3-glucan polymerization by the α-1,3-glucan synthase [126,127]. Disruption of the *amyG* gene results in a substantial decrease in the content of cell wall α-1,3-glucan and lead to hyphal dispersion or formation of tiny hyphal pellets [126,127]. Disruption or overexpression of *amyD*, which encodes GPI-anchored α-amylase, increases or decreases cell As described above, expression of α-1,3-glucan synthase genes (*agsA*, *agsB*) is controlled by MAP kinase MpkA in *A. nidulans* [18]. Single or double disruption of the two α-1,3-glucan synthase genes of *A. nidulans* has revealed that the single disruption of *agsB* and the double disruption of *agsA* and *agsB* cause complete loss of cell wall α-1,3-glucan but are not lethal [115]. *Aspergillus fumigatus* has three α-1,3-glucan synthase genes, and disruption of all the three genes (∆*ags*) is not lethal [124,125]. The germinating conidia of *A*. *fumigatus* ∆*ags* do not aggregate [124]. The hyphae of α-1,3-glucan-deficient mutants of *A. nidulans* such as ∆*agsB* and ∆*agsA*∆*agsB* are dispersed in shake-flask cultures, whereas those of the parental strain form tightly aggregated pellets [18]. In *A. nidulans*, the *agsB* gene is clustered with the α-amylase-encoding genes *amyD* and *amyG* [126]. An intracellular α-amylase AmyG hypothetically contributes to synthesis of the primer molecule for α-1,3-glucan polymerization by the α-1,3-glucan synthase [126,127]. Disruption of the *amyG* gene results in a substantial decrease in the content of cell wall α-1,3-glucan and lead to hyphal dispersion or formation of tiny hyphal pellets [126,127]. Disruption or overexpression of *amyD*, which encodes GPI-anchored α-amylase, increases or decreases cell wall α-1,3-glucan, respectively [126]. The hyphae of the *amyD* overexpression strain show a phenotype similar to those of the α-1,3-glucan-deficient mutants [126,128]. These

results suggest that AmyD represses α-1,3-glucan biosynthesis. Overexpression of AmyD without the C-terminal GPI-anchor in *A. nidulans* scarcely affects cell wall α-1,3-glucan, suggesting the importance of the GPI anchor for correct cellular localization and function of AmyD [129]. In *A. fumigatus*, treatment with α-1,3-glucanase removes α-1,3-glucan from conidia and leads to their dispersion in medium, indicating the involvement of α-1,3-glucan in conidial aggregation [130]. Dispersion of hyphae in *A. nidulans* in α-1,3-glucan-deficient mutants and that of germinating conidia in *A. fumigatus* α-1,3-glucan-deficient mutants suggests that α-1,3-glucan functions as an aggregation factor for hyphae and conidia. Interestingly, *A. nidulans* ∆*agsB* and ∆*agsA*∆*agsB* mutants produce considerably more hyphal cells than the wild-type strain does under submerged culture conditions, implying that the dispersed hyphal cells of the α-1,3-glucan deficient mutants can be used for fermentation of valuable products. The α-1,3-glucan deficient mutants of *A. nidulans* show better production of endogenous penicillin and α-amylase than the wild type [131].

In the α-1,3-glucan-deficient mutant of the industrial fungus *A. oryzae*, three α-1,3 glucan synthase genes are disrupted [132]. The *A. oryzae* ∆*agsA*∆*agsB*∆*agsC* mutant forms smaller hyphal pellets than the parental wild-type strain, suggesting that α-1,3-glucan is also an aggregation factor in *A. oryzae*. This mutant produces more recombinant protein than the wild-type strain [133]. Jeennor et al. [134] reported that the disruption of the *ags1* gene (probably *agsB* in Miyazawa's work [133]) in *A. oryzae* significantly improves lipid production in a stirred-tank bioreactor. The disruptant of the *Aspergillus luchuensis agsE* gene, an ortholog to *A. nidulans agsB*, shows better protoplast formation than the wild-type strain when treated with the cell wall lytic enzyme Yatalase [135]. The *A. fumigatus* mutants in which the *ags1* gene, an ortholog of *A. nidulans agsB*, is disrupted, form smaller hyphal pellets than the wild type [136]. Taken together, α-1,3-glucan is an aggregation factor for hyphae and conidia in *Aspergillus* fungi.

#### *5.2. Phenotypes of Galactosaminogalactan-Deficient Mutants*

In *Aspergillus* species, GAG is one of the components of the extracellular matrix and is essential for biofilm formation [137]. In the background of the defect of α-1,3-glucan biosynthesis (∆*agsA*∆*agsB*∆*agsC*) in *A. oryzae*, disruption of the *sphZ* and *ugeZ* genes (AG∆-GAG∆), which are speculative GAG biosynthetic genes of *A. oryzae*, leads to dispersion of hyphae under submerged culture conditions, suggesting that GAG also contributes to aggregation in *A. oryzae* [138]. A simultaneous defect of α-1,3-glucan and GAG biosynthesis also leads to hyphal dispersion in *A. fumigatus* [136]. In *B. cinerea* and *Cochlioborus heterostrophus*, GAG also contributes to hyphal aggregation [139]. Recently, Mei et al. [140] reported that the insect pathogenic fungus *Metarhizium robertsii* has GAG biosynthetic genes, and defects of GAG biosynthesis lead to hyphal dispersion. In ascomycetes, GAG biosynthetic genes are found in some *Pezizomycotina*, and only in *Trichosporon asahii* in basidiomycetes [141]. Expression of GAG biosynthetic genes is thought to be regulated by transcription factors such as StuA, MedA, and SomA in *A. fumigatus* [142–144]. Since disruption of the *agdZ* gene increases GAG secretion in *A. oryzae* and *A. fumigatus* (Miyazawa et al., unpublished results), some mechanisms might sense adhesion of hyphae and subsequently downregulate GAG biosynthesis.

#### *5.3. Improvement of Productivity Using a Mutant Lacking both α-1,3-Glucan and GAG*

Regulation of macromorphology such as hyphal pellets and pulp form has been a key issue in fermentation using filamentous fungi [145]. Macromorphology of filamentous fungi is controlled by adjusting culture conditions such as agitation speed, pH, and medium composition [146,147]. Recently, addition of microparticles such as titanate and talc to liquid culture media has been found to promote the formation of micro-pellets that can improve productivity of fermentation of filamentous fungi [148]. We here illustrate our strategy for improving productivity with cell wall mutants of *A. oryzae* (Figure 2). Miyazawa et al. [138] showed that hyphae of the AG∆-GAG∆ mutant are fully dispersed under submerged culture conditions, and production of recombinant polyesterase CutL1 is significantly higher in AG∆-GAG∆ than

in the parental wild-type strain in shake-flask culture [138]. Ichikawa et al. [149] showed that the production of secreted CutL1 was higher in AG∆-GAG∆ than in the wild type or mutants lacking α-1,3-glucan (AG∆) or GAG (GAG∆) in batch culture in a 5 L lab-scale bioreactor. The apparent viscosity of the AG∆-GAG∆ culture tended to be lower than that of the wild-type strain culture at each agitation speed examined (200–600 rpm), suggesting that the lack of α-1,3-glucan and GAG in the hyphae improves culture rheology, increasing recombinant protein production [149]. Sakuragawa et al. [150] reported that the AG∆-GAG∆ strain produces more recombinant cellulase CBHI than the wild-type strain in a 250 mL bioreactor. The AG∆-GAG∆ strain shows rapid glucose consumption, increased mycelial dry weight, and higher respiration activity in comparison with the wild-type strain. The levels of metabolites of glycolysis and TCA cycle are lower in AG∆-GAG∆ than in the wild type in liquid culture, suggesting that AG∆-GAG∆ shows higher metabolic flux than the wild type [150]. Since the production of beneficial compounds from fungal cells is attributable to complex physiological events, the mechanisms underlying the productivity of AG∆-GAG∆ in the bioreactor are presently being analyzed. Further improvement of the productivity is expected to be achieved by conferring stress susceptibility to the AG∆-GAG∆ mutant and fine tuning the culture conditions through the screening for stress factors and multi-omics analyses in the cultivation. higher in AGΔ-GAGΔ than in the wild type or mutants lacking α-1,3-glucan (AGΔ) or GAG (GAGΔ) in batch culture in a 5 L lab-scale bioreactor. The apparent viscosity of the AGΔ-GAGΔ culture tended to be lower than that of the wild-type strain culture at each agitation speed examined (200–600 rpm), suggesting that the lack of α-1,3-glucan and GAG in the hyphae improves culture rheology, increasing recombinant protein production [149]. Sakuragawa et al. [150] reported that the AGΔ-GAGΔ strain produces more recombinant cellulase CBHI than the wild-type strain in a 250 mL bioreactor. The AGΔ-GAGΔ strain shows rapid glucose consumption, increased mycelial dry weight, and higher respiration activity in comparison with the wild-type strain. The levels of metabolites of glycolysis and TCA cycle are lower in AGΔ-GAGΔ than in the wild type in liquid culture, suggesting that AGΔ-GAGΔ shows higher metabolic flux than the wild type [150]. Since the production of beneficial compounds from fungal cells is attributable to complex physiological events, the mechanisms underlying the productivity of AGΔ-GAGΔ in the bioreactor are presently being analyzed. Further improvement of the productivity is expected to be achieved by conferring stress susceptibility to the AGΔ-GAGΔ mutant and fine tuning the culture conditions through the screening for stress factors and multi-omics analyses in the cultivation.

strategy for improving productivity with cell wall mutants of *A*. *oryzae* (Figure 2). Miyazawa et al. [138] showed that hyphae of the AGΔ-GAGΔ mutant are fully dispersed under submerged culture conditions, and production of recombinant polyesterase CutL1 is significantly higher in AGΔ-GAGΔ than in the parental wild-type strain in shake-flask culture [138]. Ichikawa et al. [149] showed that the production of secreted CutL1 was

*J. Fungi* **2022**, *8*, x FOR PEER REVIEW 15 of 24

**Figure 2.** Improvement of productivity with the *Aspergillus oryzae* mutant lacking both α-1,3-glucan and GAG (AGΔ-GAGΔ). (**A**) Growth of the wild-type and AGΔ-GAGΔ strains in liquid culture. Although the wild type forms pellets of several millimeters, the AGΔ-GAGΔ hyphae are fully dispersed. This unique macromorphology of AGΔ-GAGΔ results in increased production of secreted recombinant polyesterase CutL1 and recombinant cellulase CBHI. Conidia (1.0 × 105/mL) of each strain were inoculated into 50 mL of YPD (2% peptone, 1% yeast extract and 2% glucose) medium in a 200 mL Erlenmeyer flask and rotated at 120 rpm at 30 °C. Magnified images (bottom right) were taken under a stereomicroscope. (**B**) AGΔ-GAGΔ culture has improved rheological properties. The wild type and AGΔ-GAGΔ expressing recombinant *cutL1* gene were cultured in YPDS (6% peptone, 1% yeast extract, 6% glucose and 20 mM **Figure 2.** Improvement of productivity with the *Aspergillus oryzae* mutant lacking both α-1,3-glucan and GAG (AG∆-GAG∆). (**A**) Growth of the wild-type and AG∆-GAG∆ strains in liquid culture. Although the wild type forms pellets of several millimeters, the AG∆-GAG∆ hyphae are fully dispersed. This unique macromorphology of AG∆-GAG∆ results in increased production of secreted recombinant polyesterase CutL1 and recombinant cellulase CBHI. Conidia (1.0 <sup>×</sup> <sup>10</sup>5/mL) of each strain were inoculated into 50 mL of YPD (2% peptone, 1% yeast extract and 2% glucose) medium in a 200 mL Erlenmeyer flask and rotated at 120 rpm at 30 ◦C. Magnified images (bottom right) were taken under a stereomicroscope. (**B**) AG∆-GAG∆ culture has improved rheological properties. The wild type and AG∆-GAG∆ expressing recombinant *cutL1* gene were cultured in YPDS (6% peptone, 1% yeast extract, 6% glucose and 20 mM succinate buffer) in a 5 L lab-scale bioreactor. Left panels: Chinese ink was dropped onto the culture surface at 60 h, and diffusion was imaged at 6 s. Right panel: Apparent viscosity of the culture at 36 h. Torque values were measured with a mixing torquemeter, and apparent viscosity was calculated from the *Np*-*Re* diagram at the indicated agitation speeds.

#### *5.4. Improvement of Productivity by Mutations in Cell Wall-Related Genes*

Both extracellular hydrolytic enzymes such as amylases and proteases and cell wall synthesizing enzymes are packaged in vesicles and delivered from the Golgi to the hyphal tip of filamentous fungi [151]. Delivery of cell wall synthesizing enzymes to the hyphal tip balances necessity to secrete extracellular enzymes for nutrient acquisition [151]. Since secretion of enzymes and cell wall biogenesis are linked, perturbation to cell wall biogenesis seems to considerably affect enzyme secretion [151].

The *A. niger* SH2 strain is widely used in industrial enzyme production [151,152]. In the SH2 genome sequence, Yin et al. [152] found frame-shift mutations and non-synonymous SNPs in genes of CWI signaling, β-1,3-glucan synthesis and chitin synthesis and suggested that they affect hyphal development and hyphal fragmentation during industrial fermentation. Sun et al. [153] constructed *A. niger* mutants with the silenced chitin synthase gene *chsC*. The mutants showed shorter hyphae with lower proportion of dispersed mycelia, decreased viscosity and improved oxygen and mass transfer efficiency, which consequently improved production of citric acid [153]. Yin et al. [154] evaluated citrate production by *A. niger* H915-1 (an industrial producer) and by A1 and L2 ("degenerated" isolates of H915-1) strains. The H915-1 forms bulbous hyphae with short, swollen branches during citrate fermentation, and has the highest citrate titer, whereas A1 forms fewer compact pellets and L2 forms mycelial clumps [154]. Yin et al. [154] indicated that these differences in morphology may influence medium viscosity and hyphal respiration [154]. For citrate generation, the tight pellet form but not the diffuse filamentous form is preferred [154]. Liu et al. [155] reported that silencing of the *chs4* gene encoding class III chitin synthase in *Penicillium chrysogenum* by RNA interference causes formation of a smaller pellet, hyper-branched hyphae, and improves penicillin production. To find *N. crassa* mutants with decreased viscosity in submerged culture, Lin et al. [156] screened 90 morphological mutants and found two such mutants. The causing gene *gul*-*1* encodes an mRNA-binding protein. Disruption of this gene downregulates GPI-anchored cell wall proteins, upregulates non-GPI cell wall proteins, and alters expression of the hydrophobin gene. Disruption of *gul*-*1* in the hyper-cellulase–producing strain significantly decreases culture viscosity compared to the parental strain. Fiedler et al. [157] analyzed the transcriptomics of *A. niger* cells treated with inhibitors of synthesis of chitin (CFW), glucan (caspofungin), sphingolipids (aureobasidin A), and ergosterol (fenpropimorph), and of calcium/calcineurin signaling (FK506), which directly or indirectly interfere with CWI. The analysis suggests that (i) the CWI PKC pathway as a main compensatory response is induced by caspofungin via RhoB and by aureobasidin A via RhoD, followed by activation of the MAPKK MkkA and the TF RlmA; (ii) RlmA is the main TF for protection against CFW, but it cooperates with MsnA and CrzA for protection against caspofungin and aureobasidin A; (iii) aureobasidin A, but not fenpropimorph, induces cell wall stress.

Overall, the macromorphology of filamentous fungi closely relates to productivity. Although several components regulated by the CWI PKC pathway in the production strains have been revealed, how to regulate the CWI PKC pathway to improve productivity is scarcely understood. Combining the screening of phenotypic mutants and analysis of the mechanisms underlying cellular physiology as described by Lin et al. [156] could lead to a breakthrough technology to further improve fungal productivity.

#### **6. Conclusions and Perspectives**

The cell wall of filamentous fungi is constantly exposed to the environment and is closely involved in interactions with other microorganisms, plants and animals. The fungal cell wall, as well as those of bacteria and plants, is mainly composed of polysaccharides, but these polysaccharides and their structures are quite different from those of bacteria and plants. Although the PKC is conserved in all eukaryotes, CWI PKC pathway has evolved independently in fungi and varies even at the species level. Perturbing CWI signaling is an effective strategy for controlling fungal growth. Chemical compounds that target certain signaling factors of CWI signaling can be used to control pathogens of plants and

animals. Effective antifungal drugs targeting the cell wall biosynthesis of filamentous fungi are now on the market, and the screening for and consequent development of such chemicals are underway. Since the genomic information of filamentous fungi is continuously accumulated and artificial intelligence (AI)-based analyses are advancing in various fields, the development of antifungal drugs targeting CWI signaling will be further accelerated by utilizing AI technology in the analysis of genomic information.

The studies of CWI signaling have revealed that polysaccharides such as α-1,3-glucan and GAG function as adhesive factors for hyphae in aspergilli and cause the formation of hyphal pellets. Regulation of the display of these polysaccharides on the cell surface enables filamentous fungi to control their macromorphology such as pellets and pulp forms. Filamentous fungi are extensively used for large-scale industrial cultivation in submerged culture for production of proteins and low-molecular-weight chemicals. However, the capacity of production by filamentous fungi does not reach that by the unicellular fungus *S. cerevisiae* or bacteria *Escherichia coli* and *Bacillus subtilis*, because of the unstable macromorphology of filamentous fungi during liquid cultivation. Several attempts have been made to control the hyphal morphology in filamentous fungi to improve the cultivation characteristics, but the fundamental technology to control hyphal pellet formation has not been established. Modifying polysaccharide contents of the cell surface has led to strains with dispersed hyphae and normal growth, which ensures the efficient acquisition of nutrients and dissolved oxygen. Further analysis of the mechanisms of cell wall biogenesis in filamentous fungi will generate knowledge that will lead to the development of antifungal agents and may also lead to innovative technology for industrial cultivation using filamentous fungi. Therefore, studies on the cell wall biogenesis of filamentous fungi should be continuously promoted, so that the ensuing fruitful achievements can contribute to the improvement of human life.

**Author Contributions:** A.Y., K.M. and K.A. wrote the manuscript. M.K. checked the manuscript. K.A. supervised this research. All authors have read and agreed to the published version of the manuscript.

**Funding:** This work was supported by the Institute for Fermentation, Osaka, Japan (Grant numbers K-2019-002 to A.Y. and M.K. and L-2018-2-014 to K.A.). This work was also supported by a project JPNP20011 (K.A.) commissioned by the New Energy and Industrial Technology Development Organization (NEDO), the Japan Society for the Promotion of Science (JSPS) KAKENHI Grant Numbers JP20H02895, JP26292037 (K.A.), JP18K05384 (A.Y.), JP18J11870 and JP20K22773 (K.M.). This work also supported by ISHIZUE 2021 of Kyoto University Research Development Program (A.Y.) and the Kyoto University Foundation (M.K.).

**Institutional Review Board Statement:** Not applicable.

**Informed Consent Statement:** Not applicable.

**Conflicts of Interest:** The authors declare no conflict of interest.

## **References**


**Chibuike Ibe 1,\* and Carol A. Munro <sup>2</sup>**


**Abstract:** *Candida* species are part of the normal flora of humans, but once the immune system of the host is impaired and they escape from commensal niches, they shift from commensal to pathogen causing candidiasis. *Candida albicans* remains the primary cause of candidiasis, accounting for about 60% of the global candidiasis burden. The cell wall of *C. albicans* and related fungal pathogens forms the interface with the host, gives fungal cells their shape, and also provides protection against stresses. The cell wall is a dynamic organelle with great adaptive flexibility that allows remodeling, morphogenesis, and changes in its components in response to the environment. It is mainly composed of the inner polysaccharide rich layer (chitin, and β-glucan) and the outer protein coat (mannoproteins). The highly glycosylated protein coat mediates interactions between *C. albicans* cells and their environment, including reprograming of wall architecture in response to several conditions, such as carbon source, pH, high temperature, and morphogenesis. The mannoproteins are also associated with *C. albicans* adherence, drug resistance, and virulence. Vitally, the mannoproteins contribute to cell wall construction and especially cell wall remodeling when cells encounter physical and chemical stresses. This review describes the interconnected cell wall integrity (CWI) and stress-activated pathways (e.g., Hog1, Cek1, and Mkc1 mediated pathways) that regulates cell wall remodeling and the expression of some of the mannoproteins in *C. albicans* and other species. The mannoproteins of the surface coat is of great importance to pathogen survival, growth, and virulence, thus understanding their structure and function as well as regulatory mechanisms can pave the way for better management of candidiasis.

**Keywords:** fungi; cell wall; cell wall proteins; signaling pathways; stress tolerance

#### **1. Introduction**

*Candida albicans* is abundantly found in mammals. It often resides on the skin and mucosal layers of individuals as part of their normal flora. *C. albicans* causes a range of infections from superficial to life-threatening and systemic, dependent upon the host's immune system [1] *C. albicans* uses an arsenal of pathogenic mechanisms to subdue or evade host immune responses [2,3]. The mannosylated surface protein coat is covalently linked to the skeletal cell wall polysaccharides and plays a vital role in mediating *C. albicans* interaction with the host. The proteins are not only important in maintaining cell wall integrity, masking the polysaccharide rich layer, therefore preventing recognition by dectin-1, but also contribute to virulence of this pathogen in many ways. They mediate adherence to host cells and indwelling medical devices, enable invasion of epithelial cells, facilitate biofilm formation, protect *C. albicans* against immune attack, coordinate communication between host cells and *C. albicans*, and are important in nutrient scavenging including zinc and iron [3]. Given the important roles of the cell surface proteins at every stage of *C. albicans* infection process, research has been focused on expanding our understanding of their biology and structure as well as their function in the cell wall [4]. This area is,

**Citation:** Ibe, C.; Munro, C.A. Fungal Cell Wall Proteins and Signaling Pathways Form a Cytoprotective Network to Combat Stresses. *J. Fungi* **2021**, *7*, 739. https://doi.org/ 10.3390/jof7090739

Academic Editors: María Molina and Humberto Martín

Received: 29 July 2021 Accepted: 4 September 2021 Published: 8 September 2021

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**Copyright:** © 2021 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/).

however, rapidly expanding as the cell surface proteins have the potential to be a unique drug/vaccine target [5–7]. Proteomics analysis of purified cell wall material has shown that the walls contain about 20 different types of covalently bound cell wall proteins (CWPs) at any time and the protein profiles can change dramatically depending on the growth conditions [8]. In addition, the presence of particular cell surface proteins morphologically depends and correlates with either *C. albicans* yeast or hyphal form [8]. The aim of this review is to discuss the characteristics and functions of covalently bound CWPs, and how they are important for fitness and virulence, and enable the fungus to cope with host infection-induced stress conditions. The review will also discuss the regulatory mechanisms that control expression of cell wall-related genes and relate what is known in *C. albicans* and other *Candida* species.

#### **2. Function of Cell Wall Proteins**

Based on the existing model of the cell wall, it is made up of an inner polysaccharide rich layer and the outer protein coat [9–11]. A 3-D nanoscalar model of the *C. albicans* cell wall has been developed to probe accurate thickness and structure of the cell wall [4,10]. The investigators used an optimized 3-D electron tomogram and computer vision technique to make accurate measurements of cell wall thickness [4]. The scalar model developed gave a more refined prediction of the thickness of each cell wall layer and the precise structure of some of the wall components [4]. The inner layer of the cell wall is composed mainly of β-glucans (β-1,3-glucan and β-1,6-glucans), chitin microfibrils, and a small amount of mannosylated proteins is distributed throughout the inner layer [4]. Chitin (β-1,4-*N*-acetyl glucosamine) and β-glucans (β-1,3-glucan) are the main structural polysaccharides of the cell wall [12]. β-1,3-glucan forms a three-dimensional network comparable to a flexible wire spring, which explains the elastic nature of the cell wall and provides the platform for the attachment of β-1,6-glucan, CWPs, and chitin [13]. Chitin is covalently cross-linked to the β-1,3-glucan network and contributes to the rigidity and physical strength of the fungal cell wall [12]. The outer coat is made up of glycan fibrils post-translationally attached to CWPs that are vertically arranged perpendicular to the inner layer [4].

The outer coat of mannoproteins determines cell wall permeability and surface charge [9]. Restriction of cell wall permeability is due to the densely packed CWPs, the presence of bulky protein sidechains, and the formation of disulfide bridges between CWPs [12,14,15]. This feature protects the structural polysaccharides against enzyme degradation and dectin-1 receptor recognition [15,16]. The use of genomic and proteomic techniques has advanced our knowledge of the nature and abundance of these surface proteins. CWPs have a unique structure, they generally contain: an N terminus with a secretory motif and a C terminus [17]. They bear serine/threonine-*O*-manno-oligosaccharide and/or asparagine-*N*-glycan and may contain internal repeats and/or a glycosylphosphatidylinositol, GPI anchor attachment sequences [18,19]. The most abundant cell proteins are the GPI-modified proteins, which receive a GPI anchor during their passage through the secretory pathway [20–24] and constitute about 88% of the total wall mannosylated protein classes [25] (Table 1). The second class of CWPs are those with internal repeats, PIR-CWPs [18,26] (Table 1).


**Table 1.** Characteristics of specific *Candida albicans* surface proteins.

A 3-D electron tomogram was used to determine the structure of the outer coat of mannosylated proteins. The scalar architectural model of mannosylated proteins gave a more precise detail of their structure, location and molecular size including measurements of their length and branching [4,71]. The cell wall through the outer proteins mediates host pathogen-interaction. The scalar architectural model may be useful in investigating the structure–function relationships that support the fungal infection strategy [4,10].

CWPs have both enzymatic and structural functions and their population may differ in their abundances depending on environmental conditions, developmental stage and phase of the cell cycle [9]. During cell wall synthesis, the cell wall polysaccharides, chitin and β-1,3-glucan are synthesized by enzymes localized in the plasma membrane and are extruded out to the cell exterior and are then acted upon by wall-localized cell wall remodeling enzymes [17]. CWPs modify these cell wall polysaccharides and cross-link them, thus maintaining cell wall integrity [9]. The cross-linking between cell wall macromolecules extruded into the wall space is catalyzed by carbohydrate active cell wall remodeling enzymes, hydrolases, transferases, and transglycosidases that are located in the cell wall space [17,72]. Some of these enzymes include β-1,3-glucanosyltransferases, e.g., Phr family (see Table 1), which are a Gas-like family, Bgl2 (GH17), and Crh family representing chitinglucanosyltransferases, these are cell wall-localized GPI anchored proteins [17,50,51,73–76]. *C. albicans phr1*∆/∆ and *phr2*∆/∆ mutants showed hypersensitivity to cell wall stressors such as Calcofluor white, CFW [45]. In *C. albicans*, synthetically lethal GPI-anchored proteins such as Dfg5 and Dcw1 (glycoside hydrolase (GH) family 76) are required for the incorporation of mannosylated proteins into the cell wall [55].

Structural surface proteins with no enzymatic activities such as flocculins (e.g., Flo1, Pga24), agglutinins (e.g., Als1, Rbt1, Hwp1), or β-1,3-glucan cross connectors (e.g., Pir1) that can form a scaffold for the attachment of other wall components, are important for cell:cell interactions and wall integrity [27,30,31,33,77–81]. Ssr1, a structural protein has been shown to contribute to normal cell wall architecture [82]. Pga59 is thought to be associated with the formation of a coat around the cell wall that can restrict cell wall permeability [64]. CWPs are also associated with virulence, biofilm formation, and coping with stress in fungi [33,36,61,67,68,83–85]. The following are some examples. The *ALS* gene family encodes eight GPI modified cell surface glycoproteins with peptide binding ability Ig-fold domain at the N terminus [86]. The Ig-fold mediates adhesion to fibronectin and other specific host proteins [87], and cell to cell aggregation through Als to Als interaction (Nobile et al., 2008). Heterologous expression of Als proteins in a nonadherent *S. cerevisiae* strain demonstrated that the Als proteins promote attachment to different surfaces (Nobile et al., 2008). The *als3*∆/∆ mutant has reduced virulence in a murine model of oropharyngeal candidiasis [31]. Hwp1 N terminus contains a secretory signal sequence rich in proline and glutamine that is cross-linked by host transglutaminase to epithelial cells enabling the attachment of *C. albicans* to human buccal epithelial cells [39,88,89]. *C. albicans hwp1*∆ mutant has reduced ability to bind to human buccal epithelial cells and has poor translocation from the mouse intestine into the bloodstream, demonstrating a role for Hwp1 in disseminated candidiasis [31]. Attachment to host cells by *C. albicans* can also be due to morphology-independent covalently bound wall proteins, Hyr1, Ecm33, Iff4, and Eap1, covalently bound wall proteins, Phr1, and cell-surface associated proteases, Sap9 and Sap10 [90,91].

*C. albicans* can use endocytosis (through interaction of Als3 with host cadherins) or active penetration to invade the host cell [31]. After *C. albicans* adhesion to the host cell surface and hyphal germination and growth, there are hyphal-induced hydrolytic enzymes that facilitate host cell degradation. They particularly aid active penetration into host cells and damage tissues [92].

*C. albicans* expresses ten secreted aspartyl proteinase (Sap) isoenzymes. Each mature Sap protein contains two aspartic acid residues conserved within the active site and a conserved cysteine residue that plays a structural role. Sap1–8 are secreted and released to the environment, whereas Sap9 and Sap10 are cell surface bound [63,93,94]. Sap proteins have been linked to the ability of *C. albicans* to adhere to and damage host tissue as well as the ability to evade the host immune response [95]. Sap9 and Sap10 have proteolytic activity on non-basic, basic, and dibasic peptides and have targeted Cht2, Ecm33, Pga4, Ywp1, Als2, Rhd3, Rbt5, and glucan cross-linked protein, Pir1 as substrates. *C. albicans sap9*∆/∆ and *sap10*∆/∆ mutants demonstrated reduction in cell wall-associated Cht2 activity suggesting a direct influence of Sap9 and Sap10 activity on Cht2 function and a role in maintaining cell wall integrity [96].

During *C. albicans* infection, Als family, and Eap1 adhesin, are involved in the development of biofilms, an important virulent attribute. The fungus forms biofilms when it encounters solid surfaces such as indwelling medical devices, where fungal cells are encapsulated in a dense extracellular matrix, which sequesters antifungal drugs promoting drug resistance and persistence in the host [97,98]. Biofilm formation by *C. albicans* has been shown to be under the positive regulatory control of the transcription factor, Bcr1. Bcr1 regulates the expression of Als1, Als3, and Hwp1 [34–36]. These proteins in addition to Als2 are associated with various stages of biofilm formation in *C. albicans* [28,99].

The cell wall during growth requires continuous remodeling of its macromolecular network [17]. During cell wall stress, a fungus can also rapidly remodel its wall and adapt the composition of the new cell wall [52,73,100]. For example, in exposure to cell damaging antifungal drugs, *C. albicans* triggers cell wall rescue mechanisms that influence the expression of wall biosynthetic genes and CWPs [4,60,101]. Rescuing the cell wall requires stress signals that activate the cell wall integrity (CWI) pathways. Cell surface proteins that function as mechanosensors primarily are responsible for activating these CWI pathways. These proteins (Wsc1-3 and Mtl1) act like linear nanosprings that detect and transmit cell wall damage or stress [102–105] to the downstream receptors in the signaling pathways. The sensors have an overall similar structure in that they contain in their sequences: short C terminal cytoplasmic domains, a single transmembrane domain, and a periplasmic ectodomain that is rich in Ser/Thr residues [106]. The Ser/Thr-rich regions are highly *O*-mannosylated, accounting for extension and stiffening of the proteins. Thus, these polypeptides have been proposed as mechanosensors that act as rigid probes of the extracellular matrix [106,107]. Functionally, signals are received and transmitted through the highly *O*-mannosylated extracellular domains and phosphatidylinositol (PI)- 4,5-bisphosphate, which recruits the N terminal domains of the Rom1/2-guanine nucleotide exchange factors through the plasma membrane, the sensors stimulate nucleotide exchange on Rho1 [102,105]. The various effectors of Rho1 include β-1,3-glucan synthase, β-1,3 glucan synthase activity, and Pkc1-activated MAPK cascade [104].

In summary, CWPs have a wide range of diverse functions that contribute to virulence, to maintenance of wall structure to ensure cellular integrity remains intact, and to sensing and transmitting signals from the environment. Many CWPs have been functionally characterized and their amino acid sequences are known, but only a handful have had their structures fully elucidated. Structure has a functional implication and understanding CWP structure can increase our knowledge of their functions, including roles in cell wall biogenesis.

#### **3. Fungal Cell Wall Remodeling and Signaling Pathways That Are Activated in Response to Stress**

*C. albicans* has been shown to grow at a high concentration of caspofungin a phenomenon called paradoxical growth. Paradoxical growth, in *C. albicans* is associated with induced cell aggregation and an increase in cell volume and cell wall chitin content [108]. In *C. auris*, however, it only induced an increase in cell wall chitin content [108]. Genes encoding Fks1 and Fks2 harboring the single nucleotide polymorphisms hot spot regions have been identified in *C. auris* [108]. The Fks2 carries the F635Y mutation that confers intrinsic echinocandin resistance on *Candida glabrata* [108]. Interestingly, *C. auris* RNA-seq data showed that paradoxical growth activates genes encoding cell membrane proteins and GPI-modified proteins required for cell wall damage response, chitin synthase, and

MAPKs such as Mkc1, and Hog1 involved in maintaining cell wall integrity [108]. Fungal pathogens activate a lot of pathways to successfully adapt to caspofugin stress. *J. Fungi* **2021**, *7*, x FOR PEER REVIEW 6 of 23

> Deletion of cell wall biosynthetic pathway genes in fungi often results in increased susceptibility of the cell wall to wall perturbing agents as well as alterations in chitin and β-1,3-glucan contents and linkages in the cell wall, synthesis of new wall proteins, and changes in the crosslinking to alternative wall polysaccharides [109,110]. Inhibition of β-1,3-glucan synthesis has been associated with altered crosslinking of chitin to β-1,6-glucan-GPI-modified proteins in the cell wall [109]. The amount of chitin→β-glucan←GPI-CWP complexes in the cell wall increased to 40% in wall defective mutants, indicating this is a repair mechanism protecting the cell wall from degrading enzymes and other stresses [109]. Most cell wall restructuring processes do not involve activation of the signaling pathways. For example, the carbon-source-induced alteration in osmotic tolerance in *C. albicans* was shown to be independent of the CWI pathways, but rather mediated by alterations in the architecture and biophysical properties of the cell wall [111]. However, during the cell wall response to most stressors, signals that indicate weaknesses in the wall are received by the surface sensors and transmitted leading to activation of the corresponding CWI pathways. In *Saccharomyces cerevisiae* and *C. albicans*, signaling pathways are activated in response to a wide range of stresses such as CFW, harsh temperatures, oxygen starvation, host immune response during infection and antifungal such as echinocandins, altered nutrient levels, and carbon source [109,112–114]. Cell wall stress response is mediated through the protein kinase C, PKC cell integrity mitogen-activated protein (MAP) kinase cascade, and its downstream transcription factors [112,114,115] (Figure 1). Other MAP kinase cascades, the high osmolarity glycerol response, HOG, and Candida ERK-like kinase, Cek1 mediated pathways, have also been shown to play a role in the cell wall reconstruction process [112,116,117] (Figure 1). MAP kinase defective *C. albicans* mutants display attenuated virulence in infection models showing that MAP kinase pathways are also important for virulence [118–120]. β-1,3-glucan contents and linkages in the cell wall, synthesis of new wall proteins, and changes in the crosslinking to alternative wall polysaccharides [109,110]. Inhibition of β-1,3-glucan synthesis has been associated with altered crosslinking of chitin to β-1,6-glucan-GPI-modified proteins in the cell wall [109]. The amount of chitin→β-glucan←GPI-CWP complexes in the cell wall increased to 40% in wall defective mutants, indicating this is a repair mechanism protecting the cell wall from degrading enzymes and other stresses [109]. Most cell wall restructuring processes do not involve activation of the signaling pathways. For example, the carbon-source-induced alteration in osmotic tolerance in *C. albicans* was shown to be independent of the CWI pathways, but rather mediated by alterations in the architecture and biophysical properties of the cell wall [111]. However, during the cell wall response to most stressors, signals that indicate weaknesses in the wall are received by the surface sensors and transmitted leading to activation of the corresponding CWI pathways. In *Saccharomyces cerevisiae* and *C. albicans*, signaling pathways are activated in response to a wide range of stresses such as CFW, harsh temperatures, oxygen starvation, host immune response during infection and antifungal such as echinocandins, altered nutrient levels, and carbon source [109,112–114]. Cell wall stress response is mediated through the protein kinase C, PKC cell integrity mitogen-activated protein (MAP) kinase cascade, and its downstream transcription factors [112,114,115] (Figure 1). Other MAP kinase cascades, the high osmolarity glycerol response, HOG, and Candida ERK-like kinase, Cek1 mediated pathways, have also been shown to play a role in the cell wall reconstruction process [112,116,117] (Figure 1). MAP kinase defective *C. albicans* mutants display attenuated virulence in infection models showing that MAP kinase pathways are also important for virulence [118–120].

**Figure 1.** Signaling pathways that regulate cell wall remodeling of *S. cerevisiae* and *C. albicans*. The Hog1, Cek1, and Pkc MAP kinase cascades and the Ca2+/calcineurin signaling pathway control a sensors of the MAP kinase cascades include Wsc family, Dfi1, Sho1, and Sln1, detect signals report- **Figure 1.** Signaling pathways that regulate cell wall remodeling of *S. cerevisiae* and *C. albicans*. The Hog1, Cek1, and Pkc MAP kinase cascades and the Ca2+/calcineurin signaling pathway control a number of

number of cellular processes including cell wall synthesis and maintenance. Upstream membrane

cellular processes including cell wall synthesis and maintenance. Upstream membrane sensors of the MAP kinase cascades include Wsc family, Dfi1, Sho1, and Sln1, detect signals reporting weakened cell wall or alterations in the wall, and convey the signal to the downstream components of the pathway. The PKC pathway plays an important role in response to caspofungin and activates Rho1, a regulatory sub-unit of β-1,3-glucan synthase. Rhb1, an Rheb-related GTPase, activate the CWI MAP kinase Mkc1 in response to cell wall stress. An Rhb1 deletion mutant is hypersensitive to cell wall stress and to rapamycin [121]. Rho1 activates protein kinase C, which phosphorylates and activates Bck1 in the MAP kinase cascade. Bck1 in turn activates the MAP kinase kinases Mkk1/2, which then phosphorylate Mkc1, which may hypothetically target Rlm1 in *C. albicans*. Although the Mkc1–Rlm1 relationship has been shown in *S. cerevisiae*, there is no evidence in *C. albicans* that Rlm1 is downstream of the Pkc pathway. A number of transcription factors contribute to the echinocandin stress response including Cas5 and Bcr1 [54]. In *C. albicans*, Cas5 is activated through an unknown mechanism involving dephosphorylation by Glc7 phosphatase [122]. Cas5 interacts with Swi4 and Swi6 to activate Cas5-dependent gene transcription leading to the upregulation of genes involved in cell biogenesis/integrity and cellular metabolism [122]. Cas5 and Efg1 have been shown to interact in response to caspofungin stress. Efg1 regulates the transcriptional response to cell wall damage by caspofungin [123]. *C. albicans efg1*∆/∆ mutant is hypersensitive to caspofungin [123,124]. Cas5 and Efg1 coregulate the expression of caspofungin-inducible genes. Cek1 pathway impinges on cell wall regulation and has also been implicated in systemic candidiasis [119,125]. *C. albicans* Dfi1, a homologue of *S. cerevisiae* Mid2/Mtl1 is known to partly activate the MAP kinase Cek1 and confer tolerance to caspofungin, CR, and CFW [126]. A Dfi1 deletion mutant is severely affected in invasive filamentation and virulence in a murine infection model. Msb2 in cooperation with Sho1 is also thought to play a role in Cek1 activation [127]. It is predicted that the transcription factor, Cph1 a homologue of *Sc*Stel2, is downstream of the Cek1 mediated pathway [112,119,127]. Cph1 is associated with regulation of filamentation [127]. The Rlm1 and Bcr1 transcription factors control the expression of a number of cell wall-related genes [34,128] with Bcr1 playing a dominant role in the regulation of biofilm formation by controlling expression of several important adhesins. In *C. albicans*, the Rlm1 activation mechanism is unknown, but once localized in the nucleus, activated Rlm1 leads to the upregulation of genes involves in cell wall biogenesis/integrity, macromolecular localization, and organelle localization [129]. Putative Rlm1 binding motifs in the promoters of *CHS2* and *CHS8* contribute to their cell wall stress-activated regulation [10,130,131]. In *S. cerevisiae*, Pkc1 is involved in targeting Chs3 to the plasma membrane in response to heat shock [129,132]. Significant re-wiring of signaling pathways is evident in *C. albicans*, compared to the *S. cerevisiae* paradigm, for example, the role of the Sko1 transcription factor in response to caspofungin is independent of Hog1 MAP kinase, but involves the Psk1 PAK kinase [133] and Rlm1. In *C. albicans*, Sko1 regulates the expression of some genes involved in cell wall biogenesis and remodeling, and osmoadaptation [133]. Sko1 binding motif has been identified for regulating Sko1-dependent genes. Sko1 also binds to its motif to promote self-activation. The calcineurin pathway is activated by calcium that may enter the cells through membrane-localized channels Cch1 and Mid1 or a third minor channel Fig1. Alternatively, the pathway may be activated by calcium released from intracellular stores. Ca2+ binds to and activates calmodulin (Cmd1) that in turn activates the phosphatase calcineurin. The calcineurin is made up of two sub-units, Cna1 and Cnb1. Calcineurin dephosphorylates the transcription factor Crz1, which moves into the nucleus and induces expression of genes through binding to CDREs (calcium dependent response elements) within their promoter sequences. Two Crz1 DNA binding motifs have been identified in some genes regulated by Crz1. Adapted from [112,114,126]. CR = Congo red, CS = caspofungin, CFW calcofluor white, CWM = cell wall matrix, PM = plasma membrane.

There is some redundancy in the regulatory networks responding to echinocandininduced cell wall damage where more than one transcription factor controls overlapping sets of downstream target genes to control changes in the cell wall [54,122,133,134]. Three transcription factors, Cas5, Sko1, and Rlm1 have been implicated in echinocandin-induced cell wall damage signaling [54,133].

Cas5 has been shown to be involved in cell wall remodeling in *C. albicans* during cell growth, morphology, and virulence [54,122,135,136]. *C. albicans cas5*∆/∆ mutants and including mutants with a missense mutation in Cas5 DNA-binding domain is hypersensitive to caspofungin and other cell wall stressors such as CFW [54,122]. A *cas5*∆/∆ deletion mutant has also been shown to have attenuated virulence in both murine and invertebrate models of systemic candidiasis [135]. Genome-wide microarray studies showed that Cas5 regulates about 50% of the highly expressed caspofungin-inducible genes, including some cell wall integrity genes [54]. Studies using RNA polymerase II chromatin immunoprecipitation and sequencing analyses showed that the number of caspofungin-inducible genes is markedly higher and genes with cell wall-associated functions were markedly overrepresented [122]. Furthermore, Cas5 was found to regulate over 60% of caspofungin-inducible genes, including those involved in cell wall integrity [122].

Information on the upstream regulation of Cas5 is limited in *C. albicans*, but available data suggest that Cas5 is dephosphorylated by phosphatase Glc7 following caspofungininduced cell wall damage [122]. The study further showed that upon dephosphorylation of Cas5, it is activated and interacts with Swi4 and Swi6 to activate the transcription of Cas5-dependent genes [122]. This leads to the upregulation of genes involved in cell wall synthesis/integrity and cell metabolism [122].

Cas5 together with Efg1 regulate the transcriptional response to cell wall damage by caspofungin [123]. Efg1 is a member of the APSES family of basic helix-loop-helix transcriptional regulators that is proposed to function downstream of the cAMP/protein kinase A (PKA) pathway to induce a hyphal transcription program [137,138]. Likewise, Efg1 is important for transcriptional responses to echinocandins and *C. albicans efg1*∆/∆ mutant is hypersensitive to caspofungin [124]. Efg1 also required for the induction of *CAS5* in response to cell wall damage by caspofungin [125]. Deletion of *EFG1* in a *cas5*∆/∆ mutant exacerbates caspofungin hypersensitivity and make caspofugin-resistant *C. albicans* sensitive again. The ectopical expression of *CAS5* could not salvage the growth defect of *C. albicans efg1*∆/∆ mutant treated with caspofungin [123]. Genome wide transcription profiling of *C. albicans cas5*∆/∆ and *efg1*∆/∆ mutants using RNA-Seq showed that Cas5 and Efg1 can coregulate the expression of caspofungin-inducible genes and can also independently regulate some genes [123]. Using yeast two-hybrid and in vivo immunoprecipitation, Cas5 and Efg1 were shown to interact and bind to the promoter of some caspofungin-inducible genes to coordinately activate their expression [123].

Efg1 has also been shown to regulate Czf1 expression [139,140]. Czf1, a *C. albicans* zinc finger cluster transcription factor, is required for white-opaque switching and filamentation [141]. Efg1 and Czf1 interact in a yeast two-hybrid experiment [140] and coordinate responses to farnesol during quorum sensing and white-opaque thermal dimorphism [142]. In the screen of a library of genetically activated forms of zinc cluster transcription factors, hyperactive Czf1 was found to have a cell wall associated function in *C. albicans* [143]. Hyperactive Czf1 drives the expression of many CWPs with cell wall associated functions that can induce a physical change in the cell wall architecture and rescue the hypersensitivity of different CWI partway deletion mutants to cell wall perturbing agents [143]. In addition, *C. albicans czf1*∆/∆ mutant is hypersensitive to caspofungin [143].

Downstream of the Pkc pathway is the transcription factor, Rlm1. Rlm1 has been extensively studied in *S. cerevisiae* where it is the main transcriptional regulator of the Pkc CWI pathway [144,145]. However, our understanding of the function of the protein is limited in *C. albicans*. *C. albicans rlm1*∆/∆ mutant is hypersensitive to CFW and Congo red [54] and analysis of mutant cell wall composition compared to wild type showed marked reduction in mannan composition and an increase in chitin levels [128]. This suggested that Rlm1 is involved in caspofungin induced CWI signaling. These characteristics of *rlm1*∆/∆ in *C. albicans* have not been observed in *S. cerevisiae*, showing divergence of these orthologues [128]. In *C. glabrata,* which is more closely related to *S. cerevisiae* than *C. albicans*, *rlm1*∆/∆, *mkk1*∆/∆, and *bck1*∆/∆ mutants are sensitive to caspofungin, but not to CFW or Congo red [146] and the full influence of this pathway on cell wall regulation is yet to be studied. Genome-wide microarray studies in *C. albicans* showed that Rlm1 only induced the expression of five genes under basal condition and only two of these

genes were caspofungin-inducible [54]. Another genome-wide study demonstrated that Rlm1 regulated the expression of 773 genes under basal conditions [128] and some of the highly upregulated genes have cell wall associated function. These data suggest that Rlm1 may have a more general regulatory role in controlling cell wall associated gene during non-stressed physiological activities. Genome-wide ChIP Seq data revealed that Rlm1-target genes encode proteins that have cell wall-associated function [134]. Rlm1 bound to the upstream intergenic regions of 25 genes and 18 of the genes were highly caspofungin-inducible [134]. Furthermore, a *rlm1*∆/∆ mutant attenuated virulence in a murine model of systemic candidiasis [128].

Orthologues of Pkc pathway are conserved in *C. albicans*; however, it is not known if the Mkc1 directly or indirectly activates Rlm1. Genomic, biochemical, and cellular data suggest circuit rewiring in Rlm1 and Sko1 CWI signaling [134]. Sko1 function has been extensively studied in *S. cerevisiae* and shown to be part of the MAP kinase high osmolarity glycerol, Hog, signaling pathway with a role in osmotic and oxidative stress responses [147]. The Hog pathway in *C. albicans* is associated with pathogenicity traits and it is involved in the control of both pathogenic and commensal state programs [148]. Sko1 function as the regulator of osmotic stress is conserved in *C. albicans* and it is phophorylated by the MAP kinase Hog1 following osmotic shock [133]. However, Sko1 regulates genes in *C. albicans* whose orthologues in *S. cerevisiae* are not involved in osmotic stress response, therefore showing circuitry rewiring [149]. Sko1 function in regulating the oxidative stress response is also conserved in *C. albicans* [150].

A *sko1*∆/∆ mutant is hypersensitive to caspofungin, but not to Congo red, CFW, or SDS [148,150], suggesting Sko1 may not have such a global role in cell wall architecture as Rlm1 or Cas5. Microarray and RT-qPCR data demonstrated that Sko1 regulates 81 caspofungin-inducible genes and 26 of these genes are upregulated by Sko1 [133]. Several of the genes regulated by Sko1 have cell wall-associated function (*PGA13* and *CRH11*), and cell metabolism functions [133,134].

The upstream regulatory mechanisms controlling Sko1 expression in *C. albicans* are more complex than in *S. cerevisiae*, where the Hog pathway principally regulates Sko1 transcriptional activity. Caspofungin-induced Sko1 activity is independent of Hog pathway function [133]. RT-qPCR data demonstrated that caspofungin markedly induces *SKO1* transcription and this requires the glucose-partitioning PAS kinase, Psk1 [133], but Psk1 does not regulate transcription directly. Microarray data indicate that Rlm1 regulates *SKO1* expression under basal conditions [128]. Furthermore, caspofungin-induced *SKO1* expression is markedly reduced in *rlm1*∆/∆ mutant but not in *cas5*∆/∆ mutant. It is unknown if Psk1 binds directly to Rlm1 to regulate its activation of *SKO1* expression. However, a DNA-binding consensus has been identified in the Sko1 promoter sequence for regulating Sko1 inducible genes and also for autoactivation of *SKO1* [134].

The Hog pathway has not been well studied in other *Candida* species. However, in clinical strains of *C. auris*, Hog1 and Ssk1 have been shown to have variable activities, which suggested some sort of genetic flexibility with effects on cell wall function and stress adaptation [151]. A *C. auris ssk1*∆/∆ *hog1*∆/∆ mutant had altered tolerance to caspofungin and amphotericin B, with increased echinocandin susceptibility [151]. The mutant also had altered cell wall mannan content and altered hyper-resistance to cell wall stressors [151]. Targeting these two signaling components of the Hog pathway may provide options for an effective combination therapy or enhancement of echinocandin susceptibility.

Phosphotransferase regulator Ypd1 and phosphatase Ptp2 have been identified as the Sko1 targets following caspofungin treatment of *C. albicans* [134]. Both Ypd1 and Ptp2 are known to inhibit the Hog pathway, indicating that Sko1 blocks the Hog pathway following caspofungin treatment [152,153]. There is also cross communication between the Hog1 and Cek1 pathways under basal condition [154] and *C. albicans hog1*∆/∆ mutants have constitutively higher levels of Cek1 phosphorylation [117].

The Cek1 pathway is involved in cell pathogenesis and participate in cell wall construction [155,156]. Cell surface signals that activate the Cek1 pathway are transmitted

by membrane bound sensor: Sho1, Msb2, and Opy2 [154–156], and mediated through Cph1 and Tec1 [157,158]. Signals through the sensors trigger stimulus through Cst20 to the Ste11-Hst7-Cek1 MAPK cascade [119]. Deletion of any of these downstream elements as well as Cph1 does not affect filamentation [159]. Cek1 has also been shown to target another transcription factor, Ace2 to upregulate genes encoding protein *O*-mannosyltransferases in response to defective protein *N*- or *O*-glycosylation activities [160]. Cell surface proteins are post-translationally modified to maintain cell wall structure. Genes encoding components in the Cek1 pathway, *MSB2*, *CST20*, *HST7*, *CEK1*, and *ACE2* are Ace2 targets, indicating Ace2-mediated transcriptional upregulation of pathway genes under *N*-glycosylation stress [160].

In *C. albicans* and most other fungi, cell damage through the inhibition of β-1,3-glucans synthesis triggers compensatory chitin synthesis [101,114,161–163]. We have shown that Pkc, Hog, and Ca2+ signaling pathways co-ordinately regulate chitin synthesis in response to cell wall stress [72,110,163]. These pathways regulate *CHS* gene expression and chitin synthesis individually and in concert, leading to rearrangement of wall macromolecules in response to cell wall stresses [110]. A *lacZ* reporter gene was fused to the putative promoters of each of the *CHS* genes of *C. albicans* to monitor the expression of *CHS* genes when treated with cell wall perturbing agents such as CFW and showed that exogenous Ca2+ , which induces the calcineurin pathway, activated all the *CHS* genes in a Crz1-dependent manner [110]. Crz1 is the downstream transcription factor in the Ca2+/calcineurin signaling pathway. Treating *C. albicans* cells with CFW, which activates the Pkc pathway, results in a three-fold increase in chitin content [101]. However, hyper-stimulation of *CHS* gene expression was observed when Pkc and Ca2+ pathways were simultaneously activated, and this resulted in increased chitin in the cell wall [110]. In *S. cerevisiae* and *C. albicans*, the Pkc and Hog MAP kinase cascades and the Ca2+/calcineurin pathway have been shown to regulate CWPs, such as Sed1, Pst1, Crh1, Cwp1, Ssr1, Yps1, Pir1, and Pir3, involved in cell wall remodeling activities [51,134,145,164–167].

The Ca2+/calcineurin signaling pathway is implicated in the activation of cell wall remodeling processes in response to damage to the cell wall [52,101] (Figure 1). The proposed model for Crz1 regulation in *C. albicans* is that the influx of Ca2+ activates calcineurin that then dephosphorylates and activates Crz1. The activated Crz1 enters the nucleus and binds to one or both Crz1 binding motifs in the promoter of target genes leading to their expression [168]. Crz1 has been shown to regulate the expression of 34 genes involved in cell wall biosynthesis in response to calcium stress and 12 of these genes encode proteins that are covalently bound to the cell wall: *CRH11*, *UTR2*, *PGA1*, *PGA6*, *PGA13*, *PGA23*, *PGA39*, *PGA52*, *PGA20*, *ECM331*, *PHR2*, *DFG5* [168]. Microarray and RNA sequencing data have reveal that Crz1 binds in vitro and in vivo to two identified motifs (calcineurin dependent response element, CDRE) in the promoter of some of the target genes [53,168] to induce their expression. The promoter of 79 genes regulated by Crz1 have two binding motifs for Crz1, while 104 Crz1-regulated genes have only one motif. Meanwhile, 36 Crz1 regulated genes have no discernible Crz1 binding motifs [168]. This suggests that the expression of Crz1 target genes is differentially regulated. It has been shown that Crz1 binds to two motifs in the promoter region of *UTR2* to induce expression in response to calcium stress [168].

*C. albicans* lacking calcineurin is markedly attenuated in virulence in a murine model of systemic candidiasis and cannot survive in the presence of cell membrane stressors [169–171]. *C. albicans* lacking Crz1, the major target of calcineurin is partially virulent in a murine model of systemic candidiasis, indicating the existence of other calcineurin targets that are important for virulence [168,172,173].

Another determinant of caspofungin sensitivity is the transcription factor, Cup9, which is required for normal caspofungin tolerance in hyphae alone and activates the expression of CWPs with cell wall function [174]. *C. albicans cup9*∆/∆ mutant is hypersensitensive to caspofungin stress. RNA-seq data from *C. albicans cup9*∆/∆ mutant with or without caspofungin demonstrated that Cup9 has a narrow rather than global effect in the cell wall damage response and activates proteins such as *PGA31* and *IFF11* with a known role in cell wall integrity [174].

Generally, these signaling pathways have not been studied in detail in other *Candida* species; however, in *C. glabrata*, 3 genes: *SLT2*, *YPK2*, and *YPK1*, whose protein products are involved in cell wall maintenance are associated with in vivo and in vitro echinocandins tolerance [175]. A *C. glabrata* strain lacking these three genes was susceptible to caspofungin treatment in a murine model of gastrointestinal candidiasis [175]. Furthermore, genes encoding ortholoques of kinases in the cell wall signaling pathway *SLT2*, *MKK1*, *BEM2*, and *SW14* were identified in *C. glabrata* as well as genes encoding ortholoques of calcineurin pathway membrane components: *CCH1* and *MID1* [146]. Mutants lacking any of these genes were hypersensitive to caspofungin [146]. Deletion of genes representing all stages of CWI pathway from surface sensing to transcription regulation resulted in various degrees of susceptibility to caspofungin and cell wall degrading enzymes [146]. Although more studies are required to understand caspofungin-induced cell wall stress responses in other *Candida* species, available data suggest similarity in signaling pathways, the response strategies deployed, and the wall proteins involved in maintaining cell wall integrity.

#### **4. Cell Wall Remodeling in Response to Thermal Stress**

The fungal response to heat shock has been well characterized [176–178]. Temperature stress signals are thought to be sensed by signaling mucins. Signaling mucins are transmembrane glycoproteins that receive and transmit surface signals to signaling pathways (Figure 2). Signaling mucin, Msb2 is known to regulate environmental stress, cell wall biogenesis, and the Cek1 and Pkc pathways in most fungi [178,179]. Msb2 is a global regulator of temperature stress in *C. albicans* [113]. Msb2 is required for fungal survival and hyphae formation at 42 ◦C. Msb2 also regulates temperature-dependent activation of genes involved in MAP kinase and unfolded protein response pathways (Figure 2) [113].

Generally, the temperature stress response is controlled by an essential protein, the heat shock transcription factor, Hsf1, which is phosphorylated upon sudden temperature rise [180]. Following temperature rise from 30 to 42 ◦C, Hsf1 is phosphorylated rapidly within 60 s and upon adaptation, downregulated [181]. Under normal growth conditions, Hsf1 binds as a trimer to heat shock elements (HSEs) in the promoters of target heat shock protein (*HSP*) genes [182]. When *S. cerevisiae* or *C. albicans* cells experience an acute heat shock, Hsf1 is hyper-phosphorylated and activated, resulting in the transcriptional induction of the target *HSP* genes, thus stimulating cellular adaptation to the thermal insult [183]. Most heat shock proteins, Hsp, are molecular chaperones that promote client proteins folding, assembly, or cellular localization. They also often target unfolded or damaged proteins for degradation [184]. In *C. albicans*, Hsf1 interacts with Hsp such as Hsp90 under steady-state conditions, and upon thermal shocks, this interaction is strengthened, suggesting existence of a Hsf1-Hsp90 autoregulatory circuit [177]. Hsp90 is localized to the nucleus during elevated temperatures. It is possible that the Hsf1-Hsp90 regulon is critical for the maintenance of thermal homeostasis, not merely for adaptation to acute heat shocks. This suggests that the Hsf1-Hsp90 interaction is important for regulation of short-term responses to heat shock (Figure 2).

**Figure 2.** Hsp90 acts as a biological transistor, modulating Hsf1 and the MAPKs transcription factors in response to thermal fluctuations. Msb2 plays a vital role in thermotolerance in *C. albicans*. The protein transmits heat shock signals through unknown mechanisms that induce downstream targets such as the Pkc pathways in response to high temperatures. Hsf1 activation is required for thermotolerance. The MAP kinase signaling pathways are also required to promote thermotolerance through remodeling the cell wall [117,185]. Because Hsp90 coordinates much of this activity, Hsf1, Hog1, Mkc1, and Cek1 are all thought to be Hsp90 client proteins [177,181]. Fluctuations in ambient temperature affect interactions between Hsp90 and Hsf1, and probably affect Hsp90 interactions with the MAP kinase transcription factors [181], thus modulating the role of the signaling pathways and thermal adaptation outcome. Temperature upshifts activate Hsf1, which induces the expression of protein chaperones (HSPs), including Hsp90, which promotes shorter term thermal adaptation. It is thought that Hsp90 then down-regulates Hsf1 and modulates MAP kinase signaling, to alter cell wall architecture, which leads to long term thermotolerance in *C. albicans*. Adapted from [177]. Broken lines indicate unconfirmed regulatory mechanisms. **Figure 2.** Hsp90 acts as a biological transistor, modulating Hsf1 and the MAPKs transcription factors in response to thermal fluctuations. Msb2 plays a vital role in thermotolerance in *C. albicans*. The protein transmits heat shock signals through unknown mechanisms that induce downstream targets such as the Pkc pathways in response to high temperatures. Hsf1 activation is required for thermotolerance. The MAP kinase signaling pathways are also required to promote thermotolerance through remodeling the cell wall [117,185]. Because Hsp90 coordinates much of this activity, Hsf1, Hog1, Mkc1, and Cek1 are all thought to be Hsp90 client proteins [177,181]. Fluctuations in ambient temperature affect interactions between Hsp90 and Hsf1, and probably affect Hsp90 interactions with the MAP kinase transcription factors [181], thus modulating the role of the signaling pathways and thermal adaptation outcome. Temperature upshifts activate Hsf1, which induces the expression of protein chaperones (HSPs), including Hsp90, which promotes shorter term thermal adaptation. It is thought that Hsp90 then down-regulates Hsf1 and modulates MAP kinase signaling, to alter cell wall architecture, which leads to long term thermotolerance in *C. albicans*. Adapted from [177]. Broken lines indicate unconfirmed regulatory mechanisms.

Cell wall integrity is compromised at elevated temperatures. Temperature affects cell wall polysaccharide composition and the incorporation levels of covalently anchored proteins [186]. Yeasts cells are thought to adapt to heat stress in the longer term by activating the Hog1, Mkc1, and Cek1 MAP kinase pathways, which contribute to thermotolerance [177,186] (Figure 2). These MAP kinase pathways, even though they contribute to thermal adaptation in the longer term through cell wall remodeling, are not essential for Hsf1 activation. Genetic depletion of Hsp90 affects cell wall remodeling activities, suggesting that Hog1, Mkc1, and Cek1 may be client proteins of Hsp90. Hsp90 is thought to be able to integrate both the short term and longer-term molecular responses that underpin thermotolerance [177] (Figure 2). Cell wall integrity is compromised at elevated temperatures. Temperature affects cell wall polysaccharide composition and the incorporation levels of covalently anchored proteins [186]. Yeasts cells are thought to adapt to heat stress in the longer term by activating the Hog1, Mkc1, and Cek1 MAP kinase pathways, which contribute to thermotolerance [177,186] (Figure 2). These MAP kinase pathways, even though they contribute to thermal adaptation in the longer term through cell wall remodeling, are not essential for Hsf1 activation. Genetic depletion of Hsp90 affects cell wall remodeling activities, suggesting that Hog1, Mkc1, and Cek1 may be client proteins of Hsp90. Hsp90 is thought to be able to integrate both the short term and longer-term molecular responses that underpin thermotolerance [177] (Figure 2).

In *S. cerevisiae*, MAP kinase pathways have been shown to contribute to thermotolerance *[132,187]*, through localization of Chs3 to the plasma membrane in response to heat shock *[129].* Each of these MAP kinase pathways is known to contribute to cell wall remodeling and mutations that interfere with cell wall synthesis increase sensitivity of *C. albicans* to elevated temperatures*.* For example, the deletion of certain protein mannosyltransferases of the PMT family, or the inactivation of *OCH1* can increase susceptibility to temperature [188,189]. Furthermore, deletion of *SSR1* causes elevated susceptibility to temperatures [60]. In *S. cerevisiae*, MAP kinase pathways have been shown to contribute to thermotolerance [132,187], through localization of Chs3 to the plasma membrane in response to heat shock [129]. Each of these MAP kinase pathways is known to contribute to cell wall remodeling and mutations that interfere with cell wall synthesis increase sensitivity of *C. albicans* to elevated temperatures. For example, the deletion of certain protein mannosyltransferases of the PMT family, or the inactivation of *OCH1* can increase susceptibility to temperature [188,189]. Furthermore, deletion of *SSR1* causes elevated susceptibility to temperatures [60].

In a study, thermal upshift was shown to cause reduced secretion of chitinases and have a huge impact on cell wall *N* mannan composition [190]. Analysis of the cell wall phospholipomannan moiety revealed reduction in *N* mannan composition of β-1,2-mannose [190]. *C. albicans* is more susceptible to cell wall stressors when grown at 42 °C [186]. In a study, thermal upshift was shown to cause reduced secretion of chitinases and have a huge impact on cell wall *N* mannan composition [190]. Analysis of the cell wall phospholipomannan moiety revealed reduction in *N* mannan composition of β-1,2-mannose [190]. *C. albicans* is more susceptible to cell wall stressors when grown at

42 ◦C [186]. Coping with this thermal stress leads to increased phosphorylation of Mkc1, which mediates activation of the CWI pathways. Consequently, the levels of Sap9, the chitin transglycosylases Crh11 and Utr2, and the cell wall maintenance protein, Ecm33, increased, and cells reinforce their walls with chitin through increased chitin synthesis and reduced chitin degradation [186]. Ecm33 is required for growth at high temperatures and *S. cerevisiae* and *C. albicans ecm33*∆/∆ disruptant strains exhibit a temperature sensitive growth defect [191,192].

The Mkc1, Hog1, and Cek1 signaling pathways and associated cell wall remodeling mannoproteins have been proposed to promote longer term thermotolerance through the maintenance of a robust cell wall (Figure 2).

#### **5. Echinocandin-Induced Cell Wall Remodeling in Yeast**

β-1,3-glucan is a hallmark component of most yeast cell walls and is synthesized by β-1,3-glucan synthase. The protein has an integral membrane catalytic subunit, Fks [193]. *C. albicans* has three *FKS* genes, but the main activity is from the *FKS1* gene product, Fks1. Fks1 is essential and found in association with the regulatory subunit, Rho1 GTPase [194]. Rho1 is required to activate Fks1 for β-1,3-glucan synthesis (Figure 1). Echinocandins non-competitively inhibit β-1,3-glucan synthesis by inhibiting the catalytic function of Fks1, leading to a weak cell wall [195]. Echinocandins are fungicidal against *Candida* species and resistance to the drug has been predominantly associated with point mutations in the *FKS1* gene. However, most yeast have been shown to withstand caspofungin treatment, becoming more tolerant to the drug both in vivo and in vitro by inducing the upregulation of chitin synthesis, the second wall structural polysaccharide [162,163].

Chitin is synthesized by chitin synthase enzymes and *C. albicans* has four chitin synthase proteins comprising of *Chs1*, *Chs2*, *Chs3*, and *Chs8*. Elevated cell wall chitin is a cell wall rescue mechanism shown to be orchestrated by the CWI pathways [101,110,196]. Pkc, Hog, and Ca2+ signaling pathways have been shown to control the expression of *CHS2* and *CHS8* through binding motifs in their promoter sequences [131]. Hyper-stimulation of *CHS* gene expression was observed when the three signaling pathways were activated at the same time and this leads to elevated cell wall chitin content [110]. Cell wall mutants with higher basal chitin contents are also less susceptible to caspofungin [60,197]. Chitin synthase proteins can also synthesize alternative septa that restore *C. albicans* capacity to bud during cell wall stress [198].

Genome wide studies have been carried out to study the response of fungal cells to echinocandin drugs treatment and to identify genes whose upregulation is required for adaptive growth in the presence of sub-MIC concentrations of echinocandins. DNA microarrays studies identified genes that are activated in *S. cerevisiae* and *C. albicans* when they are challenged with sub-MIC concentrations of caspofungin [54]. The induced genes include those genes that are typically upregulated following the activation of the Pkc pathway. In *C. albicans* and *S. cerevisiae*, some of the Pkc pathway signature genes: *CRH11*/*CRH1*, *ECM331*/*PST1*, *DFG5*, encode GPI anchored cell surface proteins that have been implicated in cell wall biogenesis or repair [51,55,199–202]. Pga31, a predicted GPI anchored wall protein, is upregulated during caspofungin stress, and *pga31*∆/∆ mutants have thinner cell walls, reduced chitin content, and are hypersensitive to caspofungin [60]. Cas5 regulates the expression of some CWPs in response to caspofungin, including Crh11, Ecm331, Pga13, and Pga23 [54]. Pga13 plays a role in cell wall architecture [203] and may be required for cell wall repair.

The phosphorylated form of the Pkc pathway component Mkc1/Slt2 and phosphorylated form of Cek1 have been detected in *S. cerevisiae* and *C. albicans* [155] following caspofungin challenge. Furthermore, *C. albicans mkc1*∆/∆ mutant is hypersensitive to caspofungin [101]. This suggests that the Pkc pathway is a major signaling pathway for triggering cell wall macromolecule rearrangement in response to caspofungin stress in *S. cerevisiae* and *C. albicans* [101,196].

#### **6. Cell Wall Remodeling and Protein Abundance**

In an analysis of the cell wall proteome of *C. albicans* growing on minimal medium without stress using liquid chromatography-mass spectrometry, LC-MS revealed 21 covalently bound CWPs. Out of the 21 CWPs identified, 19 had predicted GPI anchor sequence with cell wall associated function [204]. In other studies, the proteomics technique was used to study the impact of carbon source on the *C. albicans* cell wall proteome and secretome when cells were grown in minimal medium containing 2% glucose, lactate, or glucose plus lactate [73,205]. The results revealed higher amounts of predicted GPI anchored CWPs with functions in cell wall biogenesis/integrity in the secretomes and proteomes. Major differences were seen in the profiles of secreted and CWPs in lactate and glucose-grown *C. albicans* cells. Many of the differences suggested that specific cellular processes associated with the cell surface such as cell wall remodeling, adherence, and biofilm formation, may be affected by the change in carbon source [73]. The secretome and proteome of lactate grown cells had increased levels of proteins involved in the remodeling of β-glucan [73]. Lactate grown cells were more adherent, and consequently, more virulent in in vivo models of systemic candidiasis and vaginitis, and display increased resistance to caspofungin as well as other stressors [111]. Lactate signaling regulates glucan masking and modulates the immune response [206]. Furthermore, elevated stress resistance did not correlate with increased activation of the CWI pathways, thus the observed phenotypes may be due to the alteration in the architecture as well as the biochemical and biophysical properties of the cell wall [111]. However, Hog1 or Mkc1 signaling pathways mediate expression of CWPs that promote cell wall elasticity required for adaptation to hyperosmotic stress [52]. Interestingly, alterations in the cell wall in response to different media or carbon sources have been shown to involve changes in the molecular weight of mannoproteins [207]. Mannoproteins from *C. albicans* cultivated on blood or serum have increased molecular weight, when compared with mannoproteins from cells grown on YPD at 30 and 37 ◦C [207].

A microarray study using DAY185 *C. albicans* strain with or without caspofungin treatment identified 216 caspofungin-inducible genes with an expression change of at least two-fold following 1-h caspofungin treatment [54]. A core set of 34 caspofungin stress inducible genes included genes that are known to be involved in cell wall remodeling such as *PGA13*, *CRH11*, and *PHR1* [54]. In addition, *C. albicans* grown in vagina-simulative medium, aerated with a gas mixture reflecting the gas composition in the vaginal environment had five CWPs [Als3, Hwp1, Sim1, Tos1, Utr2) in the wall that were absent in the YPD grown control [38]. However, O<sup>2</sup> restriction led to higher levels of the non-GPI protein Pir1, β-1,3-glucan cross-linking protein, and of the GPI anchor protein, Hwp1, an adhesion protein [38].

Environmental pH has also been shown to greatly alter the fungal cell wall proteome. Klis lab used a system that mimics mucosal surfaces to investigate the influence of host pH on *C. albicans* cell wall proteome [208]. At pH 4.0, yeast cells and pseudohyphae were predominantly seen while at pH 7.0, hyphal growth was mainly seen. Relative quantitation of <sup>15</sup>N-labelled CWPs using ESI-FT-MS revealed the identity of 21 covalently linked CWPs, most of which are GPI anchored, excluding Tos1, Mp65, and Pir1. At pH 7.0, Als1, Als3, Hyr1, Phr1, Rbt1, Sod5, and Tos1 were identified, while only the transglycosidase, Phr2 was found at pH 4.0. Furthermore, at pH 4.0, 12 out of the 21 CWPs were overexpressed, whereas at pH 7.0, 9 proteins were overexpressed. The proteome of the *C. albicans* cell wall is constantly reshuffled to enable cells to adapt to prevailing environmental conditions. The consequences of not adapting to that changing environment is cell death. This is why the cell wall, and its components, are attractive targets for developing more effective diagnostics and therapeutics.

#### **7. Perspective**

The covalently bound CWPs in the protein coat are indispensable for the survival of *C. albicans* in the environment and during infection. They also play a major role in the development of biofilms and are regulated by signaling pathways that help remodel the cell wall during stress. However, our knowledge of their structure, which may influence their function regarding structure-function relation is limited and our understanding of their exact function in many cases is still poor. This calls for a continued functional analysis of fungal CWPs. The regulatory mechanisms associated with the construction of the cell wall protein coat are not well understood. The precise mechanism of coupling these proteins to cell wall and their method of interaction with wall polysaccharide and other proteins in the cell wall, which may affect their localization and hence their function, are still not clear. Understanding the function and regulatory mechanisms of these CWPs will ultimately inform our knowledge of fungal pathogenesis and host-pathogen interactions.

CWPs have carbohydrate-binding motifs and may thus be involved in cell wall synthesis and remodeling, in biofilm formation, or even in the interaction with host cell receptors or other environmental signals. Most importantly, our knowledge of the exact roles CWPs play in CWI pathways, their downstream signaling activities, and the extent of their involvement in the cross interactions between the pathways during cell wall stress is relatively unexploited. The cell wall proteome can change significantly in response to specific environmental stress, including during infection. The fungal cell wall proteome changes associated with infection conditions need more extensive studies, as the cell wall in vivo is likely to be very different to the wall generated under laboratory growth conditions. Finally, the relative and absolute quantitation of CWPs under host-related conditions and an extensive understanding of their exact structure and functions will be vital in identifying the most suitable diagnostic, therapeutic, and vaccine candidates.

**Author Contributions:** Conceptualization, C.I. and C.A.M.; original draft preparation, C.I.; review and editing, C.I. and C.A.M. All authors have read and agreed to the published version of the manuscript.

**Funding:** This research was funded by Abia State University and the University of Aberdeen.

**Institutional Review Board Statement:** Not applicable.

**Informed Consent Statement:** Not applicable.

**Conflicts of Interest:** The authors declare no conflict of interest.

#### **References**

