**1. Introduction**

Cryptococcosis is a fungal infectious disease ubiquitously distributed around the world [1]. Two fungal species, *Cryptococcus neoformans* and *C. gattii*, are the main infectious agents causing cryptococcal meningitis in both immunocompetent and immunocompromised humans [1–4]. This disease is the major cause of morbidity and mortality among people living with advanced HIV and annually accounts for 15% of all HIV-related deaths globally [5,6]. The burden of HIV-associated cryptococcal disease in Sub-Saharan Africa is alarming, as 73% of deaths in the world are reported in this region [5,6]. Apart from these opportunistic pathogens, the genus *Cryptococcus* contains species with biotechnological potential (Table 1). Among the cryptococcal species, *C. amylolentus* is closely

related to the pathogenic *C. neoformans* and is extensively used for comparative studies to identify the pathogenic traits in *C. neoformans* [7].

The genus *Cryptococcus* belongs to *Tremellomycetes*, a fungal class in the subphylum *Agaricomycotina*, which contains organisms adapted to different niches and/or having different lifestyles (Table 1). Some of the organisms in this class are now regarded as emerging opportunistic human pathogens and some species are adapted to extreme ecological niches, such as cold regions (Table 1). Despite being fungi, *Naematella encephala* and *Tremella mesenterica* Fries exhibit fungal parasitism. The diverse lifestyles or characteristics of some species of *Tremellomycetes* are summarized in Table 1.


**Table 1.** Some species of *Tremellomycetes* and their well-known characteristics.


**Table 1.** *Cont*.

In countering cryptococcosis, three classes of antifungal agents are available: polyenes (such as amphotericin B), azoles (such as fluconazole) and the pyrimidine analogue to flucytosine [1]. The gold standard induction treatment includes giving amphotericin B along with flucytosine [23]. However, this combination therapy has substantial side effects and the need for intravenous medications poses a problem, as these are not readily available in developing countries, which are most affected by cryptococcosis [24]. To overcome this problem, a combination of fluconazole along with flucytosine has been recommended after initial therapy with amphotericin B and flucytosine [1,23].

Fluconazole binds to the fungal cytochrome P450 monooxygenase (CYP/P450) enzyme 14α-demethylase, named CYP51, which converts lanosterol into ergosterol, an essential component of the fungal cell membrane [25]. *C. neoformans* also has *CYP51* and quite a number of studies have indicated that the development of drug resistance to fluconazole is due to the mutations in the *CYP51* gene and to the elevated levels of CYP51 in cryptococcal species [26–30]. In addition to *C. neoformans*, drug resistance in other species of *Tremellomycetes* has also been reported owing to mutations in *CYP51* [31,32]. Recent studies have demonstrated that the new anti-cryptococcosis drug named VT-1129 that is in the pipeline strongly binds and inhibits CYP51 of *C. neoformans* and *C. gattii* [33–35].

Despite the importance of CYPs as drug targets, to date, the CYP repertoire in cryptococcal species or in other species of *Tremellomycetes* has not been elucidated. A few studies reported the CYP contingent of *C. neoformans* and *T. mesenterica* Fries with the purpose of comparing the CYP profiles with wood-degrading fungi [22,36,37]. Thus, in this study we present a comparative analysis of CYPs in species of *Tremellomycetes*.

#### **2. Results and Discussion**

#### *2.1. Pathogenic Cryptococcal Species Have Few CYPs in Their Genomes*

Genome-wide data mining of CYPs in 16 cryptococcal species revealed the presence of 112 CYPs in their genomes (Figure 1). *C. curvatus* and *C. terricola* have the highest number of CYPs (16 CYPs each), and *C. gattii* VGIV IND107 has the lowest number of CYPs (Figure 1). An interesting pattern was observed when comparing the CYP count among cryptococcal species. Almost 50% fewer CYPs were found in pathogenic cryptococcal species compared to non-pathogenic cryptococcal species (Figure 1). This suggests that adaptation to survive in a host (mainly animals) that has a rich source of simple nutrients might have led to the loss of CYPs. The same phenomenon was observed in fungal species belonging to the subphylum *Saccharomycotina*, where species lost a considerable number of CYPs owing to their adaptation to simpler carbon sources [38].

**Figure 1.** Comparative analysis of CYPs in the species of *Tremellomycetes*.

The comparison of cryptococcal species' CYP count with other species belonging to the same subphylum *Agaricomycotina*, especially the well-studied wood-degrading fungi, is not logical, since the wood-degrading species have quite a large number of CYPs in their genomes [22]. As cryptococcal species fall under *Tremellomycetes*, in this study, a comprehensive comparative analysis of CYPs in *Tremellomycetes* was carried out (Figure 1). As shown in Figure 1, the comparison of CYPs among species of *Tremellomycetes* indicated that pathogenic cryptococcal species have a lower number of CYPs compared to other species of *Tremellomycetes*. Fungal parasites such as *T. mesenterica* Fries and *N. encephela* have eight and 10 CYPs in their genomes, somewhat lower than non-pathogens. It is interesting to note that the species belonging to the genus *Trichosporon* have the highest number of CYPs in their genomes, both pathogenic and non-pathogenic (Figure 1). It is well-known that most of the species belonging to this genus are considered commensals of the human skin and gastrointestinal tract, and these species are now increasingly causing superficial and invasive diseases in immunocompromised individuals and intensive care unit patients [18,39]. This indicates that these organisms have a long way to go to adapt better, similar to the cryptococcal species, and thus, in this process they may lose CYPs as well.

#### *2.2. New CYP Families Were Found in Tremellomycetes*

A total of 203 CYPs were found in 23 species of *Tremellomycetes* (Figure 2 and Supplementary Dataset 1). Sixteen CYPs were found to be pseudo/false positives, as they lack one of the CYP characteristic motifs and/or short fragments (listed in Supplementary Dataset 1). Thus, these CYPs

were not included in the study. The annotation of CYPs as per International P450 Nomenclature Committee rules [40–42] in combination with phylogenetic analysis (Figure 2) revealed that 203 *Tremellomycetes* CYPs could be grouped into 38 CYP families and 72 CYP subfamilies (Figure 2 and Supplementary Dataset 2, sheet 1). Phylogenetic analysis of CYPs is critical in assigning the CYP family and subfamily for the CYPs that have a borderline percentage identity of around 40–41% (for a family) and 55–56% (for a subfamily) with the named fungal CYPs.

**Figure 2.** Phylogenetic analysis of CYPs from the species of *Tremellomycetes*. CYP families that are highly populated in species of *Tremellomycetes* are indicated in different colors. A high-resolution phylogenetic tree is provided in Supplementary Figure S1.

A total of 23 new CYP families, named CYP5215, CYP5216, CYP5393, CYP5394, CYP5698-CYP5702, CYP5878-CYP5882, and CYP5886-CYP5894A1, were identified in species of *Tremellomycetes. Kockovaella imperatae* NRRL Y-17943 have the highest number of new CYP families (six CYP families: CYP5888-CYP5893) followed by *Naganishia vishniacii* (five new CYP families: CYP5698-CYP5702), *C. terricola* (three new CYP families: CYP5879-CYP5881), *T. mesenterica* Fries (two new CYP families: CYP5393 and CYP5394), *T. oleaginosus* IBC0246 (CYP5878 and CYP5882) and two new CYP families (CYP5886 and CYP5887) were found in *T. asahii* var. *asahii* CBS 2479 and *T. asahii* var. *asahii* CBS 8904. Three species of *Tremellomycetes* have only one new CYP family: *C. neoformans* var. *grubii* H99 (CYP5215), *C. neoformans* var. *neoformans* B-3501A (CYP5216), and *N. encephela* UCDFST 68-887.2 (CYP5894).

#### *2.3. Four CYP Families Are Conserved in Pathogenic Cryptococcal Species*

CYP family-level comparative analysis revealed that among 38 CYP families, the CYP5139 family was found to be dominant in species of *Tremellomycetes* with 51 members, following the CYP51 and CYP61 families each with 23 members, the CYP5216 family with 14 members, the CYP5215 family with 13 members, and the CYP505 family with 12 members (Figure 3). Analysis of CYP family conservation revealed that three CYP families, namely CYP5139, CYP51, and CYP61, are conserved in all 23 species of *Tremellomycetes* (Figure 4). CYP family comparison among pathogenic cryptococcal species revealed conservation of two more CYP families, CYP5215 and CY5216, in all species except *C. gattii* VGIV IND107, which does not have CYP5215 (Figure 4). These two CYP families are also present in fungal parasites, *T. mesenterica* Fries (both families) and *N. encephela* UCDFST 68-887.2 (only CYP5216 family), and non-pathogenic *C. amylolentus* CBS 6273 (Figure 4). The CYP family CYP5231 found in *N. vishniacii* is also present in the fungal species, *Melampsora laricis-populina* and *Puccinia graminis*, belonging to the class *Pucciniomycotina*, where this family is bloomed in both species [36]. The presence of the CYP5126 family only in pathogenic or parasitic *Tremellomycetes* indicates that this CYP family might be playing a role in the adaptation of these organisms to their host. The analysis of CYP subfamilies revealed that the CYP5139 family has 17 CYP subfamilies, indicating the blooming of members in this family. The same was observed for quite a number of CYP families in other fungi [36,37].

**Figure 3.** The CYP family-level comparative analysis in the species of *Tremellomycetes*. The numbers next to the family bar indicate the total number of CYPs. The data on the number of CYPs in each CYP family, along with subfamilies, are presented in Supplementary Dataset 2, sheet 1.

**Figure 4.** Heat map representing the presence or absence of cytochrome P450 families in 23 species of *Tremellomycetes*. The data have been represented as −3 for gene absence (green) and 3 for gene presence (red). Twenty-three species of *Tremellomycetes* form the horizontal axis and CYP families form the vertical axis. The data used in the generation of Figure 4 are presented in Supplementary Dataset 2, sheet 2.
