*5.3. Autotransporters*

Numerous virulence factors of bacterial pathogens contain domains or motifs related to adhesion to biotic or abiotic surfaces. A comprehensive search for conserved adhesion-associated domains/motifs in the *B. suis* 1330 genome and subsequent phylogenetic analyses revealed the presence of three clearly separated groups of adhesins: (1) monomeric autotransporters (BRA0173, BR2013, BRA1148), (2) trimeric autotransporters (BR0072 and BR1846), and (3) Ig-like domain containing-proteins (BR2009 and BR2012) [75]. Other proteins with no clear associated functions were also identified but not described in this work. Group 2 also included the protein BR0049. We have recently shown that BR0049 is certainly not an adhesin, but is required for the correct insertion of proteins from the autotransporter families (see below) [53]. Group 3 comprised orthologous proteins of the *B. abortus* BigA and BigB proteins described above.

### 5.3.1. Monomeric Autotransporters

As mentioned above, *B. abortus* binds in a dose-dependent manner to components of the ECM, such as fibronectin and vitronectin [27]. In an attempt to identify *B. suis* genes encoding proteins that might be involved in the binding of brucellae to fibronectin, Posadas et al. [67] panned an M13 phage display library of the *B. suis* 1330 genome against immobilized fibronectin. Several recombinant phages showed a ffinity to immobilized fibronectin. However, only one expressed a portion of a protein that was predicted to be exposed on the cell surface. This protein corresponded to one of the monomeric autotransporters described above (BRA1148) and was named as BmaC for *Bucella* monomeric autotransporter. This protein exhibits all the characteristics of this protein family. BmaC is a large 340 kDa-protein with a long N-terminal cleavable 72 amino acid signal peptide and several adhesion-related motifs within the passenger domain, an extended pectin lyase virulence factor domain, and several passenger-associated-transport-repeats (PATR) (Figure 1). The portion of BmaC expressed on the phage also exhibits a ffinity (although much less) for type I collagen, suggesting that this very large protein might contribute to the interaction of *B. suis* with other ECM ligands.

Several lines of evidence have shown that BmaC of *B. suis* 1330 mediates the binding of *B. suis* to epithelial cells through cellular fibronectin. The attachment of *B. suis* to HeLa cells was inhibited by the φ-BRA1148 recombinant phage in a dose-dependent manner. A *bmaC* deletion mutant was impaired in the attachment to immobilized fibronectin and to the surface of HeLa and A549 epithelial cells (Table 1). Furthermore, the *bmaC* defective mutant was outcompeted by the wild-type strain in co-infection experiments, and anti-BmaC and anti-fibronectin antibodies significantly inhibited the binding of *B. suis* to HeLa cells [67]. Immunofluorescence microscopy showed that all bacteria with a detectable fluorescent signal displayed BmaC at only one pole, indicating that BmaC is polarly exposed on the cell surface (Figure 2). This is not surprising since several monomeric autotransporters are exposed on the bacterial surface at one pole [76,77]. Confocal microscopy analysis showed the presence of some small GFP-labelled bacterial aggregates on the surface of HeLa cells, mostly in cell boundaries. Single bacteria were found interacting through one of their poles with the cell surface on both the cell body and cell protrusion. Occasionally, polar BmaC was located at the pole interacting with the cell. These observations suggested that the polar localization of BmaC could be relevant in the interaction with host cells in vivo [67].

The monomeric autotransporter proteins encoded by BR0173 and BR2013 (BmaA and BmaB, respectively) of *B. suis* 1330, although much smaller, share significant sequence similarities with BmaC (Figure 1). It was reported that a mutant of *B. suis* 1330, deficient in BmaB (previously called OmaA), is cleared from spleens of BALB/c mice faster than the wild-type strain (between the third and the ninth week post infection), suggesting that BmaB is required during the chronic phase of infection [78]. A recent study [68] indicated that the *bmaB* locus from all *B. abortus* strains analyzed and both the *bmaA* and *bmaC* loci from all *B. melitensis* strains seem to correspond to pseudogenes, while, in *B. suis*, all the Bma proteins could be functional in several strains of this species. In line with these observations, gain or loss of function studies indicated that, at least in *B. suis* strain 1330, BmaA, BmaB, and BmaC proteins contribute, to a greater or lesser degree, to bacterial adhesion among di fferent cell types, such as epithelial (HT-29 and Caco2), synoviocytes, osteoblasts, and trophoblasts (Table 1, Figure 2). These observations show that there are variations in the repertoire of functional adhesins in *Brucella* spp. and open the possibility that these adhesins are involved in host preferences. BmaB was also found at the new pole generated after cell division [68].

### 5.3.2. Trimeric Autotransporters

As mentioned above, the search for conserved adhesion-associated domains/motifs in *B. suis* 1330 identified a group of trimeric autotransporters, including BR0072 and BR1846, which were named BtaE and BtaF, respectively. The *B. suis* BtaE trimeric autotransporter is a 740 amino acid protein that harbors several regions corresponding to the head and the neck subdomains in addition to a connector region and the β-barrel translocator domain (Figure 1). Di fferent genetic approaches showed that BtaE of *B. suis* is involved in the adhesion to ECM components and host cells [69]. The BtaE-defective strain exhibited a decreased ability to adhere to HeLa and A549 epithelial cells and was outcompeted by the wild-type *B. suis* strain for the adhesion to HeLa cells (Table 1). Expression of BtaE in a "non-adherent" *E. coli* strain increased the binding of this heterologous bacterium to immobilized hyaluronic acid and fibronectin. On the other hand, *btaE* deletion impaired bacterial adhesion to hyaluronic acid but had no effect in the adhesion to fibronectin, suggesting that other fibronectin-binding adhesins (such as BmaC) could compensate somehow for the absence of BtaE. The adhesion of the wild-type strain to HeLa

cells decreased in the presence of hyaluronic acid, while this compound had almost no effect in the attachment of the *btaE* mutant to these cells, supporting the hypothesis that BtaE mediates the binding of *Brucella* to hyaluronic acid. Therefore, BtaE could also participate in *Brucella* dissemination to different target tissues such as cartilage, heart, and bone, which may result in brucellosis complications. In vivo experiments using the mouse model indicate that the BtaE adhesin is necessary for a successful infection. In effect, a significantly lower number of bacteria were recovered from spleens of animals inoculated through the intragastric route with the *btaE* mutant compared to those inoculated with the wild-type strain [69].

In a subsequent work, it was shown that the BtaE orthologue of *B. abortus* 2308 is also involved in adhesion to epithelial cells. Compared to *B. suis* BtaE, the ortologue of *B. abortus* is much larger and contains a higher number of repetitive adhesion motifs. Furthermore, the *btaE* gene of *B. suis* and *B. abortus* are under the regulation of different mechanisms (see below) [79]. The *btaE* deletion mutant of *B. abortus* 2308 showed a significant reduction in the adhesion to HeLa cells when compared with the wild-type strain, demonstrating that the BtaE variant of *B. abortus* 2308 contributes to the interaction of *Brucella* with the host cell surface to a similar extent to that observed for the *B. suis* 1330 orthologue [69].

The regulation of *btaE* at a promoter level was analyzed by Sieira et al. [79]. Comparison of *btaE* promoter sequences among different *Brucella* species revealed that a novel HutC binding site in the promoter region of *btaE* from *B. abortus* 2308 was generated de novo recently in the evolution of the genus. HutC, which is a regulator of the histidine metabolism, also acts as a co-activator contributing to modulation of expression of the *Brucella virB* operon [80]. Moreover, additional transcriptional factors (MdrA and IHF) binding sites were identified in the *btaE* promoter of *B. abortus*. The target-DNA sequences were confirmed by EMSA and DNAseI footprinting assays.

The HutC binding site is not present in the *btaE* promoters of other *Brucella* strains since it is interrupted by a cytosine. In effect, an electrophoretic mobility shift assay showed that HutC is not able to interact with the *btaE* promoter of *B. suis* 1330, even though the IHF and MdrA showed a binding pattern similar to that observed for the *btaE* promoter of *B. abortus* 2308. Based on these findings, it was proposed that, as a result of the cis regulatory gain of function, the *btaE* promoter acquired the ability to fine-tune its transcriptional output in response to changes in environmental parameters such as nutrient availability. Thus, differential *btaE* expression might generate phenotypic diversity at the regulatory level of adhesins, which might contribute to reciprocal selection between *Brucella* species and their mammalian hosts.

The BtaF trimeric autotransporter of *B. suis* 1330, encoded by the BR1846 annotated locus, is a small protein that harbors an N-terminal peptide signal, a 170 amino acid passenger domain, and a YadA-like C-terminal translocator region (β-barrel translocator domain) (Figure 1). Unlike BtaE, the BtaF protein does not show the presence of conserved adhesion motifs. Analysis with the TA Domain Annotation (daTAA) server [81] showed that most of the passenger domains correspond to a coiled coil stalk but none associated with the "head" structural region [70]. In addition, a careful analysis of the annotated *btaF* upstream region indicated that the ORF starts earlier, adding 33 additional amino acids at the N-terminal sequence. Using a new version of the program to predict the structural features of trimeric autotransporters and the alternative ORF, it was possible to identify a region at the N-terminus that would correspond to the head, even though the structure would be different from those described so far [82].

BtaF of *B. suis* 1330 has shown to be promiscuous in its ability to bind to different substrates. The heterologous expression of this small trimeric protein markedly increased the adhesion of non-adherent *E. coli* to HeLa cells and various substrates such as fibronectin, fetuin (a sialic acid-rich protein), hyaluronic acid, and collagen I and also to an abiotic surface such as polystyrene [70] (Table 1, Figure 2). In agreemen<sup>t</sup> with these observations, the *btaF* deletion mutant of *B. suis* showed a significant reduction in the ability to bind to fetuin, hyaluronic acid, and collagen I, and in the adhesion to an abiotic surface, even though the adhesion to fibronectin was not affected. Again, as it was observed for the *btaE* mutant, overlapping functions with other adhesins (such as Bma proteins and BtaE) may account for the lack of a Δ*btaF* phenotype toward fibronectin. The BtaF-defective strain showed a significant reduction in the attachment to HeLa and A549 epithelial cells.

Notably, BtaF was required for complete virulence in mice infected through the oral route (intragastric administration) [70]. The strain lacking BtaF showed a reduction of about one log in the number of bacteria recovered from spleen at early stages of infection. The absence of both trimeric adhesins (BtaE and BtaF) resulted in a more severe phenotype in vivo compared with the attenuation observed for the single mutants. It is possible that some of the functions might be shared or complementary between BtaE and BtaF, while others could be exclusive to BtaF or BtaE. An indirect ELISA assay on sera from healthy and sick pigs infected with *B. suis* suggested that both adhesins are expressed in vivo in the natural host (swine), supporting the role of these adhesins in the infection process. Recently, it was shown that BtaF is also required for virulence in mice after inoculation via a respiratory (intratracheal administration) route [71]. In this case, the splenic load of the deletion mutant was significantly reduced at 7-days and 30-days post-infection as compared to the wild-type strain.

Smooth *Brucella* strains prevent detection by complement partly due to a distinctive structure of its LPS [83,84]. However, it was proposed that, in addition to the LPS, other surface factors mediate the varied sensitivity of *Brucella* species to the bactericidal action of serum [84]. In addition, it has been shown that, in contrast to human serum, components present in murine normal serum do not opsonize smooth *B. abortus* [85]. The *btaF* mutant showed a significantly reduced survival in the presence of 50% porcine serum compared with the wild-type strain [70]. Furthermore, an *E. coli* strain expressing BtaF showed a more than ten-fold increase in the survival percentage in 8% porcine serum as compared with the control strain. Both strains showed similar levels of survival in heat-inactivated porcine serum, suggesting that BtaF is involved in the resistance to complement-mediated serum killing.

Similar to BmaC and BmaB, BtaE and BtaF adhesins were found to be polarly localized on the bacterial surface [69,70] (Figure 2). Again, the trimeric adhesins were detected in a low proportion of bacteria but, in all cases, the signal showed unipolar localization and, in some cases, sub-polar localization. As it was observed for BmaC and BmaB, they were found at the same pole as AidB-YFP (a new pole marker) [86] and at the opposite of the PdhS-eGFP labeling (old pole marker) [87]. It was proposed that the new pole generated after the asymmetric division would be functionally di fferentiated for adhesion. An attractive hypothesis is that the initial adhesion of *Brucella* to the host cell would be mediated by adhesins located at the new pole and that adhesin expression only occurs in an infectious bacterial subpopulation. Various cellular mechanisms such as asymmetric division, polar growth, and polar functions generate two functionally di fferentiated cells [88,89]. Polar localization could be a way of increasing the adhesive power by concentrating the adhesins in a particular region. In fact, host invasion by a bacterial pole can facilitate entry because of the bacterial shape [90]. It is important to note that polar adherence to surfaces is a conserved mechanism shared by several *Alphaproteobacteria* [91,92].

### 5.3.3. Autotransporters Insertion in the Outer Membrane

Autotransporter translocation into the outer membrane is assisted by the BAM machinery and associated chaperones. More recently, it was shown that the TAM system, made up of TamA and TamB, is also required for the correct insertion of autotransporters from the *Gammaproteobacteria* group (see above) [93].

As mentioned, during the construction of the phylogenetic tree, the BR0049 protein came out as a possible adhesin from the autotransporter family likely due to some structural similarities with this family of proteins. However, our in silico analysis and other reports [94] indicated that BR0049 and its orthologues from other *Alphaproteobacteria* are phylogenetically related to members of the TamB family from *Gammaproteobacteria*. TamB is a large protein mostly periplasmic but inserted in the inner membrane through a non-cleavable signal peptide. BR0049 of *Brucella* spp. shares a relatively low identity (around 22%) with TamB from *Gammaproteobacteria*, but, similarly to this protein, it contains a membrane anchor signal at the N-terminus, which is followed by a region with an abundant β-helix

structure, and, at the C-terminus, a short β-barrel structure within the conserved DUF490 domain. As it was proposed for TamB from *Gammaproteobacteria*, it was demonstrated that BR0049 is required for the correct insertion in the OM of the *B. suis* BmaB monomeric autotransporter. In addition, BR0049 was required for complete virulence in mice infected through the intragastric route [53]. The BR0049 mutant showed an increased sensitivity to polymyxin B, lysozyme, and Triton X-100, and, thus, BR0049 was named as MapB (Membrane altering protein). Several results indicated that MapB of *Brucella* plays functions that go beyond that of assisting in autotransporter assembly, suggesting that the TAM machinery would be involved in cell envelope biogenesis.

### *5.4. Brucella Adhesins as Vaccine Candidates*

Vaccination is a key health measure in the control and prevention of infectious diseases. In the case of brucellosis, it is necessary to control bacterial dissemination by vaccination of natural hosts as well as vaccination of people professionally exposed to *Brucella* spp. infection. Commercially available *Brucella* vaccines approved for use in animals are based on attenuated strains. These vaccines have serious disadvantages such as producing abortion in pregnan<sup>t</sup> females, being virulent for humans [95–97], and inducing immune responses that interfere with animal serological diagnosis [98]. Thus, there is a need to develop safer and more e fficient vaccines [99]. In this sense, acellular vaccines provide grea<sup>t</sup> advantages, mainly in terms of safety, not only in its production but also in its administration. Nevertheless, the selection of appropriate vaccine candidates requires the study of host-pathogen interaction.

An essential step in establishing a successful infection is the adhesion of microorganisms to eukaryotic cells, resulting in colonization of the tissue involved. Therefore, the molecules involved in this initial interaction have been widely studied as targets for the development of vaccines against various pathogens such as *E. coli* [100,101], *Haemophilus ducreyi* [102], and *Neisseria meningitidis* [103,104] among others. Despite the extensive knowledge of adhesins' role in the pathogenicity of various bacteria, this group of proteins was not studied well in *Brucella* spp. in terms of immunogenicity and its potential as vaccine candidates.

Al-Mariri et al. studied the immunogenicity and protective e fficacy of a DNA vaccine encoding SP41 adhesin from *B. melitensis* in BALB/c mice [105]. Intramuscular (i.m.) administration of pCISP41, a plasmid construct for SP41 expression in mammalian cells, induced SP41-specific serum immunoglobulin G (IgG) antibodies. Moreover, spleen cells from pCISP41-vaccinated mice showed significant T cell proliferation after in vitro stimulation with recombinant SP41 (rSP41) and lysed *B. melitensis*. Splenocytes from pCISP41-immunized animals also responded to rSP41 and bacterial lysate stimulation secreting high levels of gamma interferon (IFN-γ), even though no interleukin-5 (IL-5) was detected. This suggests a predominant T-helper-1 (Th1) immune response.

After an intraperitoneal challenge with *B. melitensis* 16M, mice immunized with pCISP41 exhibited a reduction of 1.25 and 1.14 log in their spleen burden when compared with control mice at 4-weeks and 8-weeks post-challenge, respectively. Nevertheless, vaccination with attenuated *B. melitensis* Rev-1 strain induced better protection levels than pCISP41 vaccination at both time points, achieving a reduction in spleen burden of 1.79 and 3.17 log, respectively. Although SP41 has been shown to be involved in adhesion to epithelial cells, no studies have been made to evaluate the potential of mucosal or systemic vaccination with this adhesin to protect against *Brucella* infection acquired through mucosae.

*Brucella* infection is frequently acquired through the oral and respiratory routes, and adhesins are anticipated to have a relevant role in these infectious processes. As mentioned above, our results showed that BtaF adhesin from *B. suis* is necessary for complete virulence of *B. suis* after both oral and intratracheal infection [70,71]. In line with these results, we assessed the immunogenic and protective potential of recombinant BtaF when administered intranasally with the mucosal adjuvant Bis-(3',5')-cyclic dimeric adenosine monophosphate (c-di-AMP) [71]. To this end, the trimeric form of the BtaF passenger domain fused at the C-terminus to the GCN4tri sequence to facilitate trimerization was successfully expressed and purified [71].

BALB/c mice intranasal immunization with BtaF plus c-di-AMP induced high levels of serum BtaF-specific IgG, IgA, IgG1, and IgG2a, and a mixed IgG1/IgG2a profile. In vitro, these serum antibodies reduced *B. suis* infection of human lung epithelial cells (A549 cell line), reduced bacterial binding to fetuin (a protein rich in sialic acid previously described as a ligand for BtaF), and enhanced *B. suis* phagocytosis by murine macrophages. In addition, immunization led to a significant production of specific IgA antibodies in the airways and in the gastrointestinal and genital mucosae.

BtaF immunization induced a systemic and pulmonary Th1 immune response, shown by the secretion of high levels of IFN-γ by splenocytes and lung cells. Furthermore, depletion of CD4+ or CD8+ populations from spleen cells showed that CD4+ cells were responsible for IFN-γ secretion. BtaF-vaccination also triggered the differentiation of specific CD4+ T cells to central memory cells in cervical lymph nodes, and differentiation of T cells to a Th17 profile in the spleen.

Mice vaccination with BtaF plus c-di-AMP demonstrated a high level of protection against *B. suis* oral infection, reducing the splenic burden of *B. suis* by 3.28 log. Unlike the protection achieved against oral infection, intranasal vaccination with BtaF failed to protect against respiratory infection with *B. suis* since no differences were observed in spleen or lung bacterial load between vaccinated and control mice after an intratracheal challenge.

In summary, despite extensive evidence supporting a role of *Brucella* adhesins in the infectious ability of this pathogen in vivo, only a few studies have assessed the protective efficacy of vaccination with adhesins. Such studies sugges<sup>t</sup> that adhesins hold promise as appropriate antigens for vaccination against oral infection with *Brucella*, even though some protection against systemic infection might be attained.

### **6. Summary and Future Directions**

For a long time, the process of *Brucella* adhesion to host cells has received little attention. Adhesins of *Brucella* spp. identified to date have been studied with varying degrees of depth. Some of them have been analyzed regarding their roles in the binding to components of the ECM and to different cell types as well as their in vivo role, while, in other cases, their functions have been only evaluated in vitro. Still, for various adhesins, it will be necessary to identify their ligands or cell receptors. The possible use of *Brucella* adhesins as vaccine candidates was tested only in two cases, and one of them showed encouraging results. Therefore, the in vivo role of many of the *Brucella* adhesins identified so far, and their possible applications as a basis for acellular vaccines, remains to be evaluated. It is expected that, in the future, new adhesins will be identified that mediate the initial adhesion of *Brucella* to the remarkable variety of cell types that this pathogen can invade. An interesting perspective will be to characterize the roles of the different adhesin variants from different species/strains and to determine whether they play a role in host preferences.

**Author Contributions:** Conceptualization, P.C.B. and A.Z. Literature review, critical analysis, and synthesis, M.G.B., G.S., M.C.F., F.M.G., P.C.B., and A.Z. Writing—original draft preparation, M.G.B., G.S., M.C.F., F.M.G., P.C.B., and A.Z. Supervision, P.C.B. and A.Z. All authors have read and agreed to the published version of the manuscript.

**Funding:** This work was supported by the Research Grants from Agencia Nacional de Promoción Científica (PICT 2013-0170, PICT 2016-1199, PICT-2016-2722), and from Universidad de Buenos Aires (UBACYT 20020170100036BA).

**Acknowledgments:** We are grateful to the staff of the BSL3 facility of INBIRS for expert assistance. We thank current and former members of our laboratories for all the contributions and helpful and inspiring discussions.

**Conflicts of Interest:** The authors declare no conflict of interest. The funders had no role in the writing of the manuscript, or in the decision to publish the manuscript.
