{'fraction_non_alphanumeric': 0.031341350601295094, 'fraction_numerical': 0.02730804810360777, 'mean_word_length': 5.007112691709269, 'pattern_counts': {'":': 0, '<': 1, '<?xml version=': 0, '>': 0, 'https://': 1, 'lorem ipsum': 0, 'www.': 0, 'xml': 0}, 'pii_count': 0, 'substrings_counts': 0, 'word_list_counts': {'cursed_substrings.json': 0, 'profanity_word_list.json': 0, 'sexual_word_list.json': 0, 'zh_pornsignals.json': 0}}
A genomic island in Brucella involved in the adhesion to host cells: Identification of a new adhesin and a translocation factor

Adhesion to host cells is the first step in the virulence cycle of any pathogen. In Gram‐negative bacteria, adhesion is mediated, among other virulence factors such as the lipopolysaccharides, by specific outer‐membrane proteins generally termed adhesins that belong to a wide variety of families and have different evolutionary origins. In Brucella, a widespread zoonotic pathogen of animal and human health concern, adhesion is central as it may determine the intracellular fate of the bacterium, an essential stage in its pathogenesis. In the present paper, we further characterised a genomic locus that we have previously reported encodes an adhesin (BigA) with a bacterial immunoglobulin‐like domain (BIg‐like). We found that this region encodes a second adhesin, which we have named BigB; and PalA, a periplasmic protein necessary for the proper display in the outer membrane of BigA and BigB. Deletion of bigB or palA diminishes the adhesion of the bacterium and overexpression of BigB dramatically increases it. Incubation of cells with the recombinant BIg‐like domain of BigB induced important cytoskeletal rearrangements and affected the focal adhesion sites indicating that the adhesin targets cell–cell or cell–matrix proteins. We additionally show that PalA has a periplasmic localisation and is completely necessary for the proper display of BigA and BigB, probably avoiding their aggregation and facilitating their transport to the outer membrane. Our results indicate that this genomic island is entirely devoted to the adhesion of Brucella to host cells.

In Brucella, a widespread zoonotic pathogen of animal and human health concern, adhesion is central as it may determine the intracellular fate of the bacterium, an essential stage in its pathogenesis. In the present paper, we further characterised a genomic locus that we have previously reported encodes an adhesin (BigA) with a bacterial immunoglobulin-like domain (BIg-like). We found that this region encodes a second adhesin, which we have named BigB; and PalA, a periplasmic protein neces- recognising molecular motifs with a high degree of specificity comparable to enzymes and immunoglobulins (Klemm & Schembri, 2000). There are many different families of adhesins that vary depending on the Gram nature of the bacteria, the host cell as well as the virulence cycle of the pathogen. They play pleiotropic roles, enhance competition with other bacteria and find their niche within the host, facilitating the establishment of pathogenesis and modulating the alterations of the cellular functions (Patel, Mathivanan, & Goyal, 2017).
Adhesins are responsible for pathogen persistence causing, among other symptoms, tissue inflammation in the host. Gut mucosa, lungs and bladder are some examples of inflammation mediated by adhesin immune activation (Conover et al., 2016;Mil-Homens, Pinto, Matos, Arraiano, & Fialho, 2017;Moore, Boren, & Solnick, 2011;Palmela et al., 2018). The cytoskeleton is intrinsically involved in inflammation and its plasticity is an important component of host cells. Cytoskeleton is exploited by pathogens by either triggering actin rearrangements that can cause cell invasion that lead to their own phagocytosis or by zippering, where invasion occurs first by a direct contact between the adhesin and its ligand which activates cytoskeletal components that culminate in the closure of the phagocytic cup and bacterial internalisation (Cossart & Sansonetti, 2004;Patel et al., 2017;Pizarro-Cerda & Cossart, 2006).
Many secretion mechanisms for different adhesins in a wide variety of bacteria have been described depending on their Gram classification. In Gram-negative bacteria, one of the most studied mechanisms is the chaperone-usher-assisted surface display of PapD and FimC in E. coli, consisting of an assembly platform or usher and a periplasmic chaperone, which complexes with the future adhesin avoiding proteolytic degradation and premature polymerisation (Jones et al., 1993).
Our laboratory has identified a genomic region, named Bab1_2009-Bab1_2012 gene cluster, unique to Brucella that is involved in the adhesion and invasion to host cells and important in the oral route of infection (Czibener & Ugalde, 2012). This region, with horizontally transmitted features, encodes four open reading frames present only in the genus Brucella and with no detectable homology with known proteins. Further analysis of this region determined that it encodes a powerful adhesin (BigA) with a bacterial immunoglobulin-like (Big-like) domain that targets the bacteria to the cell-cell junction membrane in polarised cells promoting the adhesion and invasion of epithelial cells through cytoskeleton modifications (Czibener et al., 2016).
In the present study, we provide new insight into the importance of the Bab1_2009-Bab1_2012 genomic cluster in adhesion, characterising two new proteins that belong to this island. One of them is a novel adhesin, which has a BIg-like domain, similar to the one present in BigA. Moreover, our results demonstrate that another gene of this region, with no apparent homology or conserved domains has a major role in the correct localisation of these adhesins in the outer membrane of Brucella. Our results indicate that the Bab1_2009-Bab1_2012 gene cluster plays a central role in the adhesion of Brucella.
2 | RESULTS 2.1 | Bab1_2012 encodes a second adhesin of the bab1_2009-bab1_2012 gene cluster As indicated above, bigA (gene bab1_2009 in B. abortus 2308 [Chain et al., 2005]) encodes an adhesin that targets the bacterium to the cell-cell interaction membrane and promotes adhesion and invasion of the host cell (Czibener et al., 2016). In order to determine if any other of the three genes encoded in this region are also adhesins, we analysed the putative proteins in search of motifs that could suggest this type of function. Interestingly, analysis of Bab1_2012 showed a weak homology (using the https://toolkit.tuebingen.mpg.de/tools/ hhpred) with bacterial immunoglobulin-like domains present in several adhesins of different pathogens and in BigA (Czibener et al., 2016).
Also, in silico analysis predicted three transmembrane segments (SMART-EMBL). To evaluate if Bab1_2012 is an adhesin, a deletion mutant was constructed and intracellular replication assays in nonphagocytic cells were performed. As can be observed in Figure 1a (Figure 2b) showed that the protein product of bab1_2012 is located in the periplasm or outer membrane, which, in combination with the result of its association with total membranes, confirms that Bab1_2012 is located in the outer membrane.
To further confirm that Bab1_2012 is exposed in the outer membrane, we developed a polyclonal antibody against the Ig-like domain of the protein and performed an immunofluorescence with non-permeabilised cells of the B. abortus expressing Bab1_2012 from a medium copy plasmid and the deletion mutant strains. As can be observed in Figure 2c, the protein was found on the bacterial surface confirming that Bab1_2012 is an exposed outer membrane protein.
Altogether, these results indicate that Bab1_2012 codes for an adhesin that mediates attachment to host cells. Consequently, we renamed this gene bigB for Bacterial Immunoglobulin Gene B.

| BigB induces cytoskeletal rearrangements of host cells
We have previously reported that treatment of cells with the recombinant bacterial immunoglobulin-like domain of BigA induces profound cytoskeletal rearrangements that results, upon longer treatments, in the detachment of the cells from the plate (Czibener et al., 2016). As BigB. Consistent with this, the constrained form observed in the treated HeLa cells proved that the cytoskeleton structure was also affected by this adhesin. Taken together, these results provide proof that BigB alters focal adhesion sites triggering cytoskeletal rearrangements in non-phagocytic cells.

| Bab1_2011 is periplasmic protein involved in the adhesion of Brucella
The Bab1_2009-Bab1_2012 genomic region encodes four open reading frames present only in the Brucella genus and with no detectable homology to other protein in databases (Czibener & Ugalde, 2012).
Since we have shown that at least two of these proteins are adhesins (BigA and BigB) this prompted us to evaluate the function of the other genes present in the region (bab1_2010 and bab1_2011). For this, we generated deletion mutants in both open reading frames and performed intracellular replication curves in HeLa cells with the resulting mutants. Deletion of bab1_2010 resulted in a strain with a reduced intracellular survival at early time points but we were not able to complement it (data not shown). Deletion of bab1_2011 resulted in a strain with a moderate but statistically significant reduction in the intracellular CFUs at 4 hr post-infection ( Figure 4a) that was complemented with the gene in trans. To determine if this reduction was the consequence of a reduced adhesion or invasion, we performed adhesion/invasion assays by immunofluorescence as indicated above. As can be observed in Figure 4b, the B. abortus Δbab1_2011 strain showed a statistically significant reduction in the adhesion capacity compared with the wild-type parental strain and, interestingly, complementation of the mutant resulted in a strain with an increased adhesion, as we have also observed for BigA and BigB. As expected, due to the reduced adhesion of the strain, invasion was also affected in the mutant strain ( Figure 4c). These results indicate that Bab1_2011 is either an adhesin per se or affects the activity of other adhesins (such as BigA and BigB).
Bab1_2011 encodes a protein of 129 amino acids conserved within the Brucella genus but with no detectable homology to other proteins and no canonical signal peptide. To gain further insight into the function of Bab1_2011, we constructed a B. abortus strain expressing a 3xFlag tagged version of the protein from a replicative plasmid (see Experimental procedures) and performed subcellular fractionation assays to determine the subcellular localisation of the protein. As can be observed in Figure 4, panels d and e, Bab1_2011 was not found associated with total membranes but partitioned in both the periplasmic plus outer membrane and the cytoplasmic fractions indicating that it is a periplasmic protein, therefore discarding Bab1_2011 to be an adhesin.
Altogether, these results strongly suggest that Bab1_2011 is a protein that probably affects the adhesion of Brucella indirectly by regulating the function or the display of the other molecules of the Bab1_2009-Bab1_2012 genomic region.

| Bab1_2011 is necessary for the insertion of BigA and BigB in the outer membrane
Our results indicate that Bab1_2011 is a periplasmic protein involved in the adhesion process and, since it is present in a locus that encodes two adhesins (BigA and BigB), we hypothesised that it could be involved in the translocation and/or insertion of these proteins in the outer membrane. To determine if this is the case, we expressed 3xFlag-tagged versions of BigA and BigB in the wild-type and Δbab1_2011 strains and analysed their subcellular localisation by performing total membrane purifications. A first unexpected observation was that BigA, in the Δbab1_2011 strain, migrated in an SDS-PAGE of total extracts as a 70 kDa protein instead of 40 kDa as it was observed in the wild-type strain (Czibener et al., 2016). (Figure 5a). This migration pattern was not altered even when higher concentrations of DTT, urea or protein alkylating agents were used strongly suggesting that BigA is probably covalently linked to itself or another protein but not through a disulfide bond. Moreover, as can be observed in Figure 5b, BigA was not found associated with total membranes as we have previously described in the wild-type strain (Czibener et al., 2016). To determine if this phenomenon was also observed with BigB, we performed the same extraction protocol with the Δbab1_2011 (BigB-3xFlag) strain and determined its membrane association. As shown in Figure 5c, in the Δbab1_2011 mutant, BigB lost its membrane localisation too although, in this case, the protein had a migration as expected according to its predicted molecular weight. Both extractions were performed in parallel with the corresponding wild-type strain.
Overall, these results indicate that Bab1_2011 has a critical role in regulating the localisation in the outer membrane of the two adhesins present in the same locus. For this reason, we renamed Bab1_2011 as palA for periplasmic adhesin locator A.
One possibility for the phenotype observed with the ΔpalA mutant is that neither BigA nor BigB could be translocated to the periplasm. To determine if this is the case, we performed periplasmic extractions with the B. abortus ΔpalA strain expressing the Flagtagged versions of the proteins and determined their localisation by western blot. Figure 5, panels d and e, shows that both BigA and BigB retain their periplasmic localisation in the mutant indicating that their translocation to this compartment is not affected. To corroborate that neither BigA nor BigB have their translocation to the periplasm affected in the ΔpalA mutant, we constructed translational fusions to the superfolder-GFP (sGFP) of both proteins and compared their localisation in the wild-type and mutant strains by confocal microscopy. As can be observed in Figure 6a,b the fusion BigA-sfGFP showed a bipolar localisation in both strains, demonstrating that although BigA in the ΔpalA mutant is located in the periplasm, the bipolar localisation is not affected in the mutant. Also, as it was previously described in Czibener et al., 2016, BigA in the wild-type strain is exposed in the membrane surface and its localisation is polar. In the case of the fusion BigB-sfGFP, the construct was unstable in the ΔpalA strain and we were not able to obtain reproducible results.
Altogether, these results indicate that the absence of PalA impairs the insertion of both BigA and BigB in the outer membrane remaining in the periplasmic space and, in the case of BigA, covalently linked to another protein or aggregated. This impairment could be the consequence of an indirect cause or because PalA interacts, either directly or in a complex, with BigA and BigB, stabilising them in the periplasm, avoiding aggregation and facilitating their transport to the outer membrane. To determine if BigA directly interacts with PalA, we performed co-immunoprecipitation assays with total extracts of B. abortus strains co-expressing PalA-3xFlag and BigA-sfGFP. As can be observed in Figure 6c, BigA-sfGFP coimmunoprecipitated with PalA demonstrating that they interact either directly or indirectly in the bacterium. In the case of BigB-sfGFP, we were not able to observe co-immunoprecipitation with PalA even though multiple attempts with different conditions of buffers and detergents were performed.

| DISCUSSION
Adhesion to tissues and host cells is the initial, and in many cases, a bottleneck step, in the virulence process of nearly all pathogens. The capacity to adhere and eventually invade target cells can determine tissue tropism, virulence cycle as well as immune response and, as a consequence, resolution of the infection. In bacteria, adhesion is mediated, among other proteins, by adhesins, a heterogeneous group of outer membrane molecules that target a wide spectrum of proteins, carbohydrates and lipids in the plasma membrane of host cells (Patel et al., 2017). Successful interaction of bacteria via these adhesins paves the way for host colonisation and pathogenesis.
While adhesion has been extensively studied in many bacteria, in Brucella, a zoonotic pathogen of animal and human health concern with an intracellular life cycle, not much has been conducted to under- Our group has identified a genomic region (genes Bab1_2009 to Bab1_2012 in B. abortus 2308) with horizontally transmitted features and demonstrated that is involved in the adhesion to host cells (Czibener & Ugalde, 2012). This region encodes four open reading frames conserved in all Brucella species but none of them has homology to known proteins. We have recently characterised one of these genes (bigA) and shown that it encodes an adhesin that targets the bacteria to the cell-cell junction membrane in confluent epithelia cell cultures (Czibener et al., 2016). BigA has an exposed bacterial immunoglobulin-like (BIg-like) domain present in many bacterial proteins whose function ranges from enzymes to pilli, fimbria and adhesins. It is proposed that these domains mainly mediate proteinprotein interactions and that its wide distribution in nature is due to its energetically favourable folding (Bodelon, Palomino, & Fernandez, 2013). Several pilli and fimbria that are central for the adhesion of the bacterium to different substrates, as well as the Intimin of enterohemorrhagic Escherichia coli or the Invasin A of Yersinia enterocolitica that mediate the invasion process of the pathogen have these BIg-like domains (Bodelon et al., 2013;Mikula, Kolodziejczyk, & Goldman, 2013).

Analysis of the other three genes present in the
Bab1_2009-Bab1_2012 genomic island showed that Bab1_2012 (BigB) also has a putative BIg-like domain although the homology is weaker than the domain present in BigA. Adhesion and invasion assays with deletion or overexpressing strains as well subcellular fractionation experiments indicated that BigB is also an adhesin located in the outer membrane of B. abortus.
Moreover, as with BigA (Czibener et al., 2016) (Soto & Hultgren, 1999). Alternatively, PalA could be part of a periplasmic secretion system that mediates the translocation of both adhesins to its final localisation in the outer membrane. In this hypothesis, absence of PalA will probably impair secretion and the adhesins would remain periplasmic, as we have observed.
Despite the function of PalA, BigA and BigB has to be translocated to the outer membrane to be functional and to achieve this, a specific mechanism has to be necessarily involved. One of the most studied mechanisms for the export of adhesins is the autotransporters, like the E. coli intimin and the invasin (Inv) and YadA from Yersinia enterocolitica (Leibiger, Schweers, & Schutz, 2019). In these autotransporters, translocation occurs by a two-step mechanism mediated by different domains present in the adhesins. First, they are targeted to the periplasm by a canonical signal peptide and, once in this compartment, they use a passenger domain that interacts with the peptidoglycan to be finally inserted in the outer membrane through a beta barrel domain (Leibiger et al., 2019;Leo, Oberhettinger, Schutz, & Linke, 2015). In these cases, there are additional proteins that act as chaperones and/or insertion complexes that help to translocate these adhesins to the outer membrane (Lazar & Kolter, 1996;Leibiger et al., 2019;Sklar, Wu, Gronenberg, et al., 2007;. BigA and BigB do not seem to belong to these families of autotransporters as they do not have the domain structure typical of these proteins. Even though we cannot discard that BigA and BigB use a similar mechanism to be transported to the outer membrane, it is highly probable they are translocated in a yet unknown way. The outcome of the interaction between Brucella and its host cells relies on the first interaction of the bacterium with the plasmatic membrane of the target cell. In this report, we further characterised the Bab1_2009-Bab1_2012 genomic island, and found that, not only encodes two adhesins, but it also regulates, through PalA, the correct display of them in the outer membrane. Further work will be required to establish how PalA achieves this, how BigB interacts with the host cell as well as the identification of the receptors for both adhesins.  :1,000). *p < .01, unpaired t-test. All assays were performed at least three times F I G U R E 2 Bab1_2012 localises in the outer membrane. (a) Western blot analysis of total membranes or soluble fraction (supernatant) prepared with the B. abortus 2308 (pBBR-Bab1_2012-3xFlag) strain. (b) Western-blot analysis of the subcellular localisation of Bab1_2012-3xFlag fusion performed with the B. abortus 2308 (pBBR-Bab1_2012-3xFlag) strain. Perip. + OM, periplasm plus outer membrane. Bab1_2012, FLAG staining; Omp19, outer membrane protein 19; GroEL, cytoplasmic chaperonin. (c) Immunofluorescence microscopy of non-permeabilised cells of B. abortus 2308 (pBBR-BigB-sfGFP) and B. abortus 2308 Δbab1_2012 with a polyclonal mouse anti Ig-Bab1_2012 plasmid digested with the same enzymes. The primers used for PCR amplification were PL4 (5 0 -CCGGAATTCATCATTCAGGAGCAAAAATG-3 0 ) and PL5 (5 0 -GATATTACGGCTTTGGGATC-3 0 ) to amplify a 500 bp upstream region and PL6 (5 0 -CAAAGCCGTAATATCTGATTTGGAGATACTTACTTT-3 0 ) and PL7 (5 0 -CGCGGATCCTAATGGCGGTGTTATC-3 0 ) to amplify a 500 bp downstream region; PL4 and PL7 were used for the overlapping PCR.
In both cases, the resulting plasmids were introduced into B. abortus 2308 by biparental mating using the E. coli S17-λpir strain.
Double recombination events (Km s Sac r ) were selected, and the gene knockout was confirmed by genomic PCR.

| Construction of pQE30 IgBigB-3xFLAG-HIS
In order to generate the poly-histidine-tagged IgBigB/3xFlag recombinant protein, a DNA fragment was first amplified from B. abortus genomic and PL2 (5 0 -CGATCATGGCTATTACCCTTGTAGA-3 0 ) and the PCR product was digested with KpnI and NcoI and cloned in pBBR1-MCS4-3xFLAG (Dohmer et al., 2014) in the same sites. The fragment was subsequently amplified using primers PL1 and PL3 (5 0 -CGATCATGGCTATTACCCTTGTAGA-3 0 ) and the PCR fragment was digested with KpnI and PstI and subcloned in the pQE30 vector in the same sites.

| Protein expression and purification
The product of this was concentrated by ultrafiltration and used as the source of recombinant protein.

| Antibody production and purification
Anti-Bab1_2012 antiserum was performed inoculating mice (a first inoculation and two boosters at 17 and 32 days after the primo injection) with 100 μg in the first and 50 μg in the boosters of the recombinant protein in Freund's adjuvant. Two weeks after the last booster, mice were evaluated for their response by western blot and the positive animals were euthanised and the serum extracted.

| Adhesion and internalisation assays
To determine adhesion and invasion, infected cells were fixed for 15 min in 4% paraformaldehyde (pH 7.4) at room temperature at 4 hr post-infection. Coverslips were washed three times with PBS, incubated for 10 min with PBS added with 50 mM NH 4 Cl and, incubated with the primary antibody rabbit anti-Brucella polyclonal antibody (dilution 1:1,500) in a PBS, 5% BSA solution. Afterwards permeabilisation with saponin was performed and then cells were washed and incubated with the other primary antibody mouse anti-M84 (anti-O-antigen) monoclonal antibody (dilution 1:1,000) (Nielsen, Kelly, Gall, Nicoletti, & Kelly, 1995) in a PBS, 5% BSA, 0.1% saponin solution followed by incubation with the secondary antibodies (Alexa Fluor 568 or 488, Molecular Probes, Invitrogen Co) in a PBS, 5% BSA, 0.1% saponin solution. The coverslips were mounted as described before. Invasion was determined as the number of bacteria positive for both labels versus the ones positive for the anti-mouse labelling. Adhesion was determined counting the number of bacteria associated per 100 cells.

| Bacterial staining
For staining of Bab1_2012 on total bacteria, a saturated culture was diluted in order to obtain 10 6 UFC ml −1 and the cells fixed in 4% paraformaldehyde for 20 min, washed with PBS and incubated in suspension with a 1:25 dilution of the anti-Bab1_2012 policlonal antibody (refer to the preceding texts) for 8 hr. After two washes with PBS, the cells were incubated with a 1:2,000 dilution of a secondary anti-mouse conjugated to Alexa 568 for 1 hr. After two washes with PBS, bacteria were deposited on polylysine-treated glasses and mounted for confocal observation.
4.10 | Analysis of protein expression and subcellular localisation 4.10.1 | Whole bacteria Brucella whole cell extracts were resuspended in Laemmli sample buffer and heated to 100 C for 5 min. Samples were submitted to SDS-PAGE (10% or 15% depending on the assay) and transferred to nitrocellulose F I G U R E 6 PalA and BigA interact directly or indirectly. (a and b) Immunofluorescence and DIC images of the wild-type and ΔpalA mutant strains expressing a BigA-super-folder GFP fusion protein (BigA-sfGFP) showing how the fusion shows a bi-polar localisation. (c) Co-immunoprecipitation (Co-IP) analysis of protein extracts from the B. abortus 2308 strain co-expressing PalA-3xFlag and BigA-sfGFP. As a negative control a B. abortus 2308 strain expressing only the BigA-sfGFP was used. Immunoprecipitation was performed with an anti-Flag monoclonal antibody coupled to sepharose and the immunoprecipitates analysed by western blot with an anti-GFP antibody. Input was the crude extracts prior to the immunoprecipitation membranes. The presence of 3xFLAG or sfGFP-tagged proteins was carried out by immunoblot analysis using mouse anti-Flag M2 monoclonal antibody (Sigma-Aldrich dilution, 1:5,000) or anti-GFP monoclonal antibody (1:2,000), and IRDye secondary anti-mouse antibody (LI-COR, Inc.).

| Periplasmic and cytoplasmic localisation assay
Fractionation assays were performed as described previously described (Dohmer et al., 2014). B. abortus strains were grown in TSB for 16-24 hr at 37 C until an A 600 of 1 was reached, and 2.5 × 10 10 bacterial cells were centrifuged for 10 min at 3,300 g. The pellets were washed with physiological solution, centrifuged for 10 min at 3,300 ×g, and resuspended in 1 ml of 0.2 M Tris-HCl (pH 7.6). One millilitre of 0.2 M Tris-HCl (pH 7.6), 1 M sucrose and 0.25% Zwitterion 3-16 solution was added to the cell suspension and incubated for 10 min at room temperature. The samples were centrifuged for 30 min at 8,000 ×g, and the pellets were separated from the supernatants and stored at −20 C until used for western blot analysis.

| Microscopy analysis of Brucella
Exponential-phase cultures of B. abortus BigB-sfGFP strains expressing the BigB gene tagged with super folder GFP fluorescent fusion protein was placed on a microscope slide that was layered with a pad of 1% agarose in phosphate-buffered saline (PBS) as previously described (Hallez et al., 2007;. For image acquisition, samples were examined on an IX81 microscope with an Olympus FV1000 confocal module (60× PLAPO objective, numerical aperture [NA] of 1.42). For each observation under the microscope, at least three fields were randomly selected for analysis. Images were processed with the Image J program (NIH, Bethesda, MD).
Detection was performed using the Odyssey imaging system (LI-COR, Inc.).