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Critical roles of a housekeeping sortase of probiotic Bifidobacterium bifidum in bacterium–host cell crosstalk

Summary Bifidobacterium bifidum YIT 10347 (BF-1) is adhesive in vitro. Here we studied the molecular aspects of the BF-1 adhesion process. We identified and characterized non-adhesive mutants and found that a class E housekeeping sortase was critical for the adhesion to mucin. These mutants were significantly less adhesive to GCIY cells than was the wild type (WT), which protected GCIY cells against acid treatment more than did a non-adhesive mutant. The non-adhesive mutants aberrantly accumulated precursors of putative sortase-dependent proteins (SDPs). Recombinant SDPs bound to mucin. Disruption of the housekeeping sortase influenced expression of SDPs and pilus components. Mutants defective in a pilin or in an SDP showed the same adhesion properties as WT. Therefore, multiple SDPs and pili seem to work cooperatively to achieve adhesion, and the housekeeping sortase is responsible for cell wall anchoring of its substrates to ensure their proper biological function.


INTRODUCTION
Bifidobacteria, a major bacterial group in the human large intestine, may provide human health benefits, but their numbers decrease upon weaning and aging (Mitsuoka, 1992;Woodmansey, 2007). Several bifidobacteria have been utilized as probiotics (Fuller, 1989), and Bifidobacterium bifidum is one of the major species used. Above all, B. bifidum has the unique ability to relieve symptoms of gastric disorders in humans (Miki et al., 2007;Gomi et al., , 2018Urita et al., 2015). The adhesion of B. bifidum in vitro is exceptionally strong among probiotics (Shibahara-Sone et al., 2016), and its mechanisms of adhesion and implications for host-bacteria crosstalk (i.e., probiotic effects) have interested many microbiologists Turroni et al., 2014).
But reductionist approaches such as biochemistry or molecular biology occasionally encounter difficulties such as false positives, overlooking interactions among multiple factors, and failure to establish causal relationships. Consequently, the overall picture of the adhesion mechanism in B. bifidum remains obscure.
We therefore aimed to (i) develop a transposon mutagenesis system and ELISA-based adhesion assay system for B. bifidum YIT 10347 (BF-1) to enable a reverse-genetic approach, (ii) isolate non-adhesive BF-1 mutants, (iii) evaluate the effects of BF-1 adhesion on acid tolerance of cultured cells, and (iv) characterize the mechanism of BF-1 adhesion. These studies revealed that a housekeeping sortase is responsible for cell wall anchoring of its substrates to ensure their proper biological function. This is the first report to present conclusive information on the mechanism of adhesion or bacterium-host cell interaction.

Impact of bacterial adhesion on cultured cells
We then compared the adhesion properties of WT and the mutant #1476 by using GCIY cells, because #1476 was constitutively non-adhesive regardless of culture conditions, as described in the following section. This mutant showed significantly less adhesion to GCIY cells than did WT ( Figure 1A), consistent with the results of the mucin adhesion assay.
We also evaluated the effects of BF-1 adhesion on the acid tolerance of GCIY cells. The survival ratio of acid-treated GCIY cells was significantly higher when pretreated with WT than with #1476 ( Figure 1B), suggesting that the adhesion was critical to the protective effect.

Characterization of non-adhesive mutants
The comparison of WT and #1476 in bacterial cell-GCIY cell co-incubation ( Figure 1) suggested that the housekeeping sortase is involved in the adhesion, which is critical to bacteria-host cell interaction. Next, we identified the molecules involved in adhesion by comparing the non-adhesive mutants (#1476, #1543, and #1649) with WT.
We predicted the conformation of the housekeeping sortase, the disruption of which was common to all three of the non-adhesive mutants. This sortase appeared to be a transmembrane protein possessing three membrane-spanning regions ( Figure 2A). The Tn was inserted into the sortase domain in #1476, into the second membrane-spanning region in #1543, and into the cytosolic loop in #1649. Mutants #1543 and #1649 partially recovered their adhesion when cultured in m-ILS medium. In contrast, #1476 was non-adhesive in both m-ILS and MRS media ( Figure S4). These results suggest that the site of Tn insertion affected the adhesion property in different culture media, although the underlying mechanism remains unclear. None of the non-adhesive mutants could grow on mucin ( Figure 2B), indicating that the housekeeping sortase is responsible for anchoring and processing of proteins that have a role to play in adhesion or in mucin degradation.
Cell membrane proteins extracted from the mutants listed in Table 1 were subjected to SDS-PAGE. Aberrantly accumulated proteins ranging 110-260 kDa were detected specifically in the three non-adhesive ll OPEN ACCESS 2 iScience 24, 103363, November 19, 2021 iScience Article mutants ( Figure 2C). Three proteins, BF1_0041 (BBPR_1514, b-N-acetylglucosaminidase), BF1_0740 (BBPR_0460, polysaccharide-degrading enzyme), and BF1_1579 (BBPR_1438, lacto-N-biosidase), were identified by proteomic analysis of these bands. Each of them had an N-terminal signal peptide and a C-terminal [L/I]SXTG motif followed by a membrane-spanning region and additional positively charged residues ( Figure 2D).

Sortase complementation of a non-adhesive mutant
The B. breve tuf promoter-containing sequence and BF1_0427 were subcloned into an E. coli-Bifidobacterium shuttle vector to yield a complementation plasmid for the housekeeping sortase. We used this construct to produce a complemented strain from #1476 ( Figure 3A). Adhesion was recovered upon complementation ( Figure 3B); antibody reactivity was almost the same among WT, #1476, the vector control, and the complemented strain ( Figure 3C). Aberrantly accumulated proteins disappeared upon complementation ( Figure 3D).

Genome survey for putative sortase-dependent proteins
We surveyed the BF-1 genome and found several similar proteins but possessing a VAXTG motif instead of the [L/I]SXTG motif. Thus, we extensively searched for proteins having the signal peptide at the N-terminus and an [L/I/V][S/A]XTG motif, a membrane-spanning region, and positively charged residues at the C-terminus. We found 26 such sortase-dependent proteins (SDPs), most of which were glycoside hydrolases (GHs) ( Table 2). These proteins were also found in B. bifidum PRL2010. Molecular weights of these proteins

Biophysical interaction between mucin and SDPs
To evaluate the contribution of SDPs to mucin adhesion, we prepared recombinant SDPs and investigated the binding of each SDP to mucin by surface plasmon resonance (SPR) assay. Of the 26 SDPs identified, 5 formed inclusion bodies or showed no expression, so 21 recombinant SDPs were used.
Next, we developed an SPR assay system to evaluate adhesion to mucin, using WGA and SBA as positive controls and bovine serum albumin as a negative control ( Figure S5). In the SPR assay, each SDP appeared to have a unique affinity to mucin ( Figure 4A). BF1_0030, BF1_0032, BF1_0041, BF1_0056, BF1_0510, BF1_0575, and BF1_0740 were selected as representative of SDPs for kinetic analysis. Of these, BF1_0030 (BBPR_1503), BF1_0041 (BBPR_1514), and BF1_0056 (BBPR_nagZ2) showed typical binding/ dissociation curves and dose responses ( Figure 4B), as did the lectins WGA and SBA, suggesting that multiple SDPs are involved in BF-1 adhesion to mucin.

Effects of sortase disruption on BF-1 transcriptome
To explore the molecular mechanism of BF-1 adhesion, we compared transcriptomes among WT and nonadhesive mutants cultured in MRS or m-ILS medium ( Figure S6). In all non-adhesive mutants regardless of culture medium, BF1_0322-0324 (Pil1 cluster [BBPR_1820-1822 in B. bifidum PRL2010]), BF1_1117-1120 (BBPR_937-940), and BF1_1439 (BBPR_1289) were down-regulated 5-fold, and BF1_0001 (BBPR_1472) was up-regulated 5-fold. Because pilins are regarded as adhesion factors , we examined the effects of housekeeping sortase disruption on the expression of BF-1 pilins. Pil1 was highly expressed in WT but was remarkably down-regulated in the non-adhesive mutants. Pil2 (BBPR_1707-1709, no BF1_0221 in B. bifidum PRL2010) was moderately expressed in WT and was down-regulated approximately 2-fold in the non-adhesive mutants. Pil3 (BBPR_0282-0284) had relatively low expression, and its expression was not changed by housekeeping sortase deficiency ( Figure 5A). These results indicate a strong interaction between the housekeeping sortase and pili, especially Pil1 and Pil2. BF-1 also has Type IVb tight adherence (Tad) pili (O'Connell et al., 2011), but the orthologous operon (BF1_0265-0271) was not influenced by housekeeping sortase disruption (data not shown). iScience Article Next, we evaluated the effects of housekeeping sortase disruption on SDP expression. SDPs with low expression in WT were up-regulated in all non-adhesive mutants, and such up-regulation was more remarkable in m-ILS medium than in MRS medium. Most of the up-regulated SDPs were GHs ( Figure 5B, left). The aberrant proteins identified by proteomic analysis-BF1_0041, BF1_0740, and BF1_1579-were also remarkably up-regulated in the non-adhesive mutants. The expression of other SDPs was almost the same among the non-adhesive mutants and WT, or they were weakly down-regulated in the non-adhesive mutants. Most of these SDPs were not GHs ( Figure 5B, right). We suspected that the housekeeping sortase interacted with pili and/or SDPs, and we presumed that pili and/or SDPs were the adhesion factors; therefore, we examined the adhesion properties of pilin-disrupted or SDP-disrupted mutants. Because we had identified mutants with Tn inserted into pilin or SDP genes in our evaluation of the Tn mutagenesis system ( Figure   iScience Article presence of multiple adhesion factors and that a single-gene disruption does not critically affect BF-1 adhesion.

DISCUSSION
By using reverse genetics (i.e., the Tn mutagenesis system) and high-throughput screening (i.e., the ELISAbased mucin adhesion assay), we were able to isolate non-adhesive BF-1 mutants ( Table 1). Adhesion of BF-1 and the mutants to mucin-producing GCIY cells was consistent with their adhesion to mucin; therefore, the main ligand would be mucin of GCIY cells. Adhesion was important for increasing acid tolerance of GCIY cells following BF-1 treatment ( Figure 1B). Oral administration of B. bifidum OLB 6378 to rats activates Toll-like receptor-2 (TLR-2) to reduce apoptosis in the intestinal epithelium in necrotizing enterocolitis (Khailova et al., 2010), and cell-surface b-glucan/galactan of B. bifidum PRI1 induces regulatory T cells through a partially TLR-2-mediated mechanism (Verma et al., 2018). BF-1 exerts anti-ulcer effects via increasing the levels of epidermal growth factor and basic fibroblast growth factor in vitro and in vivo (Nagaoka et al., 1994). BF-1 also represses the expression of inflammatory cytokines induced by Helicobacter pylori infection via the NF-kB signaling pathway in GCIY cells (Shirasawa et al., 2010). Such effects might be related to the GCIY responses we observed.
Genes encoding class C sortases adjacent to genes encoding pilus components (pilins) have been studied in several bifidobacterial species (Milani et al., 2017). Genes encoding housekeeping sortases (class E) are not clustered with genes for their substrates. Housekeeping sortases appear to be involved in pilus attachment and/or formation of aerial hyphae in high-G + C bacterial species (Spirig et al., 2011;Bradshaw et al., 2015). As a result of the first-ever Tn mutagenesis system for B. bifidum that we originally developed, we were able to examine all the clones positively selected by antibiotic resistance. Consequently, 2,685 clones could be examined to discover the critical role of the housekeeping sortase in adhesion to mucin and/or to cultured cells.
Mucin adhesion was closely linked to mucin utilization ( Figure 2B), suggesting that activation of SDPs involved in mucin degradation would require processing by the housekeeping sortase, anchoring to the iScience Article cell wall, or both. Mucin adhesion of #1543 and #1649 changed depending on culture medium ( Figure S4). Several SDPs may be induced by lactose, the main substrate in m-ILS (but not MRS, which contains glucose), and partially processed in #1543 or #1649, in which the housekeeping sortase domain was intact ( Figure 2A).
Our results suggest that SDPs and pili cooperatively confer the bacterial adhesion property and compensate for one another. Indeed, most of the SDPs had lectin-like domains, bacterial IgG-like domains, and carbohydrate-binding domains (data not shown). In addition, pilin-disrupted or SDP-disrupted mutants would likely be present in the Tn-disruptant library (>2,500 clones), but none of the identified pilin- iScience Article disrupted or SDP-disrupted mutants were non-adhesive. These circumstantial lines of evidence indirectly support the assumption that a single SDP or pilin disruption is not critical to BF-1 adhesion.
Based on our results and previous reports Nishiyama et al., 2017), we propose a mechanism of BF-1 adhesion ( Figure 6). SDPs with an [L/I/V][S/A]XTG motif, mainly GHs, are secreted, processed by the housekeeping class E sortase, and displayed on the cell wall. Consequently, these SDPs are involved in adhesion ( Figure 6A). Pilus shaft components (Fim A/P) are polymerized and assembled with pilus tip components (Fim B/Q) by the adjacent class C sortases (Foroni et al., 2011). The produced pilus precursors iScience Article are processed and attached to the cell surface by the housekeeping class E sortase. The displayed pili are involved in adhesion ( Figure 6B). On the whole, SDPs and pili each exert adhesion activity, and the housekeeping sortase is a key enzyme in BF-1 adhesion.
Generally, housekeeping class E sortases recognize LAXTG as a sorting signal (Kattke et al., 2016), but BF1_0427 presumably recognizes [L/I/V][S/A/P]XTG, which deviates from motifs recognized by known class E sortases. Further structural studies will be necessary to reveal the usage of these unique sorting signals.
Bifidobacterium bifidum is unique among bifidobacterial species. For instance, by being able to utilize human milk oligosaccharides, B. bifidum is one of the major bacterial species among the first colonizers of the newborn's gastrointestinal tract (Duranti et al., 2019), and by being the only bifidobacterial species able to assimilate mucin (Pechar et al., 2014), it contributes to cross-feeding of other bacteria including bifidobacterial species (Egan et al., 2014;Turroni et al., 2015;Bunesova et al., 2018;Gotoh et al., 2018;Nishiyama et al., 2018;O'Connell et al., 2018;Centanni et al., 2019). Such characteristics might arise from the SDPs specific to B. bifidum (i.e., GHs). The reason why B. bifidum has developed such SDPs in evolution (e.g., adaptation to the environment of the gastrointestinal tract) remains unclear. Studies into why this is so will provide good opportunities to integrate microbial ecology and pan-genomics. The housekeeping sortase and SDPs would provide a new approach to studies on bifidobacteria.
In conclusion, the housekeeping sortase in B. bifidum is a key molecule both in adhesion to mucus and in bacterium-host cell interaction.

Limitations of the study
Because BF-1 is adhesive to the gastric mucus in vivo (Shibahara-Sone et al., 2016), we used reagent-grade porcine stomach mucin for the large-scale screening. Yet, the bifidobacterial habitat is the large intestine; therefore, it might be better to check the adhesion properties of non-adhesive mutants using mucins purified from the human colon or a human colon cell line. The adhesion mechanism presented in this paper is based on in vitro study. As the next step, the adhesion mechanism in vivo could be investigated using animal models, including gnotobiotic mice or rats.
Microarray analysis revealed that housekeeping sortase would functionally interact with pili, but the other effects of sortase disruption remain obscure. Identification of other genes up-regulated or down-regulated in the non-adhesive mutants might provide insights into the pleiotropic effects of housekeeping sortase disruption. Although multiple SDPs or pili are assumed to be involved in BF-1 adhesion, the contribution of each SDP or pilus remains unclear, because their levels and adhesive activity have not been characterized in detail. An SDP-deletion or pilus-deletion mutant generated by genome editing would be useful for investigating the adhesion system.

Materials availability
All materials are available from the corresponding author upon reasonable request.

Data and code availability
Complete genome sequence and microarray data have been deposited and are publicly available as of the date of publication. Accession numbers are listed in the key resources table. This study did not generate any computational codes. Any additional information required to reanalyze the data reported in this paper is available from the lead contact upon request.
To count viable bacteria (CFUs), serial dilutions of the samples were plated on agar plates containing MRS medium supplemented with 0.05% L-cysteine HCl and cultured at 37 C. Preparation of the dilutions and cultures was carried out anaerobically.
Escherichia coli JM109 (Takara Bio, Shiga, Japan) were used as competent cells for plasmid construction; they were aerobically cultured at 37 C in Luria-Bertani medium (Difco Laboratories).

Assay of bacterial adhesion to mucin
Mucin (porcine stomach mucin, Type III, bound sialic acid 0.5%-1.5%; Sigma-Aldrich) was serially diluted (0.0005-1 mg/well) with 50 mM sodium carbonate buffer (pH 9.6) and immobilized in 96-well flat-bottom plates (Nunc MaxiSorp) at 4 C overnight. The immobilized mucin was blocked with PBS containing 1% gelatin (#1706537, Bio-Rad Laboratories). After removal of the blocking buffer, a bacterial suspension (OD 660 nm = 1.0) in PBS containing 1% gelatin was added to the plates and incubated at 37 C for 1 h, shaken at 1,200 rpm with a microplate shaker (Certomatâ MT, B. Braun, Melsungen, Germany). After removal of the bacterial suspension, the plates with adhering bacteria were washed three times with PBS containing 0.1% Tween 20 (Bio-Rad Laboratories). Bacteria adhering to the immobilized mucin were measured by using a rabbit anti-BF-1 custom polyclonal antibody (Eurofins, Tokyo, Japan) as primary antibody and horseradish peroxidase (HRP)-conjugated goat anti-rabbit Ig (SouthernBiotech, Birmingham, AL, USA) as iScience Article

Microarray analysis
A custom microarray (Agilent Technologies) based on the genome sequence of BF-1 was used for microarray analysis. According to the manufacturer's protocol, the microarray images were scanned by a Sure-Scan microarray scanner (Agilent Technologies) and analyzed by Feature Extraction 12.0 (Agilent Technologies). Microarray data were analyzed by GeneSpring 14 (Agilent Technologies). The array format and data were submitted to the Gene Expression Omnibus (GEO: GSE175843). Because bacterial transcriptomes are reproducible, no replications were performed; instead, we examined all three mutants to discover common gene expression profiles. The expression of genes shown in Figure 5 was verified by RT-qPCR.

QUANTIFICATION AND STATISTICAL ANALYSIS
Statistical significance was determined with Welch t test for two groups and one-way ANOVA with post hoc Tukey honestly significant difference (HSD) test for more than two groups in R software (https://www.Rproject.org/). Differences were considered statistically significant at p values of %0.05.