Structural basis for the role of Serine-Rich Repeat Proteins from Lactobacillus reuteri in gut microbe-host interactions

Lactobacillus reuteri, a Gram-positive bacterial species inhabiting the gastrointestinal tract of vertebrates displays remarkable host adaptation. Previous mutational analyses of rodent strain L. reuteri 100-23C identified a gene encoding a predicted surface-exposed serine-rich repeat protein (SRRP100-23) that was vital for L. reuteri biofilm formation in mice. SRRPs have emerged as an important group of surface proteins on many pathogens but no structural information is available in commensal bacteria. Here we report the 2.00 Å and 1.92 Å crystal structures of the binding regions (BRs) of SRRP100-23 and SRRP53608 from L. reuteri ATCC 53608, revealing a unique “β-solenoid” fold in this important adhesin family. BRSRRP53608 boundto host epithelial cells and DNA at neutral pH and recognised polygalacturonic acid (PGA), rhamnogalacturonan I or chondroitin sulfate A at acidic pH. Mutagenesis confirmed the role of the BR putative binding site in the interaction of BRSRRP53608 with PGA. Long molecular dynamics simulations showed that SRRP53608 undergoes a pH-dependent conformational change. Together, these findings shed new mechanistic insights into the role of SRRPs in host-microbe interactions and open new avenues of research into the use of biofilm-forming probiotics against clinically important pathogens

Serine-Rich Repeat Protein adhesins from Lactobacillus reuteri display strain specific glycosylation profiles

Lactobacillus reuteri is a gut symbiont inhabiting the gastrointestinal tract of numerous vertebrates. The surface-exposed Serine-Rich Repeat Protein (SRRP) is a major adhesin in Gram-positive bacteria. Using lectin and sugar nucleotide profiling of wild-type or L. reuteri isogenic mutants, MALDI-ToF-MS, LC-MS and GC-MS analyses of SRRPs, we showed that L. reuteri strains 100-23C (from rodent) and ATCC 53608 (from pig) can perform protein O-glycosylation and modify SRRP100-23 and SRRP53608 with Hex-Glc-GlcNAc and di-GlcNAc moieties, respectively. Furthermore, in vivo glycoengineering in E. coli led to glycosylation of SRRP53608 variants with α-GlcNAc and GlcNAcβ(1→6)GlcNAcα moieties. The glycosyltransferases involved in the modification of these adhesins were identified within the SecA2/Y2 accessory secretion system and their sugar nucleotide preference determined by saturation transfer difference NMR spectroscopy and differential scanning fluorimetry. Together, these findings provide novel insights into the cellular O-protein glycosylation pathways of gut commensal bacteria and potential routes for glycoengineering applications

Genetic and protein determinants involved in adhesive processes of human commensal bacterium Streptococcus salivarius

Afin de caractériser les mécanismes moléculaires sous-jacents au processus d’adhésion des bactéries commensales, nous avons utilisé Streptococcus salivarius comme modèle. Streptococcus salivarius est une bactérie pionière dans la colonisation des surfaces orales chez le nouveau né, et devient par la suite un composant majoritaire du microbiote oral de l'adulte avec un rôle écologique majeur. Nous avons développé une méthode pour identifier, par des tests de criblage phénotypique, les gènes impliqués dans l’adhésion de S. salivarius aux surfaces bactériennes ou de l’hôte. Notre approche a permis d’identifier un ensemble de gènes codant pour des protéines de surfaces, des glycosyltransférases, des transporteurs qui sont impliqués dans les phénomènes d’auto-agréation et / ou de co-agrégation avec d’autres espèces et / ou l’adhésion aux protéines de l’hôte.En particulier, nous avons montré que le système SecA2Y2, qui comprend des gènes codant pour des protéines dédiées à la glycosylation et l'export de protéines de surface riche en sérine (SRRPs), participe aux processus d’agrégation, de formation de biofilms, à l'adhésion in vitro aux protéines de l’hôte et in vivo à la colonisation du tractus digestif de souris. Alors que toutes les bactéries contenant un système similaire possèdent un substrat unique au système, une SRRP, le locus génétique secA2Y2 comprend trois SRRPs qui présentent des rôles complémentaires dans les phénotypes précédement cités. SrpB est spécifiquement impliquée dans la liaison aux cellules epitheliales, tandis que SrpC participe à l’adhésion aux protéines de la matrice extracellulaire et le mucus. De manière atypique, nous avons démontré que le processus de maturation des SRRPs est supporté par glycosyltransférases extra-cluster. Cette étude est le premier rapport indiquant la présence dans une bactérie de trois SRRPs, qui présentent des rôles complémentaires dans l'interaction bactéries-hôte. Bien que le système SecA2Y2 soit principalement associé à la virulence des bactéries pathogènes, il semble être clairement impliqué dans les caractères de commensalité de S. salivarius, tels que la colonisation de ses niches écologiques orales et intestinales. Ce travail offre de nouvelles perspectives sur les mécanismes de colonisation des bactéries commensales.To characterize molecular mechanisms underlying adhesion of commensal bacteria, we used Streptococcus salivarius (SSAL) as a model. SSAL is among the most important pioneer colonizers of neonatal oral mucosal surfaces, and later becomes a predominant component of the human adult oral microbiota with pre-eminent ecological role. We developed a method to identified, through phenotypic screening assays, genes involved in SSAL adhesion to host or bacterial surfaces. In particular, we showed that the SecA2Y2 system, which comprises genes devoted to glycosylation and export of surface Serine Rich Repeat Proteins (SRRPs), participates to bacterial aggregation, biofilm formation, in vitro adhesion and colonization of mice. While all bacteria containing a similar system possess only one SRRP, the SSAL secA2Y2 locus comprises three SRRPs with complementary role in line with the previous phenotypes. Interestingly, SrpB is specifically involved in the binding to epithelial cells, while SrpC to the extracellular matrix and mucus proteins. We showed that these interactions require glycosylation of both bacterial SRPs and host surfaces. Surprisingly, we demonstrated that this essential process is shared by glycosyltransferases located in other genomic regions. This work is the first report showing the presence in a bacterium of three SRPs, which display complementary roles in bacterial-host interaction. While the SecA2Y2 system is mostly associated to virulence in pathogenic bacteria, it appears to be involved in the expression of commensal traits in SSAL, such as its colonization and its resilience to oral and intestinal niches. This work may offer new insights into the mechanisms of niche establishment (host, microbial communities) of commensal bacteria

[i]Streptococcus thermopilus[/i] biofilm lifestyle : A remnant of commensal ancestral life?

Microorganisms have a long history of use in food production and preservation. Their adaptation to food environments has profoundly modified their features, mainly through genomic flux. Streptococcus thermophilus, one of the most frequent starter culture organisms consumed daily by humans emerged recently from a commensal ancestor. As such, it is a useful model for genomic studies of bacterial domestication processes. Many streptococcal species form biofilms, a key feature of the major lifestyle of these bacteria in nature. However, few descriptions of S. thermophilus biofilms have been reported. An analysis of the ability of a representative collection of natural isolates to form biofilms revealed that S. thermophilus was a poor biofilm producer and that this characteristic was associated with an inability to attach firmly to surfaces. The identification of three biofilm-associated genes in the strain producing the most biofilms shed light on the reasons for the rarity of this trait in this species. These genes encode proteins involved in crucial stages of biofilm formation and are heterogeneously distributed between strains. One of the biofilm genes appears to have been acquired by horizontal transfer. The other two are located in loci presenting features of reductive evolution, and are absent from most of the strains analyzed. Their orthologs in commensal bacteria are involved in adhesion to host cells, suggesting that they are remnants of ancestral functions. The biofilm phenotype appears to be a commensal trait that has been lost during the genetic domestication of S. thermophilus, consistent with its adaptation to the milk environment and the selection of starter strains for dairy fermentations

<i>Streptococcus thermophilus</i> Biofilm Formation: A Remnant Trait of Ancestral Commensal Life?

<div><p>Microorganisms have a long history of use in food production and preservation. Their adaptation to food environments has profoundly modified their features, mainly through genomic flux. <i>Streptococcus thermophilus</i>, one of the most frequent starter culture organisms consumed daily by humans emerged recently from a commensal ancestor. As such, it is a useful model for genomic studies of bacterial domestication processes. Many streptococcal species form biofilms, a key feature of the major lifestyle of these bacteria in nature. However, few descriptions of <i>S</i>. <i>thermophilus</i> biofilms have been reported. An analysis of the ability of a representative collection of natural isolates to form biofilms revealed that <i>S</i>. <i>thermophilus</i> was a poor biofilm producer and that this characteristic was associated with an inability to attach firmly to surfaces. The identification of three biofilm-associated genes in the strain producing the most biofilms shed light on the reasons for the rarity of this trait in this species. These genes encode proteins involved in crucial stages of biofilm formation and are heterogeneously distributed between strains. One of the biofilm genes appears to have been acquired by horizontal transfer. The other two are located in loci presenting features of reductive evolution, and are absent from most of the strains analyzed. Their orthologs in commensal bacteria are involved in adhesion to host cells, suggesting that they are remnants of ancestral functions. The biofilm phenotype appears to be a commensal trait that has been lost during the genetic domestication of <i>S</i>. <i>thermophilus</i>, consistent with its adaptation to the milk environment and the selection of starter strains for dairy fermentations.</p></div

Initial adhesion of natural <i>S</i>. <i>thermophilus</i> isolates to polystyrene microplates.

<p>The numbers of adherent bacteria were determined as colony-forming units on M17 solid agar plates and are expressed as percentages of the input inoculum. The columns show the mean percentages of adherent bacteria from at least three independent experiments performed in duplicate, and the error bars indicate standard deviations. Statistical analyses included one-way ANOVA and Bonferroni post hoc tests for comparisons of the mean value for each strain with that of the strain with the lowest mean value (CNRZ385). Asterisks denote statistically significant differences (<i>P</i> < 0.001).</p

The three Serine rich Proteins encoded in the secA2Y2 system of the human commensal bacterium Streptococcus salivarius promotes binding to different targets of the host

The three Serine rich Proteins encoded in the secA2Y2 system of the human commensal bacterium [i]Streptococcus salivarius[/i] promotes binding to different targets of the host. 7th international conference on Gram-positive microorganism

Role of STH8232_0171, STH8232_0714 and STH8232_1361 in biofilm formation.

<p>(A-B), Properties of biofilms formed by <i>S</i>. <i>thermophilus</i> JIM8232 and its isogenic mutants JIM9169 (STH8232_0714), JIM9170 (STH8232_1361) and JIM9171 (STH8232_0171). Panel (A) shows 3-D reconstructions of representative confocal images of biofilms and panel (B) shows biofilm biovolumes (Biofilm biovol.), substrate coverages (Subst. cover.) and mean thicknesses (Mean thick.), normalized against those for JIM8232, for each strain, as measured in nine different CLSM image stacks from three independent experiments. Scale bar, 50 μm. (C), Bacterial adhesion (after 1 h) to polystyrene wells. Adherent bacteria were counted as colony-forming units on M17 solid agar plates and the results are expressed as percentages of the input inoculum. The columns show the mean values for five independent experiments, each performed in triplicate, and the error bars indicate the standard deviations. (D), Percentage of bacteria bound to hexadecane solvent. The columns show the mean values for four independent experiments performed in duplicate, and the error bars indicate standard deviations. (E), Scanning electron microscopy of <i>S</i>. <i>thermophilus</i> JIM8232 and its isogenic mutants. Arrows indicate the cell wall polysaccharide layer. Scale bar, 0.2 μm. The significance of the differences between JIM8232 and its isogenic mutants was determined in Student’s paired <i>t</i>-tests (****, <i>P</i> < 0.0001; ***, <i>P</i> < 0.001; **, <i>P</i> < 0.01).</p