The streptococcal multidomain fibrillar adhesin CshA has an elongated polymeric architecture.

The cell surfaces of many bacteria carry filamentous polypeptides termed adhesins that enable binding to both biotic and abiotic surfaces. Surface adherence is facilitated by the exquisite selectivity of the adhesins for their cognate ligands or receptors and is a key step in niche or host colonization and pathogenicity. Streptococcus gordonii is a primary colonizer of the human oral cavity and an opportunistic pathogen, as well as a leading cause of infective endocarditis in humans. The fibrillar adhesin CshA is an important determinant of S. gordonii adherence, forming peritrichous fibrils on its surface that bind host cells and other microorganisms. CshA possesses a distinctive multidomain architecture comprising an N-terminal target-binding region fused to 17 repeat domains (RDs) that are each ∼100 amino acids long. Here, using structural and biophysical methods, we demonstrate that the intact CshA repeat region (CshA_RD1-17, domains 1-17) forms an extended polymeric monomer in solution. We recombinantly produced a subset of CshA RDs and found that they differ in stability and unfolding behavior. The NMR structure of CshA_RD13 revealed a hitherto unreported all ß-fold, flanked by disordered interdomain linkers. These findings, in tandem with complementary hydrodynamic studies of CshA_RD1-17, indicate that this polypeptide possesses a highly unusual dynamic transitory structure characterized by alternating regions of order and disorder. This architecture provides flexibility for the adhesive tip of the CshA fibril to maintain bacterial attachment that withstands shear forces within the human host. It may also help mitigate deleterious folding events between neighboring RDs that share significant structural identity without compromising mechanical stability.

The Group B Streptococcal surface antigen I/II protein, BspC, interacts with host vimentin to promote adherence to brain endothelium and inflammation during the pathogenesis of meningitis.

Streptococcus agalactiae (Group B Streptococcus, GBS) normally colonizes healthy adults but can cause invasive disease, such as meningitis, in the newborn. To gain access to the central nervous system, GBS must interact with and penetrate brain or meningeal blood vessels; however, the exact mechanisms are still being elucidated. Here, we investigate the contribution of BspC, an antigen I/II family adhesin, to the pathogenesis of GBS meningitis. Disruption of the bspC gene reduced GBS adherence to human cerebral microvascular endothelial cells (hCMEC), while heterologous expression of BspC in non-adherent Lactococcus lactis conferred bacterial attachment. In a murine model of hematogenous meningitis, mice infected with ΔbspC mutants exhibited lower mortality as well as decreased brain bacterial counts and inflammatory infiltrate compared to mice infected with WT GBS strains. Further, BspC was both necessary and sufficient to induce neutrophil chemokine expression. We determined that BspC interacts with the host cytoskeleton component vimentin and confirmed this interaction using a bacterial two-hybrid assay, microscale thermophoresis, immunofluorescent staining, and imaging flow cytometry. Vimentin null mice were protected from WT GBS infection and also exhibited less inflammatory cytokine production in brain tissue. These results suggest that BspC and the vimentin interaction is critical for the pathogenesis of GBS meningitis.

The Streptococcus gordonii Adhesin CshA Protein Binds Host Fibronectin via a Catch-Clamp Mechanism.

Adherence of bacteria to biotic or abiotic surfaces is a prerequisite for host colonization and represents an important step in microbial pathogenicity. This attachment is facilitated by bacterial adhesins at the cell surface. Because of their size and often elaborate multidomain architectures, these polypeptides represent challenging targets for detailed structural and functional characterization. The multifunctional fibrillar adhesin CshA, which mediates binding to both host molecules and other microorganisms, is an important determinant of colonization by Streptococcus gordonii, an oral commensal and opportunistic pathogen of animals and humans. CshA binds the high-molecular-weight glycoprotein fibronectin (Fn) via an N-terminal non-repetitive region, and this protein-protein interaction has been proposed to promote S. gordonii colonization at multiple sites within the host. However, the molecular details of how these two proteins interact have yet to be established. Here we present a structural description of the Fn binding N-terminal region of CshA, derived from a combination of X-ray crystallography, small angle X-ray scattering, and complementary biophysical methods. In vitro binding studies support a previously unreported two-state "catch-clamp" mechanism of Fn binding by CshA, in which the disordered N-terminal domain of CshA acts to "catch" Fn, via formation of a rapidly assembled but also readily dissociable pre-complex, enabling its neighboring ligand binding domain to tightly clamp the two polypeptides together. This study presents a new paradigm for target binding by a bacterial adhesin, the identification of which will inform future efforts toward the development of anti-adhesive agents that target S. gordonii and related streptococci.

Coassociation between Group B Streptococcus and Candida albicans Promotes Interactions with Vaginal Epithelium.

Group B Streptococcus (GBS) is a leading cause of neonatal sepsis, pneumonia, and meningitis worldwide. In the majority of cases, GBS is transmitted vertically from mother to neonate, making maternal vaginal colonization a key risk factor for neonatal disease. The fungus Candida albicans is an opportunistic pathogen of the female genitourinary tract and the causative agent of vaginal thrush. Carriage of C. albicans has been shown to be an independent risk factor for vaginal colonization by GBS. However, the nature of interactions between these two microbes is poorly understood. This study provides evidence of a reciprocal, synergistic interplay between GBS and C. albicans that may serve to promote their cocolonization of the vaginal mucosa. GBS strains NEM316 (serotype III) and 515 (serotype Ia) are shown to physically interact with C. albicans, with the bacteria exhibiting tropism for candidal hyphal filaments. This interaction enhances association levels of both microbes with the vaginal epithelial cell line VK2/E6E7. The ability of GBS to coassociate with C. albicans is dependent upon expression of the hypha-specific adhesin Als3. In turn, expression of GBS antigen I/II family adhesins (Bsp polypeptides) facilitates this coassociation and confers upon surrogate Lactococcus lactis the capacity to exhibit enhanced interactions with C. albicans on vaginal epithelium. As genitourinary tract colonization is an essential first step in the pathogenesis of GBS and C. albicans, the coassociation mechanism reported here may have important implications for the risk of disease involving both of these pathogens.

Mapping the recognition domains of pneumococcal fibronectin-binding proteins PavA and PavB demonstrates a common pattern of molecular interactions with fibronectin type III repeats.

Colonization of mucosal respiratory surfaces is a prerequisite for the human pathobiont Streptococcus pneumoniae (the pneumococcus) to cause severe invasive infections. The arsenal of pneumococcal adhesins interacts with a multitude of extracellular matrix proteins. A paradigm for pneumococci is their interaction with the adhesive glycoprotein fibronectin, which facilitates bacterial adherence to host cells. Here, we deciphered the molecular interaction between fibronectin and pneumococcal fibronectin-binding proteins (FnBPs) PavA and PavB respectively. We show in adherence and binding studies that the pneumococcal interaction with fibronectin is a non-human specific trait. PavA and PavB target at least 13 out of 15 type III fibronectin domains as demonstrated in ligand overlay assays, surface plasmon resonance studies and SPOT peptide arrays. Strikingly, both pneumococcal FnBPs recognize similar peptides in targeted type III repeats. Structural comparisons revealed that the targeted type III repeat epitopes cluster on the inner strands of both ß-sheets forming the fibronectin domains. Importantly, synthetic peptides of FnIII1 , FnIII5 or FnIII15 bind directly to FnBPs PavA and PavB respectively. In conclusion, our study suggests a common pattern of molecular interactions between pneumococcal FnBPs and fibronectin. The specific epitopes recognized in this study can potentially be tested as antimicrobial targets in further scientific endeavours.

Concerted functions of Streptococcus gordonii surface proteins PadA and Hsa mediate activation of human platelets and interactions with extracellular matrix.

A range of Streptococcus bacteria are able to interact with blood platelets to form a thrombus (clot). Streptococcus gordonii is ubiquitous within the human oral cavity and amongst the common pathogens isolated from subjects with infective endocarditis. Two cell surface proteins, Hsa and Platelet adherence protein A (PadA), in S. gordonii mediate adherence and activation of platelets. In this study, we demonstrate that PadA binds activated platelets and that an NGR (Asparagine-Glycine-Arginine) motif within a 657 amino acid residue N-terminal fragment of PadA is responsible for this, together with two other integrin-like recognition motifs RGT and AGD. PadA also acts in concert with Hsa to mediate binding of S. gordonii to cellular fibronectin and vitronectin, and to promote formation of biofilms. Evidence is presented that PadA and Hsa are each reliant on the other's active presentation on the bacterial cell surface, suggesting cooperativity in functions impacting both colonization and pathogenesis.

Composition and Activity of the Non-canonical Gram-positive SecY2 Complex.

The accessory Sec system in Streptococcus gordonii DL1 is a specialized export system that transports a large serine-rich repeat protein, Hsa, to the bacterial surface. The system is composed of core proteins SecA2 and SecY2 and accessory Sec proteins Asp1-Asp5. Similar to canonical SecYEG, SecY2 forms a channel for translocation of the Hsa adhesin across the cytoplasmic membrane. Accessory Sec proteins Asp4 and Asp5 have been suggested to work alongside SecY2 to form the translocon, similar to the associated SecY, SecE, and SecG of the canonical system (SecYEG). To test this theory, S. gordonii secY2, asp4, and asp5 were co-expressed in Escherichia coli The resultant complex was subsequently purified, and its composition was confirmed by mass spectrometry to be SecY2-Asp4-Asp5. Like SecYEG, the non-canonical complex activates the ATPase activity of the SecA motor (SecA2). This study also shows that Asp4 and Asp5 are necessary for optimal adhesion of S. gordonii to glycoproteins gp340 and fibronectin, known Hsa binding partners, as well as for early stage biofilm formation. This work opens new avenues for understanding the structure and function of the accessory Sec system.

Structural and Functional Analysis of Cell Wall-anchored Polypeptide Adhesin BspA in Streptococcus agalactiae.

Streptococcus agalactiae (group B Streptococcus, GBS) is the predominant cause of early-onset infectious disease in neonates and is responsible for life-threatening infections in elderly and immunocompromised individuals. Clinical manifestations of GBS infection include sepsis, pneumonia, and meningitis. Here, we describe BspA, a deviant antigen I/II family polypeptide that confers adhesive properties linked to pathogenesis in GBS. Heterologous expression of BspA on the surface of the non-adherent bacterium Lactococcus lactis confers adherence to scavenger receptor gp340, human vaginal epithelium, and to the fungus Candida albicans Complementary crystallographic and biophysical characterization of BspA reveal a novel ß-sandwich adhesion domain and unique asparagine-dependent super-helical stalk. Collectively, these findings establish a new bacterial adhesin structure that has in effect been hijacked by a pathogenic Streptococcus species to provide competitive advantage in human mucosal infections.

Epidemiological and Molecular Characterization of an Invasive Group A Streptococcus emm32.2 Outbreak.

An emm32.2 invasive group A streptococcus (iGAS) outbreak occurred in Liverpool from January 2010 to September 2012. This genotype had not previously been identified in Liverpool, but was responsible for 32% (14/44) of all iGAS cases reported during this time period. We performed a case-case comparison of emm32.2 iGAS cases with non-emm32.2 control iGAS cases identified in the Liverpool population over the same time period to assess patient risk factors for emm32.2 iGAS infection. The emm32.2 iGAS cases were confined to the adult population. We show that homelessness, intravenous drug use, and alcohol abuse predisposed patients to emm32.2 iGAS disease; however, no obvious epidemiological linkage between the patients with emm32.2 iGAS could be identified. Comparative whole-genome sequencing analysis of emm32.2 iGAS and non-emm32.2 control isolates was also performed to identify pathogen factors which might have driven the outbreak. We identified 19 genes, five of which had previously been implicated in virulence, which were present in all of the emm32.2 iGAS isolates but not present in any of the non-emm32.2 control isolates. We report that a novel emm32.2 genotype emerged in Liverpool in 2010 and identified a specific subset of genes, which could have allowed this novel emm32.2 genotype to persist in a disadvantaged population in the region over a 3-year period.

Porphyromonas gingivalis initiates a mesenchymal-like transition through ZEB1 in gingival epithelial cells.

The oral anaerobe Porphyromonas gingivalis is associated with the development of cancers including oral squamous cell carcinoma (OSCC). Here, we show that infection of gingival epithelial cells with P. gingivalis induces expression and nuclear localization of the ZEB1 transcription factor, which controls epithelial-mesenchymal transition. P. gingivalis also caused an increase in ZEB1 expression as a dual species community with Fusobacterium nucleatum or Streptococcus gordonii. Increased ZEB1 expression was associated with elevated ZEB1 promoter activity and did not require suppression of the miR-200 family of microRNAs. P. gingivalis strains lacking the FimA fimbrial protein were attenuated in their ability to induce ZEB1 expression. ZEB1 levels correlated with an increase in expression of mesenchymal markers, including vimentin and MMP-9, and with enhanced migration of epithelial cells into matrigel. Knockdown of ZEB1 with siRNA prevented the P. gingivalis-induced increase in mesenchymal markers and epithelial cell migration. Oral infection of mice by P. gingivalis increased ZEB1 levels in gingival tissues, and intracellular P. gingivalis were detected by antibody staining in biopsy samples from OSCC. These findings indicate that FimA-driven ZEB1 expression could provide a mechanistic basis for a P. gingivalis contribution to OSCC.

Functional regions of Candida albicans hyphal cell wall protein Als3 that determine interaction with the oral bacterium Streptococcus gordonii.

The opportunistic pathogen Candida albicans colonizes the oral cavity and gastrointestinal tract. Adherence to host cells, extracellular matrix and salivary glycoproteins that coat oral surfaces, including prostheses, is an important prerequisite for colonization. In addition, interactions of C. albicans with commensal oral streptococci are suggested to promote retention and persistence of fungal cells in mixed-species communities. The hyphal filament specific cell wall protein Als3, a member of the Als protein family, is a major determinant in C. albicans adherence. Here, we utilized site-specific in-frame deletions within Als3 expressed on the surface of heterologous Saccharomyces cerevisiae to determine regions involved in interactions of Als3 with Streptococcus gordonii. N-terminal region amino acid residue deletions Δ166-225, Δ218-285, Δ270-305 and Δ277-286 were each effective in inhibiting binding of Strep. gordonii to Als3. In addition, these deletions differentially affected biofilm formation, hydrophobicity, and adherence to silicone and human tissue proteins. Deletion of the central repeat domain (Δ434-830) did not significantly affect interaction of Als3 with Strep. gordonii SspB protein, but affected other adherence properties and biofilm formation. Deletion of the amyloid-forming region (Δ325-331) did not affect interaction of Als3 with Strep. gordonii SspB adhesin, suggesting this interaction was amyloid-independent. These findings highlighted the essential function of the N-terminal domain of Als3 in mediating the interaction of C. albicans with S. gordonii, and suggested that amyloid formation is not essential for the inter-kingdom interaction.

Streptococcus gordonii comCDE (competence) operon modulates biofilm formation with Candida albicans.

Candida albicans is a pleiomorphic fungus that forms mixed species biofilms with Streptococcus gordonii, an early colonizer of oral cavity surfaces. Activation of quorum sensing (QS; intercellular signalling) promotes monospecies biofilm development by these micro-organisms, but the role of QS in mixed species communities is not understood. The comCDE genes in S. gordonii encode a sensor-regulator system (ComDE), which is activated by the comC gene product (CSP, competence stimulating peptide) and modulates expression of QS-regulated genes. Dual species biofilms of S. gordonii ΔcomCDE or ΔcomC mutants with C. albicans showed increased biomass compared to biofilms of S. gordonii DL1 wild-type with C. albicans. The ΔcomCDE mutant dual species biofilms in particular contained more extracellular DNA (eDNA), and could be dispersed with DNase I or protease treatment. Exogenous CSP complemented the S. gordonii ΔcomC transformation deficiency, as well as the ΔcomC-C. albicans biofilm phenotype. Purified CSP did not affect C. albicans hyphal filament formation but inhibited monospecies biofilm formation by C. albicans. The results suggest that the S. gordonii comCDE QS-system modulates the production of eDNA and the incorporation of C. albicans into dual species biofilms.

Chlorhexidine hexametaphosphate nanoparticles as a novel antimicrobial coating for dental implants.

Dental implants are an increasingly popular solution to missing teeth. Implants are prone to colonisation by pathogenic oral bacteria which can lead to inflammation, destruction of bone and ultimately implant failure. The aim of this study was to investigate the use of chlorhexidine (CHX) hexametaphosphate (HMP) nanoparticles (NPs) with a total CHX concentration equivalent to 5 mM as a coating for dental implants. The CHX HMP NPs had mean diameter 49 nm and composition was confirmed showing presence of both chlorine and phosphorus. The NPs formed micrometer-sized aggregated surface deposits on commercially pure grade II titanium substrates following immersion-coating for 30 s. When CHX HMP NP-coated titanium specimens were immersed in deionised water, sustained release of soluble CHX was observed, both in the absence and presence of a salivary pellicle, for the duration of the study (99 days) without reaching a plateau. Control specimens exposed to a solution of aqueous 25 µM CHX (equivalent to the residual aqueous CHX present with the NPs) did not exhibit CHX release. CHX HMP NP-coated surfaces exhibited antimicrobial efficacy against oral primary colonising bacterium Streptococcus gordonii within 8 h. The antimicrobial efficacy was greater in the presence of an acquired pellicle which is postulated to be due to retention of soluble CHX by the pellicle.

Anti-antimicrobial peptides: folding-mediated host defense antagonists.

Antimicrobial or host defense peptides are innate immune regulators found in all multicellular organisms. Many of them fold into membrane-bound α-helices and function by causing cell wall disruption in microorganisms. Herein we probe the possibility and functional implications of antimicrobial antagonism mediated by complementary coiled-coil interactions between antimicrobial peptides and de novo designed antagonists: anti-antimicrobial peptides. Using sequences from native helical families such as cathelicidins, cecropins, and magainins we demonstrate that designed antagonists can co-fold with antimicrobial peptides into functionally inert helical oligomers. The properties and function of the resulting assemblies were studied in solution, membrane environments, and in bacterial culture by a combination of chiroptical and solid-state NMR spectroscopies, microscopy, bioassays, and molecular dynamics simulations. The findings offer a molecular rationale for anti-antimicrobial responses with potential implications for antimicrobial resistance.

Axenic culture of a candidate division TM7 bacterium from the human oral cavity and biofilm interactions with other oral bacteria.

The diversity of bacterial species in the human oral cavity is well recognized, but a high proportion of them are presently uncultivable. Candidate division TM7 bacteria are almost always detected in metagenomic studies but have not yet been cultivated. In this paper, we identified candidate division TM7 bacterial phylotypes in mature plaque samples from around orthodontic bonds in subjects undergoing orthodontic treatment. Successive rounds of enrichment in laboratory media led to the isolation of a pure culture of one of these candidate division TM7 phylotypes. The bacteria formed filaments of 20 to 200 µm in length within agar plate colonies and in monospecies biofilms on salivary pellicle and exhibited some unusual morphological characteristics by transmission electron microscopy, including a trilaminated cell surface layer and dense cytoplasmic deposits. Proteomic analyses of cell wall protein extracts identified abundant polypeptides predicted from the TM7 partial genomic sequence. Pleiomorphic phenotypes were observed when the candidate division TM7 bacterium was grown in dual-species biofilms with representatives of six different oral bacterial genera. The TM7 bacterium formed long filaments in dual-species biofilm communities with Actinomyces oris or Fusobacterium nucleatum. However, the TM7 isolate grew as short rods or cocci in dual-species biofilms with Porphyromonas gingivalis, Prevotella intermedia, Parvimonas micra, or Streptococcus gordonii, forming notably robust biofilms with the latter two species. The ability to cultivate TM7 axenically should majorly advance understanding of the physiology, genetics, and virulence properties of this novel candidate division oral bacterium.

Streptococcus pyogenes antigen I/II-family polypeptide AspA shows differential ligand-binding properties and mediates biofilm formation.

The streptococcal antigen I/II (AgI/II)-family polypeptides are cell wall-anchored adhesins expressed by most indigenous oral streptococci. Proteins sharing 30-40% overall amino acid sequence similarities with AgI/II-family proteins are also expressed by Streptococcus pyogenes. The S. pyogenes M28_Spy1325 polypeptide (designated AspA) displays an AgI/II primary structure, with alanine-rich (A) and proline-rich (P) repeats flanking a V region that is projected distal from the cell. In this study it is shown that AspA from serotype M28 S. pyogenes, when expressed on surrogate host Lactococcus lactis, confers binding to immobilized salivary agglutinin gp-340. This binding was blocked by antibodies to the AspA-VP region. In contrast, the N-terminal region of AspA was deficient in binding fluid-phase gp-340, and L. lactis cells expressing AspA were not agglutinated by gp-340. Deletion of the aspA gene from two different M28 strains of S. pyogenes abrogated their abilities to form biofilms on saliva-coated surfaces. In each mutant strain, biofilm formation was restored by trans complementation of the aspA deletion. In addition, expression of AspA protein on the surface of L. lactis conferred biofilm-forming ability. Taken collectively, the results provide evidence that AspA is a biofilm-associated adhesin that may function in host colonization by S. pyogenes.

Treponema denticola chymotrypsin-like proteinase (CTLP) integrates spirochaetes within oral microbial communities.

Treponema denticola is found ubiquitously in the human oral cavity and is mainly associated with bacterial communities implicated in the establishment and development of periodontal disease. The ability to become integrated within biofilm communities is crucial to the growth and survival of oral bacteria, and involves inter-bacterial coaggregation, metabolic cooperation, and synergy against host defences. In this article we show that the chymotrypsin-like proteinase (CTLP), found within a high-molecular-mass complex on the cell surface, mediates adherence of T. denticola to other potential periodontal pathogens, Porphyromonas gingivalis, Fusobacterium nucleatum, Prevotella intermedia and Parvimonas micra. Proteolytic activity per se did not appear to be required for the interactions, and expression of the major outer-sheath protein (Msp) was not necessary, except for binding Parv. micra. Biofilms of densely packed cells and matrix, up to 40 µm in depth, were formed between T. denticola and P. gingivalis on salivary pellicle, with T. denticola cells enriched in the upper layers. Expression of CTLP, but not Msp, was critical for dual-species biofilm formation with P. gingivalis. T. denticola did not form dual-species biofilms with any of the other three periodontal bacterial species under various conditions. Synergy between T. denticola and P. gingivalis was also shown by increased inhibition of blood clotting, which was CTLP-dependent. The results demonstrate the critical role of CTLP in interactions of T. denticola with other oral micro-organisms, leading to synergy in microbial community development and host tissue pathogenesis.

Identification of clinical isolates of α-hemolytic streptococci by 16S rRNA gene sequencing, matrix-assisted laser desorption ionization-time of flight mass spectrometry using MALDI Biotyper, and conventional phenotypic methods: a comparison.

Fifty-six α-hemolytic streptococcal isolates were identified using MALDI Biotyper MS (Bruker Daltonics), API 20 Strep (bioMérieux), and BD Phoenix (Becton, Dickinson). The gold standard for identification was 16S rRNA gene sequence analysis with 16S-23S rRNA intergenic spacer sequencing. The following percentages of isolates were correctly identified to the species level: MALDI Biotyper, 46%; BD Phoenix, 35%; and API 20 Strep, 26%.

The changing faces of Streptococcus antigen I/II polypeptide family adhesins.

Streptococcus mutans antigen I/II (AgI/II) protein was one of the first cell wall-anchored adhesins identified in Gram-positive bacteria. It mediates attachment of S. mutans to tooth surfaces and has been a focus for immunization studies against dental caries. The AgI/II family polypeptides recognize salivary glycoproteins, and are also involved in biofilm formation, platelet aggregation, tissue invasion and immune modulation. The genes encoding AgI/II family polypeptides are found among Streptococcus species indigenous to the human mouth, as well as in Streptococcus pyogenes, S. agalactiae and S. suis. Evidence of functionalities for different regions of the AgI/II proteins has emerged. A sequence motif within the C-terminal portion of Streptococcus gordonii SspB (AgI/II) is bound by Porphyromonas gingivalis, thus promoting oral colonization by this anaerobic pathogen. The significance of other epitopes is now clearer following resolution of regional crystal structures. A new picture emerges of the central V (variable) region, predicted to contain a carbohydrate-binding trench, being projected from the cell surface by a stalk formed by an unusual association between an N-terminal alpha-helix and a C-terminal polyproline helix. This presentation mode might be important in determining functional conformations of other Gram-positive surface proteins that have adhesin domains flanked by alpha-helical and proline-rich regions.