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Saliva contains many proteins that have an important role in biological process of the oral cavity and is closely associated with many diseases. Although the dog is a common companion animal, the composition of salivary proteome and its relationship with that of human are unclear. In this study, shotgun proteomics was used to compare the salivary proteomes of 7 Thai village dogs and 7 human subjects. Salivary proteomes revealed 2,532 differentially expressed proteins in dogs and humans, representing various functions including cellular component organization or biogenesis, cellular process, localization, biological regulation, response to stimulus, developmental process, multicellular organismal process, metabolic process, immune system process, apoptosis and biological adhesion. The oral proteomes of dogs and humans were appreciably different. Proteins related to apoptosis processes and biological adhesion were predominated in dog saliva. Drug-target network predictions by STITCH Version 5.0 showed that dog salivary proteins were found to have potential roles in tumorigenesis, anti-inflammation and antimicrobial processes. In addition, proteins related to regeneration and healing processes such as fibroblast growth factor and epidermal growth factor were also up-regulated in dogs. These findings provide new information on dog saliva composition and will be beneficial for the study of dog saliva in diseased and health conditions in the future.

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 pleiomorphic yeast Candida albicans is a significant pathogen in immunocompromised individuals. In the oral cavity, C. albicans is an inhabitant of polymicrobial communities, and interspecies interactions promote hyphal formation and biofilm formation. C. albicans colonizes the subgingival area, and the frequency of colonization increases in periodontal disease. In this study, we investigated the interactions between C. albicans and the periodontal pathogen Porphyromonas gingivalis . C. albicans and P. gingivalis were found to coadhere in both the planktonic and sessile phases. Loss of the internalin-family protein InlJ abrogated adhesion of P. gingivalis to C. albicans , and recombinant InlJ protein competitively inhibited interspecies binding. A mutant of C. albicans deficient in expression of major hyphal protein Als3 showed diminished binding to P. gingivalis , and InlJ interacted with Als3 heterologously expressed in Saccharomyces cerevisiae . Transcriptional profiling by RNA sequencing (RNA-Seq) established that 57 genes were uniquely upregulated in an InlJ-dependent manner in P. gingivalis - C. albicans communities, with overrepresentation of those corresponding to 31 gene ontology terms, including those associated with growth and division. Of potential relevance to the disease process, C. albicans induced upregulation of components of the type IX secretion apparatus. Collectively, these findings indicate that InlJ-Als3-dependent binding facilitates interdomain community development between C. albicans and P. gingivalis and that P. gingivalis has the potential for increased virulence within such communities. IMPORTANCE Many diseases involve the concerted actions of microorganisms assembled in polymicrobial communities. Inflammatory periodontal diseases are among the most common infections of humans and result in destruction of gum tissue and, ultimately, in loss of teeth. In periodontal disease, pathogenic communities can include the fungus Candida albicans ; however, the contribution of C. albicans to the synergistic virulence of the community is poorly understood. Here we characterize the interactions between C. albicans and the keystone bacterial pathogen Porphyromonas gingivalis and show that coadhesion mediated by specific proteins results in major changes in gene expression by P. gingivalis , which could serve to increase pathogenic potential. The work provides significant insights into interdomain interactions that can enhance our understanding of diseases involving a multiplicity of microbial pathogens. Many diseases involve the concerted actions of microorganisms assembled in polymicrobial communities. Inflammatory periodontal diseases are among the most common infections of humans and result in destruction of gum tissue and, ultimately, in loss of teeth. In periodontal disease, pathogenic communities can include the fungus Candida albicans ; however, the contribution of C. albicans to the synergistic virulence of the community is poorly understood. Here we characterize the interactions between C. albicans and the keystone bacterial pathogen Porphyromonas gingivalis and show that coadhesion mediated by specific proteins results in major changes in gene expression by P. gingivalis , which could serve to increase pathogenic potential. The work provides significant insights into interdomain interactions that can enhance our understanding of diseases involving a multiplicity of microbial pathogens.

Bacterial pathogens have evolved several ingenious mechanisms to subvert host cell biology for their pathogenesis. Bacterial attachment to the host ECM establishes a niche to grow and is considered one of the critical steps of infection. This pathogenic mechanism entails coordinated assembly of the ECM by the host to form the ECM structure and organization that are specifically recognized by bacteria for their adhesion. We serendipitously discovered that epithelial Sdc1 facilitates the assembly of FN fibrils in the corneal basement membrane and that this normal biological function of Sdc1 has detrimental consequences for the host in S. pneumoniae corneal infection. Our studies suggest that bacterial subversion of the host ECM is more complex than previously appreciated. Subversion of heparan sulfate proteoglycans (HSPGs) is thought to be a common virulence mechanism shared by many microbial pathogens. The prevailing assumption is that pathogens co-opt HSPGs as cell surface attachment receptors or as inhibitors of innate host defense. However, there are few data that clearly support this idea in vivo . We found that deletion of syndecan-1 (Sdc1), a major cell surface HSPG of epithelial cells, causes a gain of function in a mouse model of scarified corneal infection, where Sdc1 −/− corneas were significantly less susceptible to Streptococcus pneumoniae infection. Administration of excess Sdc1 ectodomains significantly inhibited S. pneumoniae corneal infection, suggesting that Sdc1 promotes infection as a cell surface attachment receptor. However, S. pneumoniae did not interact with Sdc1 and Sdc1 was shed upon S. pneumoniae infection, indicating that Sdc1 does not directly support S. pneumoniae adhesion. Instead, Sdc1 promoted S. pneumoniae adhesion by driving the assembly of fibronectin (FN) fibrils in the corneal basement membrane to which S. pneumoniae attaches when infecting injured corneas. S. pneumoniae specifically bound to corneal FN via PavA, and PavA deletion significantly attenuated S. pneumoniae virulence in the cornea. Excess Sdc1 ectodomains inhibited S. pneumoniae corneal infection by binding to the Hep II domain and interfering with S. pneumoniae PavA binding to FN. These findings reveal a previously unknown virulence mechanism of S. pneumoniae where key extracellular matrix (ECM) interactions and structures that are essential for host cell homeostasis are exploited for bacterial pathogenesis. IMPORTANCE Bacterial pathogens have evolved several ingenious mechanisms to subvert host cell biology for their pathogenesis. Bacterial attachment to the host ECM establishes a niche to grow and is considered one of the critical steps of infection. This pathogenic mechanism entails coordinated assembly of the ECM by the host to form the ECM structure and organization that are specifically recognized by bacteria for their adhesion. We serendipitously discovered that epithelial Sdc1 facilitates the assembly of FN fibrils in the corneal basement membrane and that this normal biological function of Sdc1 has detrimental consequences for the host in S. pneumoniae corneal infection. Our studies suggest that bacterial subversion of the host ECM is more complex than previously appreciated.

Candida albicans is a fungus that colonizes oral cavity surfaces, the gut, and the genital tract. Streptococcus gordonii is a ubiquitous oral bacterium that has been shown to form biofilm communities with C. albicans . Formation of dual-species S. gordonii - C. albicans biofilm communities involves interaction of the S. gordonii SspB protein with the Als3 protein on the hyphal filament surface of C. albicans . Mannoproteins comprise a major component of the C. albicans cell wall, and in this study we sought to determine if mannosylation in cell wall biogenesis of C. albicans was necessary for hyphal adhesin functions associated with interkingdom biofilm development. A C. albicans mnt1 Δ mnt2 Δ mutant, with deleted α-1,2-mannosyltransferase genes and thus defective in O -mannosylation, was abrogated in biofilm formation under various growth conditions and produced hyphal filaments that were not recognized by S. gordonii . Cell wall proteomes of hypha-forming mnt1 Δ mnt2 Δ mutant cells showed growth medium-dependent alterations, compared to findings for the wild type, in a range of protein components, including Als1, Als3, Rbt1, Scw1, and Sap9. Hyphal filaments formed by mnt1 Δ mnt2 Δ mutant cells, unlike wild-type hyphae, did not interact with C. albicans Als3 or Hwp1 partner cell wall proteins or with S. gordonii SspB partner adhesin, suggesting defective functionality of adhesins on the mnt1 Δ mnt2 Δ mutant. These observations imply that early stage O -mannosylation is critical for activation of hyphal adhesin functions required for biofilm formation, recognition by bacteria such as S. gordonii , and microbial community development. IMPORTANCE In the human mouth, microorganisms form communities known as biofilms that adhere to the surfaces present. Candida albicans is a fungus that is often found within these biofilms. We have focused on the mechanisms by which C. albicans becomes incorporated into communities containing bacteria, such as Streptococcus . We find that impairment of early stage addition of mannose sugars to C. albicans hyphal filament proteins deleteriously affects their subsequent performance in mediating formation of polymicrobial biofilms. Our analyses provide new understanding of the way that microbial communities develop, and of potential means to control C. albicans infections. In the human mouth, microorganisms form communities known as biofilms that adhere to the surfaces present. Candida albicans is a fungus that is often found within these biofilms. We have focused on the mechanisms by which C. albicans becomes incorporated into communities containing bacteria, such as Streptococcus . We find that impairment of early stage addition of mannose sugars to C. albicans hyphal filament proteins deleteriously affects their subsequent performance in mediating formation of polymicrobial biofilms. Our analyses provide new understanding of the way that microbial communities develop, and of potential means to control C. albicans infections.

Biocompatible surfaces hold key to a variety of biomedical problems that are directly related to the competition between host-tissue cell integration and bacterial colonisation. A saving solution to this is seen in the ability of cells to uniquely respond to physical cues on such surfaces thus prompting the search for cell-instructive nanoscale patterns. Here we introduce a generic rationale engineered into biocompatible, titanium, substrates to differentiate cell responses. The rationale is inspired by cicada wing surfaces that display bactericidal nanopillar patterns. The surfaces engineered in this study are titania (TiO 2 ) nanowire arrays that are selectively bactericidal against motile bacteria, while capable of guiding mammalian cell proliferation according to the type of the array. The concept holds promise for clinically relevant materials capable of differential physico-mechanical responses to cellular adhesion.

Streptococcus pyogenes (GAS) is a human pathogen that causes pharyngitis and invasive diseases such as toxic shock syndrome and sepsis. The upper respiratory tract is the primary reservoir from which GAS can infect new hosts and cause disease. The factors involved in colonisation are incompletely known however. Previous evidence in oral streptococci has shown that the AgI/II family proteins are involved. We hypothesized that the AspA member of this family might be involved in GAS colonization. We describe a novel mouse model of GAS colonization of the nasopharynx and lower respiratory tract to elucidate these interactions. We used two clinical M serotypes expressing AspA, and their aspA gene deletant isogenic mutants in experiments using adherence assays to respiratory epithelium, macrophage phagocytosis and neutrophil killing assays and in vivo models of respiratory tract colonisation and infection. We demonstrated the requirement for AspA in colonization of the respiratory tract. AspA mutants were cleared from the respiratory tract and were deficient in adherence to epithelial cells, and susceptible to phagocytosis. Expression of AspA in the surrogate host Lactococcus lactis protected bacteria from phagocytosis. Our results suggest that AspA has an essential role in respiratory infection, and may function as a novel anti-phagocytic factor.

Summary 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.

The fungus Candida albicans colonizes oral cavity surfaces and is carried by up to 60% of human populations. Biofilm development by C. albicans may be modulated by oral streptococci, such as Streptococcus gordonii, S. oralis or S. mutans, so as to augment pathogenicity. In this study we sought to determine if the cell wall-associated secreted aspartyl proteinase Sap9 was necessary for hyphal adhesin functions associated with biofilm community development. A sap9Δ mutant of C. albicans SC5314 formed biofilms that were flatter, and contained fewer blastospores and more hyphal filaments than the parent strain. This phenotypic difference was accentuated under flow (shear) conditions and in the presence of S. gordonii. Dual-species biofilms of C. albicans sap9Δ with S. oralis, S. sanguinis, S. parasanguinis, S. mutans and Enterococcus faecalis all contained more matted hyphae and more bacteria bound to substratum compared to C. albicans wild type. sap9Δ mutant hyphae showed significantly increased cell surface hydrophobicity, ∼25% increased levels of binding C. albicans cell wall protein Als3, and reduced interaction with Eap1, implicating Sap9 in fungal cell–cell recognition. These observations suggest that Sap9 is associated with protein–receptor interactions between fungal cells, and with interkingdom communication in the formation of polymicrobial biofilm communities. This study shows that the fungus Candida albicans cell wall-associated Sap9 proteinase is required for control of hyphal filament formation and separation, and for regulation of mixed-species biofilm formation by C. albicans with oral Streptococcus bacteria. Graphical Abstract Figure. This study shows that the fungus Candida albicans cell wall-associated Sap9 proteinase is required for control of hyphal filament formation and separation, and for regulation of mixed-species biofilm formation by C. albicans with oral Streptococcus bacteria.

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.