Adhesive protein-mediated cross-talk between Candida albicans and Porphyromonas gingivalis in dual species biofilm protects the anaerobic bacterium in unfavorable oxic environment.

The oral cavity contains different types of microbial species that colonize human host via extensive cell-to-cell interactions and biofilm formation. Candida albicans-a yeast-like fungus that inhabits mucosal surfaces-is also a significant colonizer of subgingival sites in patients with chronic periodontitis. It is notable however that one of the main infectious agents that causes periodontal disease is an anaerobic bacterium-Porphyromonas gingivalis. In our study, we evaluated the different strategies of both pathogens in the mutual colonization of an artificial surface and confirmed that a protective environment existed for P. gingivalis within developed fungal biofilm formed under oxic conditions where fungal cells grow mainly in their filamentous form i.e. hyphae. A direct physical contact between fungi and P. gingivalis was initiated via a modulation of gene expression for the major fungal cell surface adhesin Als3 and the aspartic proteases Sap6 and Sap9. Proteomic identification of the fungal surfaceome suggested also an involvement of the Mp65 adhesin and a "moonlighting" protein, enolase, as partners for the interaction with P. gingivalis. Using mutant strains of these bacteria that are defective in the production of the gingipains-the proteolytic enzymes that also harbor hemagglutinin domains-significant roles of these proteins in the formation of bacteria-protecting biofilm were clearly demonstrated.

Community Development between Porphyromonas gingivalis and Candida albicans Mediated by InlJ and Als3.

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 gingivalisC. 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.

Role of Candida albicans secreted aspartyl protease Sap9 in interkingdom biofilm formation.

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.

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.

O-mannosylation in Candida albicans enables development of interkingdom biofilm communities.

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 mnt1Δ mnt2Δ 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 mnt1Δ mnt2Δ 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 mnt1Δ mnt2Δ 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 mnt1Δ mnt2Δ 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.

Multiple adhesin proteins on the cell surface of Streptococcus gordonii are involved in adhesion to human fibronectin.

Adhesion of bacterial cells to fibronectin (FN) is thought to be a pivotal step in the pathogenesis of invasive infectious diseases. Viridans group streptococci such as Streptococcus gordonii are considered commensal members of the oral microflora, but are important pathogens in infective endocarditis. S. gordonii expresses a battery of cell-surface adhesins that act alone or in concert to bind host receptors. Here, we employed molecular genetic approaches to determine the relative contributions of five known S. gordonii surface proteins to adherence to human FN. Binding levels to FN by isogenic mutants lacking Hsa glycoprotein were reduced by 70 %, while mutants lacking CshA and CshB fibrillar proteins showed approximately 30 % reduced binding. By contrast, disruption of antigen I/II adhesin genes sspA and sspB in a wild-type background did not result in reduced FN binding. Enzymic removal of sialic acids from FN led to reduced S. gordonii DL1 adhesion (>50 %), but did not affect binding by the hsa mutant, indicating that Hsa interacts with sialic acid moieties on FN. Conversely, desialylation of FN did not affect adherence levels of Lactococcus lactis cells expressing SspA or SspB polypeptides. Complementation of the hsa mutant partially restored adhesion to FN. A model is proposed for FN binding by S. gordonii in which Hsa and CshA/CshB are primary adhesins, and SspA or SspB play secondary roles. Understanding the basis of oral streptococcal interactions with FN will provide a foundation for development of new strategies to control infective endocarditis.

Streptococcus gordonii modulates Candida albicans biofilm formation through intergeneric communication.

The fungus Candida albicans colonizes human oral cavity surfaces in conjunction with a complex microflora. C. albicans SC5314 formed biofilms on saliva-coated surfaces that in early stages of development consisted of approximately 30% hyphal forms. In mixed biofilms with the oral bacterium Streptococcus gordonii DL1, hyphal development by C. albicans was enhanced so that biofilms consisted of approximately 60% hyphal forms. Cell-cell contact between S. gordonii and C. albicans involved Streptococcus cell wall-anchored proteins SspA and SspB (antigen I/II family polypeptides). Repression of C. albicans hyphal filament and biofilm production by the quorum-sensing molecule farnesol was relieved by S. gordonii. The ability of a luxS mutant of S. gordonii deficient in production of autoinducer 2 to induce C. albicans hyphal formation was reduced, and this mutant suppressed farnesol inhibition of hyphal formation less effectively. Coincubation of the two microbial species led to activation of C. albicans mitogen-activated protein kinase Cek1p, inhibition of Mkc1p activation by H(2)O(2), and enhanced activation of Hog1p by farnesol, which were direct effects of streptococci on morphogenetic signaling. These results suggest that interactions between C. albicans and S. gordonii involve physical (adherence) and chemical (diffusible) signals that influence the development of biofilm communities. Thus, bacteria may play a significant role in modulating Candida carriage and infection processes in the oral cavity.

Location and activity of members of a family of virPphA homologues in pathovars of Pseudomonas syringae and P. savastanoi.

Summary virPphA is a major determinant of the pathogenicity of Pseudomonas savastanoi pv. phaseolicola to Phaseolus bean. A family of homologues of virPphA was detected in pathovars of P. savastanoi and P. syringae. We examined the structure and activity of alleles designated virPphA, virPphA(Pgy), and virPphA(Psv) from P. savastanoi pathovars phaseolicola, glycinea, and savastanoi, respectively, and avrPtoB from P. syringae pv. tomato. Sequencing showed that the virPphA(Pgy) homologue had a 48-bp central deletion in the open reading frame (ORF) compared with virPphA and virPphA(Psv), but otherwise all three P. savastanoi alleles had > 98% identity at the DNA level. By contrast, AvrPtoB from P. syringae pv. tomato strain DC3000 was predicted to have only 51% amino acid similarity with VirPphA. All ORFs have an upstream hrp-box promoter indicating potential regulation by HrpL. Each cloned homologue was introduced into the P. savastanoi pv. phaseolicola strain RW60, which lacks a native plasmid carrying virPphA as part of a pathogenicity island (PAI), and which is not pathogenic on bean. The homologues all restored virulence, as measured by the development of water-soaked lesions in bean pods, and increased bacterial populations in leaves compared with RW60 alone. RW60 harbouring virPphA or virPphA(Psv) elicited a strong hypersensitive reaction (HR) in soybean cv. Osumi; the presence of avrPtoB caused a weak HR, but virPphA(Pgy) did not affect the null reaction observed in soybean with RW60 alone. A second effector gene, avrPphD, was detected on the genomic clones carrying virPphA(Pgy) and virPphA(Psv). avrPphD was also present in both P. savastanoi pv. phaseolicola and P. syringae pv. tomato, but elsewhere in their genomes. Comparison of the genomic locations of virPphA and other effectors found in the P. savastanoi pv. phaseolicola PAI revealed the greatest divergence of the sequences surrounding virPphA to be in P. syringae pv. tomato.