Streptococcus gordonii programs epithelial cells to resist ZEB2 induction by Porphyromonas gingivalis.

The polymicrobial microbiome of the oral cavity is a direct precursor of periodontal diseases, and changes in microhabitat or shifts in microbial composition may also be linked to oral squamous cell carcinoma. Dysbiotic oral epithelial responses provoked by individual organisms, and which underlie these diseases, are widely studied. However, organisms may influence community partner species through manipulation of epithelial cell responses, an aspect of the host microbiome interaction that is poorly understood. We report here that Porphyromonas gingivalis, a keystone periodontal pathogen, can up-regulate expression of ZEB2, a transcription factor which controls epithelial-mesenchymal transition and inflammatory responses. ZEB2 regulation by P. gingivalis was mediated through pathways involving ß-catenin and FOXO1. Among the community partners of P. gingivalis, Streptococcus gordonii was capable of antagonizing ZEB2 expression. Mechanistically, S. gordonii suppressed FOXO1 by activating the TAK1-NLK negative regulatory pathway, even in the presence of P. gingivalis Collectively, these results establish S. gordonii as homeostatic commensal, capable of mitigating the activity of a more pathogenic organism through modulation of host signaling.

Identification of Small-Molecule Inhibitors Targeting Porphyromonas gingivalis Interspecies Adherence and Determination of Their In Vitro and In Vivo Efficacies.

Porphyromonas gingivalis is one of the primary causative agents of periodontal disease and initially colonizes the oral cavity by adhering to commensal streptococci. Adherence requires the interaction of a minor fimbrial protein (Mfa1) of P. gingivalis with streptococcal antigen I/II (AgI/II). Our previous work identified an AgI/II peptide that potently inhibited adherence and significantly reduced P. gingivalis virulence in vivo, suggesting that this interaction represents a potential target for drug discovery. To develop targeted small-molecule inhibitors of this protein-protein interaction, we performed a virtual screen of the ZINC databases to identify compounds that exhibit structural similarity with the two functional motifs (NITVK and VQDLL) of the AgI/II peptide. Thirty three compounds were tested for in vitro inhibition of P. gingivalis adherence and the three most potent compounds, namely, N7, N17, and V8, were selected for further analysis. The in vivo efficacy of these compounds was evaluated in a murine model of periodontitis. Treatment of mice with each of the compounds significantly reduced maxillary alveolar bone resorption in infected animals. Finally, a series of cytotoxicity tests were performed against human and murine cell lines. Compounds N17 and V8 exhibited no significant cytotoxic activity toward any of the cell lines, whereas compound N7 was cytotoxic at the highest concentrations that were tested (20 and 40 µM). These results identify compounds N17 and V8 as potential lead compounds that will facilitate the design of more potent therapeutic agents that may function to limit or prevent P. gingivalis colonization of the oral cavity.

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.

Structural insights unravel the zymogenic mechanism of the virulence factor gingipain K from Porphyromonas gingivalis, a causative agent of gum disease from the human oral microbiome.

Skewing of the human oral microbiome causes dysbiosis and preponderance of bacteria such as Porphyromonas gingivalis, the main etiological agent of periodontitis. P. gingivalis secretes proteolytic gingipains (Kgp and RgpA/B) as zymogens inhibited by a pro-domain that is removed during extracellular activation. Unraveling the molecular mechanism of Kgp zymogenicity is essential to design inhibitors blocking its activity. Here, we found that the isolated 209-residue Kgp pro-domain is a boomerang-shaped all-ß protein similar to the RgpB pro-domain. Using composite structural information of Kgp and RgpB, we derived a plausible homology model and mechanism of Kgp-regulating zymogenicity. Accordingly, the pro-domain would laterally attach to the catalytic moiety in Kgp and block the active site through an exposed inhibitory loop. This loop features a lysine (Lys129) likely occupying the S1 specificity pocket and exerting latency. Lys129 mutation to glutamate or arginine led to misfolded protein that was degraded in vivo Mutation to alanine gave milder effects but still strongly diminished proteolytic activity, without affecting the subcellular location of the enzyme. Accordingly, the interactions of Lys129 within the S1 pocket are also essential for correct folding. Uniquely for gingipains, the isolated Kgp pro-domain dimerized through an interface, which partially overlapped with that between the catalytic moiety and the pro-domain within the zymogen, i.e. both complexes are mutually exclusive. Thus, pro-domain dimerization, together with partial rearrangement of the active site upon activation, explains the lack of inhibition of the pro-domain in trans. Our results reveal that the specific latency mechanism of Kgp differs from those of Rgps.

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.

FOXO responses to Porphyromonas gingivalis in epithelial cells.

Porphyromonas gingivalis is a prominent periodontal, and emerging systemic, pathogen that redirects host cell signalling pathways and modulates innate immune responses. In this study, we show that P. gingivalis infection induces the dephosphorylation and activation of forkhead box-O (FOXO)1, 3 and 4 in gingival epithelial cells. In addition, immunofluorescence showed that FOXO1 accumulated in the nucleus of P. gingivalis-infected cells. Quantitative reverse transcription PCR demonstrated that transcription of genes involved in protection against oxidative stress (Cat, Sod2, Prdx3), inflammatory responses (IL1ß) and anti-apoptosis (Bcl-6) was induced by P. gingivalis, while small-interfering RNA (siRNA)-mediated knockdown of FOXO1 suppressed the transcriptional activation of these genes. P. gingivalis-induced secretion of interleukin (IL)-1ß and inhibition of apoptosis were also impeded by FOXO1 knockdown. Neutralization of reactive oxygen species (ROS) by N-acetyl-l-cysteine blocked the activation of FOXO1 by P. gingivalis and concomitantly suppressed the activation of oxidative stress responses, anti-apoptosis programmes and IL-ß production. Inhibition of c-Jun-N-terminal kinase (JNK) either pharmacologically or by siRNA, reduced FOXO1 activation and downstream FOXO1-dependent gene regulation in response to P. gingivalis. The results indicate that P. gingivalis-induced ROS activate FOXO transcription factors through JNK signalling, and that FOXO1 controls oxidative stress responses, inflammatory cytokine production and cell survival. These data position FOXO as an important signalling node in the epithelial cell-P. gingivalis interaction, with particular relevance to cell fate and dysbiotic host responses.

Structure and mechanism of cysteine peptidase gingipain K (Kgp), a major virulence factor of Porphyromonas gingivalis in periodontitis.

Cysteine peptidases are key proteolytic virulence factors of the periodontopathogen Porphyromonas gingivalis, which causes chronic periodontitis, the most prevalent dysbiosis-driven disease in humans. Two peptidases, gingipain K (Kgp) and R (RgpA and RgpB), which differ in their selectivity after lysines and arginines, respectively, collectively account for 85% of the extracellular proteolytic activity of P. gingivalis at the site of infection. Therefore, they are promising targets for the design of specific inhibitors. Although the structure of the catalytic domain of RgpB is known, little is known about Kgp, which shares only 27% sequence identity. We report the high resolution crystal structure of a competent fragment of Kgp encompassing the catalytic cysteine peptidase domain and a downstream immunoglobulin superfamily-like domain, which is required for folding and secretion of Kgp in vivo. The structure, which strikingly resembles a tooth, was serendipitously trapped with a fragment of a covalent inhibitor targeting the catalytic cysteine. This provided accurate insight into the active site and suggested that catalysis may require a catalytic triad, Cys(477)-His(444)-Asp(388), rather than the cysteine-histidine dyad normally found in cysteine peptidases. In addition, a 20-Å-long solvent-filled interior channel traverses the molecule and links the bottom of the specificity pocket with the molecular surface opposite the active site cleft. This channel, absent in RgpB, may enhance the plasticity of the enzyme, which would explain the much lower activity in vitro toward comparable specific synthetic substrates. Overall, the present results report the architecture and molecular determinants of the working mechanism of Kgp, including interaction with its substrates.

Noncanonical activation of ß-catenin by Porphyromonas gingivalis.

Porphyromonas gingivalis is an established pathogen in periodontal disease and an emerging pathogen in serious systemic conditions, including some forms of cancer. We investigated the effect of P. gingivalis on ß-catenin signaling, a major pathway in the control of cell proliferation and tumorigenesis. Infection of gingival epithelial cells with P. gingivalis did not influence the phosphorylation status of ß-catenin but resulted in proteolytic processing. The use of mutants deficient in gingipain production, along with gingipain-specific inhibitors, revealed that gingipain proteolytic activity was required for ß-catenin processing. The ß-catenin destruction complex components Axin1, adenomatous polyposis coli (APC), and GSK3ß were also proteolytically processed by P. gingivalis gingipains. Cell fractionation and Western blotting demonstrated that ß-catenin fragments were translocated to the nucleus. The accumulation of ß-catenin in the nucleus following P. gingivalis infection was confirmed by immunofluorescence microscopy. A luciferase reporter assay showed that P. gingivalis increased the activity of the ß-catenin-dependent TCF/LEF promoter. P. gingivalis did not increase Wnt3a mRNA levels, a finding consistent with P. gingivalis-induced proteolytic processing causing the increase in TCF/LEF promoter activity. Thus, our data indicate that P. gingivalis can induce the noncanonical activation of ß-catenin and disassociation of the ß-catenin destruction complex by gingipain-dependent proteolytic processing. ß-Catenin activation in epithelial cells by P. gingivalis may contribute to a proliferative phenotype.

Purification and characterisation of recombinant His-tagged RgpB gingipain from Porphymonas gingivalis.

Gingipain proteases are important virulence factors from the periodontal pathogen Porphyromonas gingivalis and are the target of many in vitro studies. Due to their close biochemical properties, purification of individual gingipains is difficult and requires multiple chromatographic steps. In this study, we demonstrate that insertion of a hexahistidine affinity tag upstream of a C-terminal outer membrane translocation signal in RgpB gingipain leads to the secretion of a soluble, mature form of RgpB bearing the affinity tag that can easily be purified by nickel-chelating affinity chromatography. The final product obtained high yielding high purity is biochemically indistinguishable from the native RgpB enzyme.

Porphyromonas gingivalis virulence factor gingipain RgpB shows a unique zymogenic mechanism for cysteine peptidases.

Zymogenicity is a regulatory mechanism that prevents inadequate catalytic activity in the wrong context. It plays a central role in maintaining microbial virulence factors in an inactive form inside the pathogen until secretion. Among these virulence factors is the cysteine peptidase gingipain B (RgpB), which is the major virulence factor secreted by the periodontopathogen Porphyromonas gingivalis that attacks host vasculature and defense proteins. The structure of the complex between soluble mature RgpB, consisting of a catalytic domain and an immunoglobulin superfamily domain, and its 205-residue N-terminal prodomain, the largest structurally characterized to date for a cysteine peptidase, reveals a novel fold for the prodomain that is distantly related to sugar-binding lectins. It attaches laterally to the catalytic domain through a large concave surface. The main determinant for latency is a surface "inhibitory loop," which approaches the active-site cleft of the enzyme on its non-primed side in a substrate-like manner. It inserts an arginine (Arg(126)) into the S1 pocket, thus matching the substrate specificity of the enzyme. Downstream of Arg(126), the polypeptide leaves the cleft, thereby preventing cleavage. Moreover, the carbonyl group of Arg(126) establishes a very strong hydrogen bond with the co-catalytic histidine, His(440), pulling it away from the catalytic cysteine, Cys(473), and toward Glu(381), which probably plays a role in orienting the side chain of His(440) during catalysis. The present results provide the structural determinants of zymogenic inhibition of RgpB by way of a novel inhibitory mechanism for peptidases in general and open the field for the design of novel inhibitory strategies in the treatment of human periodontal disease.

Inhibition of gingipains by their profragments as the mechanism protecting Porphyromonas gingivalis against premature activation of secreted proteases.

BACKGROUND: Arginine-specific (RgpB and RgpA) and lysine-specific (Kgp) gingipains are secretory cysteine proteinases of Porphyromonas gingivalis that act as important virulence factors for the organism. They are translated as zymogens with both N- and C-terminal extensions, which are proteolytically cleaved during secretion. In this report, we describe and characterize inhibition of the gingipains by their N-terminal prodomains to maintain latency during their export through the cellular compartments. METHODS: Recombinant forms of various prodomains (PD) were analyzed for their interaction with mature gingipains. The kinetics of their inhibition of proteolytic activity along with the formation of stable inhibitory complexes with native gingipains was studied by gel filtration, native PAGE and substrate hydrolysis. RESULTS: PDRgpB and PDRgpA formed tight complexes with arginine-specific gingipains (Ki in the range from 6.2nM to 0.85nM). In contrast, PDKgp showed no inhibitory activity. A conserved Arg-102 residue in PDRgpB and PDRgpA was recognized as the P1 residue. Mutation of Arg-102 to Lys reduced inhibitory potency of PDRgpB by one order of magnitude while its substitutions with Ala, Gln or Gly totally abolished the PD inhibitory activity. Covalent modification of the catalytic cysteine with tosyl-l-Lys-chloromethylketone (TLCK) or H-D-Phe-Arg-chloromethylketone did not affect formation of the stable complex. CONCLUSION: Latency of arginine-specific progingipains is efficiently exerted by N-terminal prodomains thus protecting the periplasm from potentially damaging effect of prematurely activated gingipains. GENERAL SIGNIFICANCE: Blocking progingipain activation may offer an attractive strategy to attenuate P. gingivalis pathogenicity.

Artificial intelligence in cardiac computed tomography.

Artificial Intelligence (AI) is a broad discipline of computer science and engineering. Modern application of AI encompasses intelligent models and algorithms for automated data analysis and processing, data generation, and prediction with applications in visual perception, speech understanding, and language translation. AI in healthcare uses machine learning (ML) and other predictive analytical techniques to help sort through vast amounts of data and generate outputs that aid in diagnosis, clinical decision support, workflow automation, and prognostication. Coronary computed tomography angiography (CCTA) is an ideal union for these applications due to vast amounts of data generation and analysis during cardiac segmentation, coronary calcium scoring, plaque quantification, adipose tissue quantification, peri-operative planning, fractional flow reserve quantification, and cardiac event prediction. In the past 5 years, there has been an exponential increase in the number of studies exploring the use of AI for cardiac computed tomography (CT) image acquisition, de-noising, analysis, and prognosis. Beyond image processing, AI has also been applied to improve the imaging workflow in areas such as patient scheduling, urgent result notification, report generation, and report communication. In this review, we discuss algorithms applicable to AI and radiomic analysis; we then present a summary of current and emerging clinical applications of AI in cardiac CT. We conclude with AI's advantages and limitations in this new field.

Disruption of gingipain oligomerization into non-covalent cell-surface attached complexes.

RgpA and Kgp gingipains are non-covalent complexes of endoprotease catalytic and hemagglutinin-adhesin domains on the surface of Porphyromonas gingivalis. A motif conserved in each domain has been suggested to function as an oligomerization motif. We tested this hypothesis by mutating motif residues to hexahistidine or insertion of hexahistidine tag to disrupt the motif within the Kgp catalytic domain. All modifications led to the secretion of entire Kgp activity into the growth media, predominantly in a form without functional His-tag. This confirmed the role of the conserved motif in correct posttranslational proteolytic processing and assembly of the multidomain complexes.

Mapping of the Neisseria meningitidis NadA cell-binding site: relevance of predicted {alpha}-helices in the NH2-terminal and dimeric coiled-coil regions.

NadA is a trimeric autotransporter protein of Neisseria meningitidis belonging to the group of oligomeric coiled-coil adhesins. It is implicated in the colonization of the human upper respiratory tract by hypervirulent serogroup B N. meningitidis strains and is part of a multiantigen anti-serogroup B vaccine. Structure prediction indicates that NadA is made by a COOH-terminal membrane anchor (also necessary for autotranslocation to the bacterial surface), an intermediate elongated coiled-coil-rich stalk, and an NH(2)-terminal region involved in cell interaction. Electron microscopy analysis and structure prediction suggest that the apical region of NadA forms a compact and globular domain. Deletion studies proved that the NH(2)-terminal sequence (residues 24 to 87) is necessary for cell adhesion. In this study, to better define the NadA cell binding site, we exploited (i) a panel of NadA mutants lacking sequences along the coiled-coil stalk and (ii) several oligoclonal rabbit antibodies, and their relative Fab fragments, directed to linear epitopes distributed along the NadA ectodomain. We identified two critical regions for the NadA-cell receptor interaction with Chang cells: the NH(2) globular head domain and the NH(2) dimeric intrachain coiled-coil α-helices stemming from the stalk. This raises the importance of different modules within the predicted NadA structure. The identification of linear epitopes involved in receptor binding that are able to induce interfering antibodies reinforces the importance of NadA as a vaccine antigen.

Human monocytes/macrophages are a target of Neisseria meningitidis Adhesin A (NadA).

Specific surface proteins of Neisseria meningitidis have been proposed to stimulate leukocytes during tissue invasion and septic shock. In this study, we demonstrate that the adhesin N. meningitidis Adhesin A (NadA) involved in the colonization of the respiratory epithelium by hypervirulent N. meningitidis B strains also binds to and activates human monocytes/macrophages. Expression of NadA on the surface on Escherichia coli does not increase bacterial-monocyte association, but a NadA-positive strain induced a significantly higher amount of TNF-alpha and IL-8 compared with the parental NadA-negative strain, suggesting that NadA has an intrinsic stimulatory action on these cells. Consistently, highly pure, soluble NadA(Delta351-405), a proposed component of an antimeningococcal vaccine, efficiently stimulates monocytes/macrophages to secrete a selected pattern of cytokines and chemotactic factors characterized by high levels of IL-8, IL-6, MCP-1, and MIP-1alpha and low levels of the main vasoactive mediators TNF-alpha and IL-1. NadA(Delta351-405) also inhibited monocyte apoptosis and determined its differentiation into a macrophage-like phenotype.

Peptide and non-peptide mimetics as potential therapeutics targeting oral bacteria and oral biofilms.

The development of the oral biofilm requires a complex series of interactions between host tissues and the colonizing bacteria as well as numerous interspecies interactions between the organisms themselves. Disruption of normal host-microbe homoeostasis in the oral cavity can lead to a dysbiotic microbial community that contributes to caries or periodontal disease. A variety of approaches have been pursued to develop novel potential therapeutics that are active against the oral biofilm and/or target specific oral bacteria. The structure and function of naturally occurring antimicrobial peptides from oral tissues and secretions as well as external sources such as frog skin secretions have been exploited to develop numerous peptide mimetics and small molecule peptidomimetics that show improved antimicrobial activity, increased stability and other desirable characteristics relative to the parent peptides. In addition, a rational and minimalist approach has been developed to design small artificial peptides with amphipathic α-helical properties that exhibit potent antibacterial activity. Furthermore, with an increased understanding of the molecular mechanisms of beneficial and/or antagonistic interspecies interactions that contribute to the formation of the oral biofilm, new potential targets for therapeutic intervention have been identified and both peptide-based and small molecule mimetics have been developed that target these key components. Many of these mimetics have shown promising results in in vitro and pre-clinical testing and the initial clinical evaluation of several novel compounds has demonstrated their utility in humans.

Proteolytic processing and activation of gingipain zymogens secreted by T9SS of Porphyromonas gingivalis.

Porphyromonas gingivalis uses a type IX secretion system (T9SS) to deliver more than 30 proteins to the bacterial surface using a conserved C-terminal domain (CTD) as an outer membrane translocation signal. On the surface, the CTD is cleaved and an anionic lipopolysaccharide (A-PLS) is attached by PorU sortase. Among T9SS cargo proteins are cysteine proteases, gingipains, which are secreted as inactive zymogens requiring removal of an inhibiting N-terminal prodomain (PD) for activation. Here, we have shown that the gingipain proRgpB isolated from the periplasm of a T9SS-deficient P. gingivalis strain was stable and did not undergo autocatalytic activation. Addition of purified, active RgpA or RgpB, but not Lys-specific Kgp, efficiently cleaved the PD of proRgpB but catalytic activity remained inhibited because of inhibition of the catalytic domain in trans by the PD. In contrast, active RgpB was generated from the zymogen, although at a slow rate, by gingipain-null P. gingivalis lysate or intact bacterial cell suspension. This activation was dependent on the presence of the PorU sortase. Interestingly, maturation of proRgpB with the catalytic cysteine residues mutated to Ala expressed in the ΔRgpA mutant strain was indistinguishable from that in the parental strain. Cumulatively, this suggests that PorU not only has sortase activity but is also engaged in activation of gingipain zymogens on the bacterial cell surface.

Roles of the host oxidative immune response and bacterial antioxidant rubrerythrin during Porphyromonas gingivalis infection.

The efficient clearance of microbes by neutrophils requires the concerted action of reactive oxygen species and microbicidal components within leukocyte secretory granules. Rubrerythrin (Rbr) is a nonheme iron protein that protects many air-sensitive bacteria against oxidative stress. Using oxidative burst-knockout (NADPH oxidase-null) mice and an rbr gene knockout bacterial strain, we investigated the interplay between the phagocytic oxidative burst of the host and the oxidative stress response of the anaerobic periodontal pathogen Porphyromonas gingivalis. Rbr ensured the proliferation of P. gingivalis in mice that possessed a fully functional oxidative burst response, but not in NADPH oxidase-null mice. Furthermore, the in vivo protection afforded by Rbr was not associated with the oxidative burst responses of isolated neutrophils in vitro. Although the phagocyte-derived oxidative burst response was largely ineffective against P. gingivalis infection, the corresponding oxidative response to the Rbr-positive microbe contributed to host-induced pathology via potent mobilization and systemic activation of neutrophils. It appeared that Rbr also provided protection against reactive nitrogen species, thereby ensuring the survival of P. gingivalis in the infected host. The presence of the rbr gene in P. gingivalis also led to greater oral bone loss upon infection. Collectively, these results indicate that the host oxidative burst paradoxically enhances the survival of P. gingivalis by exacerbating local and systemic inflammation, thereby contributing to the morbidity and mortality associated with infection.

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.

Altered von Willebrand factor subunit proteolysis and multimer processing associated with the Cys2362Phe mutation in the B2 domain.

The normal von Willebrand factor (vWF) multimer pattern results from the ADAMTS-13 cleavage of the Tyr 1605-Met 1606 bond in the A2 domain of vWF. We identified a patient with severe von Willebrand disease (vWD) homozygously carrying a Cys to Phe mutation in position 2362 of vWF with markedly altered vWF multimers and an abnormal proteolytic pattern. The proband's phenotype was characterized by a marked drop in plasma vWF antigen and ristocetin cofactor activity, and a less pronounced decrease in FVIII. The vWF multimers lacked any triplet structure, replaced by single bands with an atypical mobility, surrounded by a smear, and abnormally large vWF multimers. Analysis of the plasma vWF subunit's composition revealed the 225 kDa mature form and a single 205 kDa fragment, but not the 176 kDa and 140 kDa fragments resulting from cleavage by ADAMTS-13. The 205 kDa fragment was distinctly visible, along with the normal vWF cleavage products, in the patient's parents who were heterozygous for the Cys2362Phe mutation. Their vWF levels were mildly decreased and vWF multimers were organized in triplets, but also demonstrated abnormally large forms and smearing. Our findings indicate that a proper conformation of the B2 domain, which depends on critical Cys residues, may be required for the normal proteolytic processing of vWF multimers.