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

Interkingdom interactions on the denture surface: implications for oral hygiene

Background: Evidence to support the role of Candida species in oral disease is limited. Often considered a commensal, this opportunistic yeast has been shown to play a role in denture related disease, though whether it is an active participant or innocent bystander remains to be determined. This study sought to understand the role of Candida species alongside the bacterial microbiome in a denture patient cohort, exploring how the microbiology of the denture was affected by oral hygiene practices. Materials and methods: In vitro denture cleansing studies were performed on a complex 9-species interkingdom denture biofilm model, with quantitative assessment of retained bacterial and fungal viable bioburdens. Patient hygiene measures were also collected from 131 patients, including OHIP, frequency of denture cleansing, oral hygiene measure and patient demographics. The bacterial microbiome was analysed from each patient, alongside quantitative PCR assessment of ITS (fungal) and 16S (bacterial) bioburden from denture, mucosa and intact dentition. Results: It was shown that following in vitro denture cleansing C. albicans were unresponsive to treatment, whereas bacterial biofilms could repopulate 100-fold, but were susceptible to subsequent treatment. Within the patient cohort, oral hygiene did not impact candidal or bacterial composition, nor diversity. The levels of Candida did not significantly influence the bacterial microbiome, though an observed gradient was suggestive of a microbial composition change in response to Candida load, indicating interkingdom interaction rather than an oral hygiene effect. Indeed, correlation analysis was able to show significant correlations between Candida species and key genera (Lactobacillus, Scardovia, Fusobacterium). Conclusions: Overall, this study has shown that the denture microbiome/mycobiome is relatively resilient to oral hygiene challenges, but that Candida species have potential interactions with key oral genera. These interactions may have a bearing on shaping community structure and a shift from health to disease when the opportunity arises

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 (EMT). 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 micro RNAs. 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

Zebrafish as a new model to study effects of periodontal pathogens on cardiovascular diseases.

Porphyromonas gingivalis (Pg) is a keystone pathogen in the aetiology of chronic periodontitis. However, recent evidence suggests that the bacterium is also able to enter the bloodstream, interact with host cells and tissues, and ultimately contribute to the pathogenesis of cardiovascular disease (CVD). Here we established a novel zebrafish larvae systemic infection model showing that Pg rapidly adheres to and penetrates the zebrafish vascular endothelium causing a dose- and time-dependent mortality with associated development of pericardial oedemas and cardiac damage. The in vivo model was then used to probe the role of Pg expressed gingipain proteases using systemically delivered gingipain-deficient Pg mutants, which displayed significantly reduced zebrafish morbidity and mortality compared to wild-type bacteria. In addition, we used the zebrafish model to show efficacy of a gingipain inhibitor (KYT) on Pg-mediated systemic disease, suggesting its potential use therapeutically. Our data reveal the first real-time in vivo evidence of intracellular Pg within the endothelium of an infection model and establishes that gingipains are crucially linked to systemic disease and potentially contribute to CVD

A Novel Enzymatic System against Oxidative Stress in the Thermophilic Hydrogen-Oxidizing Bacterium Hydrogenobacter thermophilus

Rubrerythrin (Rbr) is a non-heme iron protein composed of two distinctive domains and functions as a peroxidase in anaerobic organisms. A novel Rbr-like protein, ferriperoxin (Fpx), was identified in Hydrogenobacter thermophilus and was found not to possess the rubredoxin-like domain that is present in typical Rbrs. Although this protein is widely distributed among aerobic organisms, its function remains unknown. In this study, Fpx exhibited ferredoxin:NADPH oxidoreductase (FNR)-dependent peroxidase activity and reduced both hydrogen peroxide (H2O2) and organic hydroperoxide in the presence of NADPH and FNR as electron donors. The calculated Km and Vmax values of Fpx for organic hydroperoxides were comparable to that for H2O2, demonstrating a multiple reactivity of Fpx towards hydroperoxides. An fpx gene disruptant was unable to grow under aerobic conditions, whereas its growth profiles were comparable to those of the wild-type strain under anaerobic and microaerobic conditions, clearly indicating the indispensability of Fpx as an antioxidant of H. thermophilus in aerobic environments. Structural analysis suggested that domain-swapping occurs in Fpx, and this domain-swapped structure is well conserved among thermophiles, implying the importance of structural stability of domain-swapped conformation for thermal environments. In addition, Fpx was located on a deep branch of the phylogenetic tree of Rbr and Rbr-like proteins. This finding, taken together with the wide distribution of Fpx among Bacteria and Archaea, suggests that Fpx is an ancestral type of Rbr homolog that functions as an essential antioxidant and may be part of an ancestral peroxide-detoxification system

Acquisition of Complement Inhibitor Serine Protease Factor I and Its Cofactors C4b-Binding Protein and Factor H by Prevotella intermedia

Infection with the Gram-negative pathogen Prevotella intermedia gives rise to periodontitis and a growing number of studies implies an association of P. intermedia with rheumatoid arthritis. The serine protease Factor I (FI) is the central inhibitor of complement degrading complement components C3b and C4b in the presence of cofactors such as C4b-binding protein (C4BP) and Factor H (FH). Yet, the significance of complement inhibitor acquisition in P. intermedia infection and FI binding by Gram-negative pathogens has not been addressed. Here we show that P. intermedia isolates bound purified FI as well as FI directly from heat-inactivated human serum. FI bound to bacteria retained its serine protease activity as shown in degradation experiments with 125I-labeled C4b. Since FI requires cofactors for its activity we also investigated the binding of purified cofactors C4BP and FH and found acquisition of both proteins, which retained their activity in FI mediated degradation of C3b and C4b. We propose that FI binding by P. intermedia represents a new mechanism contributing to complement evasion by a Gram-negative bacterial pathogen associated with chronic diseases

Structural and functional probing of PorZ, an essential bacterial surface component of the type-IX secretion system of human oral-microbiomic Porphyromonas gingivalis.

Porphyromonas gingivalis is a member of the human oral microbiome abundant in dysbiosis and implicated in the pathogenesis of periodontal (gum) disease. It employs a newly described type-IX secretion system (T9SS) for secretion of virulence factors. Cargo proteins destined for secretion through T9SS carry a recognition signal in the conserved C-terminal domain (CTD), which is removed by sortase PorU during translocation. Here, we identified a novel component of T9SS, PorZ, which is essential for surface exposure of PorU and posttranslational modification of T9SS cargo proteins. These include maturation of enzyme precursors, CTD removal and attachment of anionic lipopolysaccharide for anchorage in the outer membrane. The crystal structure of PorZ revealed two β-propeller domains and a C-terminal β-sandwich domain, which conforms to the canonical CTD architecture. We further documented that PorZ is itself transported to the cell surface via T9SS as a full-length protein with its CTD intact, independently of the presence or activity of PorU. Taken together, our results shed light on the architecture and possible function of a novel component of the T9SS. Knowledge of how T9SS operates will contribute to our understanding of protein secretion as part of host-microbiome interactions by dysbiotic members of the human oral cavity

Community Development between <i>Porphyromonas gingivalis </i>and <i>Candida albicans </i>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 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

Multiple reading of "A Man asleep" by Georges Perec

Moja praca licencjacka stanowi eksperyment wielokrotnej lektury przy użyciu różnych teorii literackich. Jej celem jest zaobserwowanie w jaki sposób używanie różnych metod wpływa na postrzeganie dzieła. W tym celu wybrałam cztery teorie, z których każda pozwala na zbadanie innych elementów dzieła. W pierwszym rozdziale wykorzystam narzędzia formalizmu rosyjskiego, aby zanalizować formę powieści. Szukam jednego elementu, który najintensywniej wpływa na zautomatyzowaną percepcję czytelnika. W mojej analizie, elementem tym jest narracja, która swoją szczególność zawdzięcza używaniu drugiej osoby liczy pojedynczej. W drugiej części tego rozdziału, posługuję się dekonstrukcjonizmem w celu zbadania relacji formy do treści. Aby ją przeanalizować szukam sprzeczności między nimi. Po raz kolejny tym co przyciąga uwagę jest narracja, której oryginalna funkcja i efekt końcowy uzyskany przez autora, są sprzeczne. W drugim rozdziale, korzystam z narzędzi krytyki tematycznej w celu zbadania relacji pomiędzy czytelnikiem i narratorem, ponieważ dzięki temu, można odkryć co jest tematem powieści. W przypadku Człowieka, który śpi, tematem jest depresja zaprezentowana, jako sen. Słowem kluczowym dla ostatniego rozdziału, jest właśnie sen. W trzecim rozdziale posłużę się psychoanalizą, aby zinterpretować powieść jako sen. Szukam symboli i wskazówek, po to aby dotrzeć do ukrytego sensu dzieła. Psychoanaliza pozwoli na zweryfikowanie, czy ta pozornie prosta historia jest tą, którą się wydaje. To doświadczenie wielokrotnej lektury udowadnia, że dzięki różnych teorią literackim, analiza staje się bardziej kompletna. Różne metody pozwalają na odkrycie tego, co stoi za formą i treścią. Ta analiza pokazała, że pojedyncza lektura oraz jedna teoria nie są wystarczające i że zawsze należy analizować dzieło na różnych poziomach. Dzięki tej wielokrotnej lekturze, możemy zauważyć, jak narrator gra z czytelnikiem, zostawiając mu wskazówki nie możliwie do odczytania podczas pojedynczej lektury.My bachelor thesis is an experiment of multiply reading during which I chose various literary theories. The aim is to watch how different methods affect perception of the book. I've chosen four literary theories and each of them allows me to examine different detail. In the first chapter, I'm using the tools of the Russian formalism to analyze the form of the novel. I look for the element that destroy automatism perceptual. In my analysis this element is narration, which is quite special, because of using the second person of the singular. In the second part of this chapter, I'm focused on deconstructionism so that I can observe the relation between form and the content of the book. I'm doing it searching for contradictions. In this case, once again, narration is the thing that grab the attention, because its original function and the final effect created by the author don't match to each other. In the second chapter, I'm using the tools of the thematic criticism to examine the relation between the reader and the narrator. This study allows me to find what is the theme of this novel. In A Man asleep this theme is depression which is presented as dream. The key word for my last chapter is the dream. In the third chapter I'm using psychanalyse to interpret the novel as a dream. I'm looking for the symbols and the leads to find the hidden meaning. Psychanalyse allows me to find out if this simple story is really the story that it seems to be. This experiment of multiply reading prouve that, because of using various literary theories, the analysis becomes more complete. They allow you to discover what is hidden behind the form and the content of the book. This analysis have shown that one theory and one reading isn't enough and we should always analyze the novel on the different levels. We can also notice that the narrator plays with the reader leaving hi the leads which are impossible to notice during just one lecture.Ma mémoire de licence constitue une expérience de la lecture plurielle, pendant laquelle j'utilise de différentes théories littéraires. Le but et d'observer comment différentes méthodes influencent la perception de l'œuvre. J'ai choisi quatre théories dont chacune permet d'examiner des détails différents. Dans le premier chapitre, j'utilise des outil proposés par formalisme russe pour analyser la forme du roman. Je cherche un élément qui détruit le plus l'automatisme perceptif. Dans mon analyse cet élément constitue la narration, qui est assez particulier grâce à​ l'utilisation de la deuxième personne du singulier. Dans la deuxième parti de ce chapitre, je me concentre sur déconstructionnisme pour observer la relation entre la forme et le contenu. Je le fait en cherchant des discordances. Dans ce cas, encore une fois, ce qui attire l'attention c'est la narration dont la fonction originelle et l'effet final obtenu par l'auteur ne se correspondent pas. Dans le deuxième chapitre j'utilise des outils de la critique thématique pour examiner la relation entre le narrateur et le lecteur. Grâce à​ cet étude, on peut trouver ce qui constitue le thème. Dans Un homme qui dort ce thème constitue la dépression qui est présentée comme un sommeil. Le mot clé pour le dernier chapitre est le rêve. Dans le troisième chapitre j'utilise l'approche psychanalytique pour interpréter un roman en tant qu'un rêve. Je cherche des symboles et des indications pour trouver le sens caché. Psychanalyse me permet de vérifier si cette apparemment simple histoire constitue vraiment l'histoire qu'elle semble. Cet expérience de la lecture plurielle prouve que grâce à​ l'utilisation de différentes théories, analyse devient plus complète. Elles permettent de dévoiler ce qui se cache devant la forme et le contenu. Cette analyse a montré qu'une lecture et une théorie ne sont pas suffisantes et qu'il faut toujours analyser différentes niveaux de l'œuvre. On peut apercevoir comment le narrateur jeu avec le lecteur, comment il le laisse des indications qui sont possible à ​apercevoir seulement pendant plusieurs lectures