Structural and functional insights into oligopeptide acquisition by the RagAB transporter from Porphyromonas gingivalis.
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ABSTRACT:
SUBMITTER: Madej M
PROVIDER: S-EPMC7610489 | biostudies-literature |
REPOSITORIES: biostudies-literature
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Project description:Porphyromonas gingivalis is a keystone pathogen of periodontitis. Outer membrane vesicles (OMVs) have been considered as both offense and defense components of this bacterium. Previous studies indicated that like their originating cells, P. gingivalis vesicles, are able to invade oral epithelial cells and gingival fibroblasts, in order to promote aggregation of some specific oral bacteria and to induce host immune responses. In the present study, we investigated the invasive efficiency of P. gingivalis OMVs and compared results with that of the originating cells. Results revealed that 70-90% of human primary oral epithelial cells, gingival fibroblasts, and human umbilical vein endothelial cells carried vesicles from P. gingivalis 33277 after being exposed to the vesicles for 1 h, while 20-50% of the host cells had internalized P. gingivalis cells. We also detected vesicle-associated DNA and RNA and a vesicle-mediated horizontal gene transfer in P. gingivalis strains, which represents a novel mechanism for gene transfer between P. gingivalis strains. Moreover, purified vesicles of P. gingivalis appear to have a negative impact on biofilm formation and the maintenance of Streptococcus gordonii. Our results suggest that vesicles are likely the best offence weapon of P. gingivalis for bacterial survival in the oral cavity and for induction of periodontitis.
| S-EPMC4405273 | biostudies-literature
Project description:Porphyromonas gingivalis, the major human pathogen bacterium associated with periodontal diseases, secretes virulence factors through the Bacteroidetes-specific type IX secretion system (T9SS). Effector proteins of the T9SS are recognized by the complex via their conserved C-terminal domains (CTDs). Among the 18 proteins essential for T9SS function in P. gingivalis, PorN is a periplasmic protein that forms large ring-shaped structures in association with the PorK outer membrane lipoprotein. PorN also mediates contacts with the PorM subunit of the PorLM energetic module, and with the effector's CTD. However, no information is available on the PorN structure and on the implication of PorN domains for T9SS assembly and effector recognition. Here we present the crystal structure of PorN at 2.0-Å resolution, which represents a novel fold with no significant similarity to any known structure. In agreement with in silico analyses, we also found that the N- and C-terminal regions of PorN are intrinsically disordered. Our functional studies showed that the N-terminal disordered region is involved in PorN dimerization while the C-terminal disordered region is involved in the interaction with PorK. Finally, we determined that the folded PorN central domain is involved in the interaction with PorM, as well as with the effector's CTD. Altogether, these results lay the foundations for a more comprehensive model of T9SS architecture and effector transport.
| S-EPMC8861641 | biostudies-literature
Project description:The type IX secretion system (T9SS) has been recently discovered and is specific to Bacteroidetes species. Porphyromonas gingivalis, a keystone pathogen for periodontitis, utilizes the T9SS to transport many proteins including the gingipain virulence factors across the outer membrane and attach them to the cell surface via a sortase-like mechanism. At least 11 proteins have been identified as components of the T9SS including PorK, PorL, PorM, PorN and PorP, however the precise roles of most of these proteins have not been elucidated and the structural organization of these components is unknown. In this study, we purified PorK and PorN complexes from P. gingivalis and using electron microscopy we have shown that PorN and the PorK lipoprotein interact to form a 50 nm diameter ring-shaped structure containing approximately 32-36 subunits of each protein. The formation of these rings was dependent on both PorK and PorN, but was independent of PorL, PorM and PorP. PorL and PorM were found to form a separate stable complex. PorK and PorN were protected from proteinase K cleavage when present in undisrupted cells, but were rapidly degraded when the cells were lysed, which together with bioinformatic analyses suggests that these proteins are exposed in the periplasm and anchored to the outer membrane via the PorK lipid. Chemical cross-linking and mass spectrometry analyses confirmed the interaction between PorK and PorN and further revealed that they interact with the PG0189 outer membrane protein. Furthermore, we established that PorN was required for the stable expression of PorK, PorL and PorM. Collectively, these results suggest that the ring-shaped PorK/N complex may form part of the secretion channel of the T9SS. This is the first report showing the structural organization of any T9SS component.
| S-EPMC4980022 | biostudies-literature
Project description:Ulcerative Colitis (UC) has been reported to be related to Porphyromonas gingivalis (P. gingivalis). Porphyromonas gingivalis peptidylarginine deiminase (PPAD), a virulence factor released by P. gingivalis, is known to induce inflammatory responses. To explore the pathological relationships between PPAD and UC, we used homologous recombination technology to construct a P. gingivalis strain in which the PPAD gene was deleted (Δppad) and a Δppad strain in which the PPAD gene was restored (comΔppad). C57BL/6 mice were orally gavaged with saline, P. gingivalis, Δppad, or comΔppad twice a week for the entire 40 days (days 0-40), and then, UC was induced by dextran sodium sulfate (DSS) solution for 10 days (days 31-40). P. gingivalis and comΔppad exacerbated DDS-induced colitis, which was determined by assessing the parameters of colon length, disease activity index, and histological activity index, but Δppad failed to exacerbate DDS-induced colitis. Flow cytometry and ELISA revealed that compared with Δppad, P. gingivalis, and comΔppad increased T helper 17 (Th17) cell numbers and interleukin (IL)-17 production but decreased regulatory T cells (Tregs) numbers and IL-10 production in the spleens of mice with UC. We also cocultured P. gingivalis, Δppad, or comΔppad with T lymphocytes in vitro and found that P. gingivalis and comΔppad significantly increased Th17 cell numbers and decreased Treg cell numbers. Immunofluorescence staining of colon tissue paraffin sections also confirmed these results. The results suggested that P. gingivalis exacerbated the severity of UC in part via PPAD.
| S-EPMC8484350 | biostudies-literature
Project description:Human oral and gut microbiomes are crucial for maintenance of homeostasis in the human body. Porphyromonas gingivalis, the key etiologic agent of chronic periodontitis, can cause dysbiosis in the mouth and gut, which results in local and systemic infectious inflammatory diseases. Our previous work resulted in extensive biochemical and functional characterization of one of the major P. gingivalis heme acquisition systems (Hmu), with the leading role played by the HmuY hemophore-like protein. We continued our studies on the homologous heme acquisition protein (Bvu) expressed by Bacteroides vulgatus, the dominant species of the gut microbiome. Results from spectrophotometric experiments showed that Bvu binds heme preferentially under reducing conditions using Met145 and Met172 as heme iron-coordinating ligands. Bvu captures heme bound to human serum albumin and only under reducing conditions. Importantly, HmuY is able to sequester heme complexed to Bvu. This is the first study demonstrating that B. vulgatus expresses a heme-binding hemophore-like protein, thus increasing the number of members of a novel HmuY-like family. Data gained in this study confirm the importance of HmuY in the context of P. gingivalis survival in regard to its ability to cause dysbiosis also in the gut microbiome.
| S-EPMC7956564 | biostudies-literature
Project description:UnlabelledThe CRISPR-Cas (clustered regularly interspaced short palindromic repeats/CRISPR-associated genes) system provides prokaryotic cells with an adaptive and heritable immune response to foreign genetic elements, such as viruses, plasmids, and transposons. It is present in the majority of Archaea and almost half of species of Bacteria. Porphyromonas gingivalis is an important human pathogen that has been proven to be an etiological agent of periodontitis and has been linked to systemic conditions, such as rheumatoid arthritis and cardiovascular disease. At least 95% of clinical strains of P. gingivalis carry CRISPR arrays, suggesting that these arrays play an important function in vivo. Here we show that all four CRISPR arrays present in the P. gingivalis W83 genome are transcribed. For one of the arrays, we demonstrate in vivo activity against double-stranded DNA constructs containing protospacer sequences accompanied at the 3' end by an NGG protospacer-adjacent motif (PAM). Most of the 44 spacers present in the genome of P. gingivalis W83 share no significant similarity with any known sequences, although 4 spacers are similar to sequences from bacteria found in the oral cavity and the gastrointestinal tract. Four spacers match genomic sequences of the host; however, none of these is flanked at its 3' terminus by the appropriate PAM element.ImportanceThe CRISPR-Cas (clustered regularly interspaced short palindromic repeats/CRISPR-associated genes) system is a unique system that provides prokaryotic cells with an adaptive and heritable immunity. In this report, we show that the CRISPR-Cas system of P. gingivalis, an important human pathogen associated with periodontitis and possibly also other conditions, such as rheumatoid arthritis and cardiovascular disease, is active and provides protection from foreign genetic elements. Importantly, the data presented here may be useful for better understanding the communication between cells in larger bacterial communities and, consequently, the process of disease development and progression.
| S-EPMC4507336 | biostudies-literature
Project description:Enoyl-acyl carrier protein (ACP) reductase II (FabK) is a critical rate-limiting enzyme in the bacterial type II fatty-acid synthesis (FAS II) pathway. FAS II pathway enzymes are markedly disparate from their mammalian analogs in the FAS I pathway in both structure and mechanism. Enzymes involved in bacterial fatty-acid synthesis represent viable drug targets for Gram-negative pathogens, and historical precedent exists for targeting them in the treatment of diseases of the oral cavity. The Gram-negative organism Porphyromonas gingivalis represents a key causative agent of the costly and highly prevalent disease known as chronic periodontitis, and exclusively expresses FabK as its enoyl reductase enzyme in the FAS-II pathway. Together, these characteristics distinguish P. gingivalis FabK (PgFabK) as an attractive and novel narrow-spectrum antibacterial target candidate. PgFabK is a flavoenzyme that is dependent on FMN and NADPH as cofactors for the enzymatic reaction, which reduces the enoyl substrate via a ping-pong mechanism. Here, the structure of the PgFabK enzyme as determined using X-ray crystallography is reported to 1.9 Å resolution with endogenous FMN fully resolved and the NADPH cofactor partially resolved. PgFabK possesses a TIM-barrel motif, and all flexible loops are visible. The determined structure has allowed insight into the structural basis for the NADPH dependence observed in PgFabK and the role of a monovalent cation that has been observed in previous studies to be stringently required for FabK activity. The PgFabK structure and the insights gleaned from its analysis will facilitate structure-based drug-discovery efforts towards the prevention and treatment of P. gingivalis infection.
| S-EPMC5947681 | biostudies-literature
Project description:Marginal periodontitis is not a homogeneous disease but is rather influenced by an intricate set of host susceptibility differences as well as diversities in virulence among the harbored organisms. It is likely that clonal heterogeneity of subpopulations with both high and low levels of pathogenicity exists among organisms harbored by individuals with negligible, slight, or even severe periodontal destruction. Therefore, specific virulent clones of periodontal pathogens may cause advanced and/or aggressive periodontitis. Porphyromonas gingivalis is a predominant periodontal pathogen that expresses a number of potential virulence factors involved in the pathogenesis of periodontitis, and accumulated evidence shows that its expression of heterogenic virulence properties is dependent on clonal diversity. Fimbriae are considered to be critical factors that mediate bacterial interactions with and invasion of host tissues, with P. gingivalis shown to express two distinct fimbria-molecules, long and short fimbriae, on the cell surface, both of which seem to be involved in development of periodontitis. Long fimbriae are classified into six types (I to V and Ib) based on the diversity of fimA genes encoding FimA (a subunit of long fimbriae). Studies of clones with type II fimA have revealed their significantly greater adhesive and invasive capabilities as compared to other fimA type clones. Long and short fimbriae induce various cytokine expressions such as IL-1α, IL-β, IL-6, and TNF-α, which result in alveolar bone resorption. Although the clonal diversity of short fimbriae is unclear, distinct short fimbria-molecules have been found in different strains. These fimbriae variations likely influence the development of periodontal disease.
| S-EPMC3647041 | biostudies-literature
Project description:The sialidases, which catalyze the hydrolysis of sialic acid from extracellular glycoconjugates, are a group of major virulence factors in various pathogenic bacteria. In Porphyromonas gingivalis, which causes human periodontal disease, sialidase contributes to bacterial pathogenesis via promoting the formation of biofilms and capsules, reducing the ability for macrophage clearance, and providing nutrients for bacterial colonization. Here, the crystal structure of the P. gingivalis sialidase SiaPG is reported at 2.1 Å resolution, revealing an N-terminal carbohydrate-binding domain followed by a canonical C-terminal catalytic domain. Simulation of the product sialic acid in the active-site pocket together with functional analysis enables clear identification of the key residues that are required for substrate binding and catalysis. Moreover, structural comparison with other sialidases reveals distinct features of the active-site pocket which might confer substrate specificity. These findings provide the structural basis for the further design and optimization of effective inhibitors to target SiaPG to fight against P. gingivalis-derived oral diseases.
| S-EPMC10071834 | biostudies-literature
Project description:Porphyromonas gingivalis is implicated in the etiology of chronic periodontitis. Genotyping studies suggest that genetic variability exists among P. gingivalis strains; however, the extent of variability remains unclear and regions of variability remain largely unidentified. To assess P. gingivalis strain diversity, we previously used heteroduplex analysis of the ribosomal operon intergenic spacer region (ISR) to type strains in clinical samples and identified 22 heteroduplex types. Additionally, we used ISR sequence analysis to determine the relatedness of P. gingivalis strains to one another and demonstrated a link between ISR sequence phylogeny and the disease-associated phenotype of the strains. In the current study, heteroduplex analysis of the ISR was used to determine the worldwide genetic variability and distribution of P. gingivalis, and microarray-based comparative genomic hybridization (CGH) analysis was used to more comprehensively examine the variability of major heteroduplex type strains by using the entire genome. Heteroduplex analysis of clinical samples from geographically diverse populations identified 6 predominant geographically widespread heteroduplex types (prevalence, > or = 5%) and 14 rare heteroduplex types (prevalence, <2%) which are found in one or a few locations. CGH analysis of the genomes of seven clinically prevalent heteroduplex type strains identified 133 genes from strain W83 that were divergent in at least one of the other strains. The relatedness of the strains to one another determined on the basis of genome content (microarray) analysis was highly similar to their relatedness determined on the basis of ISR sequence analysis, and a striking correlation between the genome contents and disease-associated phenotypes of the strains was observed.
| S-EPMC2756895 | biostudies-literature