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Prediction of Selected Biosynthetic Pathways for the Lipopolysaccharide Components in Porphyromonas gingivalis.

ABSTRACT: Porphyromonas gingivalis is an oral human pathogen. The bacterium destroys dental tissue and is a serious health problem worldwide. Experimental data and bioinformatic analysis revealed that the pathogen produces three types of lipopolysaccharides (LPS): normal (O-type), anionic (A-type), and capsular (K-type). The enzymes involved in the production of all three types of lipopolysaccharide have been largely identified for the first two and partially for the third type. In the current work, we use bioinformatics tools to predict biosynthetic pathways for the production of the normal (O-type) lipopolysaccharide in the W50 strain Porphyromonas gingivalis and compare the pathway with other putative pathways in fully sequenced and completed genomes of other pathogenic strains. Selected enzymes from the pathway have been modeled and putative structures are presented. The pathway for the A-type antigen could not be predicted at this time due to two mutually exclusive structures proposed in the literature. The pathway for K-type antigen biosynthesis could not be predicted either due to the lack of structural data for the antigen. However, pathways for the synthesis of lipid A, its core components, and the O-type antigen ligase reaction have been proposed based on a combination of experimental data and bioinformatic analyses. The predicted pathways are compared with known pathways in other systems and discussed. It is the first report in the literature showing, in detail, predicted pathways for the synthesis of selected LPS components for the model W50 strain of P. gingivalis.

SUBMITTER: Swietnicki W

PROVIDER: S-EPMC8003790 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:Porphyromonas gingivalis, a bacterial pathogen contributing to human periodontitis, exports and anchors cargo proteins to its surface, enabling the production of black pigmentation using a type IX secretion system (T9SS) and conjugation to anionic lipopolysaccharide (A-LPS). To determine whether T9SS components need to be assembled in situ for correct secretion and A-LPS modification of cargo proteins, combinations of nonpigmented mutants lacking A-LPS or a T9SS component were mixed to investigate in trans complementation. Reacquisition of pigmentation occurred only between an A-LPS mutant and a T9SS mutant, which coincided with A-LPS modification of cargo proteins detected by Western blotting and coimmunoprecipitation/quantitative mass spectrometry. Complementation also occurred using an A-LPS mutant mixed with outer membrane vesicles (OMVs) or purified A-LPS. Fluorescence experiments demonstrated that OMVs can fuse with and transfer lipid to P. gingivalis, leading to the conclusion that complementation of T9SS function occurred through A-LPS transfer between cells. None of the two-strain crosses involving only the five T9SS OM component mutants produced black pigmentation, implying that the OM proteins cannot be transferred in a manner that restores function and surface pigmentation, and hence, a more ordered temporal in situ assembly of T9SS components may be required. Our results show that LPS can be transferred between cells or between cells and OMVs to complement deficiencies in LPS biosynthesis and hemin-related pigmentation to reveal a potentially new mechanism by which the oral microbial community is modulated to produce clinical consequences in the human host.IMPORTANCEPorphyromonas gingivalis is a keystone pathogen contributing to periodontitis in humans, leading to tooth loss. The oral microbiota is essential in this pathogenic process and changes from predominantly Gram-positive (health) to predominantly Gram-negative (disease) species. P. gingivalis uses its type IX secretion system (T9SS) to secrete and conjugate virulence proteins to anionic lipopolysaccharide (A-LPS). This study investigated whether components of this secretion system could be complemented and found that it was possible for A-LPS biosynthetic mutants to be complemented in trans both by strains that had the A-LPS on the cell surface and by exogenous sources of A-LPS. This is the first known example of LPS exchange in a human bacterial pathogen which causes disease through complex microbiota-host interactions.

Project description:ObjectiveEphrinA2-EphA2 and ephrinB2-EphB4 critically engage in bidirectional signalling to modulate alveolar bone remodelling. The present study aimed to investigate the effects of lipopolysaccharides (LPS) derived from Porphyromonas gingivalis on ephrin/Eph signalling in periodontal ligament fibroblasts (PDLFs).Material and methodsThe primary cultured PDLFs were incubated in the absence (as a control) or presence of P. gingivalisLPS at 0.001-10 μg/mL for 24 hours. The PDLFs were then stimulated with P. gingivalisLPS at the optimal concentration (0.1 μg/mL) for different periods (6-48 hours). The expression of ephrinA2, ephrinB2, EphA2 and EphB4 was assessed by quantitative reverse-transcription real-time polymerase chain reaction and western blotting. The osteoblastic markers alkaline phosphatase, osteocalcin and Runt-related transcription factor 2 (Runx2), and the osteoclastogenesis-related factors receptor activator of nuclear factor kappa-B ligand (RANKL) and osteoprotegerin were also evaluated.ResultsThe ephrinA2 and EphA2 expression was upregulated and EphB4 expression was downregulated by stimulation of P. gingivalisLPS. EphrinA2 mRNA expression in the PDLFs was significantly upregulated from 12 to 48 hours (P<.05), whereas EphA2 exhibited no change for the first 24 hours, after which there was a significant increase at 48 hours (P<.05). EphB4 exhibited lower mRNA expression at 12 and 24 hours than did the control (P<.05), but the change was insignificant at 48 hours. In contrast, the expression of ephrinB2 remained unchanged. The expressions of ephrinA2, EphA2, ephrinB2 and EphB4 at the protein level showed a similar pattern to that at the mRNA level. The expression of Runx2 and osteocalcin significantly decreased, whereas that of RANKL/osteoprotegerin increased.ConclusionThe present study suggest that P. gingivalisLPS would contribute to a dysregulation of bone remodelling, whereby ephrinA2/EphA2 expression is stimulated and EphB4 expression is inhibited.

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Prediction of Selected Biosynthetic Pathways for the Lipopolysaccharide Components in Porphyromonas gingivalis.

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