LptO (PG0027) is required for lipid A 1-phosphatase activity in Porphyromonas gingivalis W50

ABSTRACT Porphyromonas gingivalis produces outer membrane vesicles (OMVs) rich in virulence factors, including cysteine proteases and A-LPS, one of the two lipopolysaccharides (LPSs) produced by this organism. Previous studies had suggested that A-LPS and PG0027, an outer membrane (OM) protein, may be involved in OMV formation. Their roles in this process were examined by using W50 parent and the Δ PG0027 mutant strains. Inactivation of PG0027 caused a reduction in the yield of OMVs. Lipid A from cells and OMVs of P. gingivalis W50 and the Δ PG0027 mutant strains were analyzed by matrix-assisted laser desorption ionization–time of flight mass spectrometry (MALDI-TOF MS). Lipid A from W50 cells contained bis-P-pentaacyl, mono-P-pentaacyl, mono-P-tetraacyl, non-P-pentaacyl, and non-P-tetraacyl species, whereas lipid A from Δ PG0027 mutant cells contained only phosphorylated species; nonphosphorylated species were absent. MALDI-TOF/TOF tandem MS of mono-P-pentaacyl ( m / z 1,688) and mono-P-tetraacyl ( m / z 1,448) lipid A from Δ PG0027 showed that both contained lipid A 1-phosphate, suggesting that the Δ PG0027 mutant strain lacked lipid A 1-phosphatase activity. The total phosphatase activities in the W50 and the Δ PG0027 mutant strains were similar, whereas the phosphatase activity in the periplasm of the Δ PG0027 mutant was lower than that in W50, supporting a role for PG0027 in lipid A dephosphorylation. W50 OMVs were enriched in A-LPS, and its lipid A did not contain nonphosphorylated species, whereas lipid A from the Δ PG0027 mutant (OMVs and cells) contained similar species. Thus, OMVs in P. gingivalis are apparently formed in regions of the OM enriched in A-LPS devoid of nonphosphorylated lipid A. Conversely, dephosphorylation of lipid A through a PG0027-dependent process is required for optimal formation of OMVs. Hence, the relative proportions of nonphosphorylated and phosphorylated lipid A appear to be crucial for OMV formation in this organism. IMPORTANCE Gram-negative bacteria produce outer membrane vesicles (OMVs) by “blebbing” of the outer membrane (OM). OMVs can be used offensively as delivery systems for virulence factors and defensively to aid in the colonization of a host and in the survival of the bacterium in hostile environments. Earlier studies using the oral anaerobe Porphyromonas gingivalis as a model organism to study the mechanism of OMV formation suggested that the OM protein PG0027 and one of the two lipopolysaccharides (LPSs) synthesized by this organism, namely, A-LPS, played important roles in OMV formation. We suggest a novel mechanism of OMV formation in P. gingivalis involving dephosphorylation of lipid A of A-LPS controlled/regulated by PG0027, which causes destabilization of the OM, resulting in blebbing and generation of OMVs. </jats:p

A 16S rRNA gene and draft genome database for the murine oral bacterial community

A curated murine oral microbiome database to be used as a reference for mouse-based studies has been constructed using a combination of bacterial culture, 16S rRNA gene amplicon, and whole-genome sequencing. The database comprises a collection of nearly full-length 16S rRNA gene sequences from cultured isolates and draft genomes from representative taxa collected from a range of sources, including specific-pathogen-free laboratory mice, wild Mus musculus domesticus mice, and formerly wild wood mouse Apodemus sylvaticus. At present, it comprises 103 mouse oral taxa (MOT) spanning four phyla—Firmicutes, Proteobacteria, Actinobacteria, and Bacteroidetes—including 12 novel undescribed species-level taxa. The key observations from this study are (i) the low diversity and predominantly culturable nature of the laboratory mouse oral microbiome and (ii) the identification of three major murine-specific oral bacterial lineages, namely, Streptococcus danieliae (MOT10), Lactobacillus murinus (MOT93), and Gemella species 2 (MOT43), which is one of the novel, still-unnamed taxa. Of these, S. danieliae is of particular interest, since it is a major component of the oral microbiome from all strains of healthy and periodontally diseased laboratory mice, as well as being present in wild mice. It is expected that this well-characterized database should be a useful resource for in vitro experimentation and mouse model studies in the field of oral microbiology

Hemin availability induces coordinated DNA methylation and gene expression changes in Porphyromonas gingivalis.

Periodontal disease is a chronic inflammatory disease in which the oral pathogen Porphyromonas gingivalis plays an important role. Porphyromonas gingivalis expresses virulence determinants in response to higher hemin concentrations, but the underlying regulatory processes remain unclear. Bacterial DNA methylation has the potential to fulfil this mechanistic role. We characterized the methylome of P. gingivalis, and compared its variation to transcriptome changes in response to hemin availability. Porphyromonas gingivalis W50 was grown in chemostat continuous culture with excess or limited hemin, prior to whole-methylome and transcriptome profiling using Nanopore and Illumina RNA-Seq. DNA methylation was quantified for Dam/Dcm motifs and all-context N6-methyladenine (6mA) and 5-methylcytosine (5mC). Of all 1,992 genes analyzed, 161 and 268 were respectively over- and under-expressed with excess hemin. Notably, we detected differential DNA methylation signatures for the Dam "GATC" motif and both all-context 6mA and 5mC in response to hemin availability. Joint analyses identified a subset of coordinated changes in gene expression, 6mA, and 5mC methylation that target genes involved in lactate utilization and ABC transporters. The results identify altered methylation and expression responses to hemin availability in P. gingivalis, with insights into mechanisms regulating its virulence in periodontal disease. IMPORTANCE DNA methylation has important roles in bacteria, including in the regulation of transcription. Porphyromonas gingivalis, an oral pathogen in periodontitis, exhibits well-established gene expression changes in response to hemin availability. However, the regulatory processes underlying these effects remain unknown. We profiled the novel P. gingivalis epigenome, and assessed epigenetic and transcriptome variation under limited and excess hemin conditions. As expected, multiple gene expression changes were detected in response to limited and excess hemin that reflect health and disease, respectively. Notably, we also detected differential DNA methylation signatures for the Dam "GATC" motif and both all-context 6mA and 5mC in response to hemin. Joint analyses identified coordinated changes in gene expression, 6mA, and 5mC methylation that target genes involved in lactate utilization and ABC transporters. The results identify novel regulatory processes underlying the mechanism of hemin regulated gene expression in P. gingivalis, with phenotypic impacts on its virulence in periodontal disease

Identification of an O-antigen chain length regulator, WzzP, in Porphyromonas gingivalis

The periodontal pathogen Porphyromonas gingivalis has two different lipopolysaccharides (LPSs) designated O-LPS and A-LPS, which are a conventional O-antigen polysaccharide and an anionic polysaccharide that are both linked to lipid A-cores, respectively. However, the precise mechanisms of LPS biosynthesis remain to be determined. In this study, we isolated a pigment-less mutant by transposon mutagenesis and identified that the transposon was inserted into the coding sequence PGN_2005, which encodes a hypothetical protein of P. gingivalis ATCC 33277. We found that (i) LPSs purified from the PGN_2005 mutant were shorter than those of the wild type; (ii) the PGN_2005 protein was located in the inner membrane fraction; and (iii) the PGN_2005 gene conferred Wzz activity upon an Escherichia coli wzz mutant. These results indicate that the PGN_2005 protein, which was designated WzzP, is a functional homolog of the Wzz protein in P. gingivalis. Comparison of amino acid sequences among WzzP and conventional Wzz proteins indicated that WzzP had an additional fragment at the C-terminal region. In addition, we determined that the PGN_1896 and PGN_1233 proteins and the PGN_1033 protein appear to be WbaP homolog proteins and a Wzx homolog protein involved in LPS biosynthesis, respectively

In Vitro Effect of Porphyromonas gingivalis Methionine Gamma Lyase on Biofilm Composition and Oral Inflammatory Response

Methanethiol (methyl mercaptan) is an important contributor to oral malodour and periodontal tissue destruction. Porphyromonas gingivalis, Prevotella intermedia and Fusobacterium nucleatum are key oral microbial species that produce methanethiol via methionine gamma lyase (mgl) activity. The aim of this study was to compare an mgl knockout strain of P. gingivalis with its wild type using a 10-species biofilm co-culture model with oral keratinocytes and its effect on biofilm composition and inflammatory cytokine production. A P. gingivalis mgl knockout strain was constructed using insertion mutagenesis from wild type W50 with gas chromatographic head space analysis confirming lack of methanethiol production. 10-species biofilms consisting of Streptococcus mitis, Streptococcus oralis, Streptococcus intermedius, Fusobacterium nucleatum ssp polymorphum, Fusobacterium nucleatum ssp vincentii, Veillonella dispar, Actinomyces naeslundii, Prevotella intermedia and Aggregatibacter actinomycetemcomitans with either the wild type or mutant P. gingivalis were grown on Thermanox cover slips and used to stimulate oral keratinocytes (OKF6-TERT2), under anaerobic conditions for 4 and 24 hours. Biofilms were analysed by quantitative PCR with SYBR Green for changes in microbial ecology. Keratinocyte culture supernatants were analysed using a multiplex bead immunoassay for cytokines. Significant population differences were observed between mutant and wild type biofilms; V. dispar proportions increased (p&lt;0.001), whilst A. naeslundii (p&lt;0.01) and Streptococcus spp. (p&lt;0.05) decreased in mutant biofilms. Keratinocytes produced less IL-8, IL-6 and IL-1α when stimulated with the mutant biofilms compared to wild type. Lack of mgl in P. gingivalis has been shown to affect microbial ecology in vitro, giving rise to a markedly different biofilm composition, with a more pro-inflammatory cytokine response from the keratinocytes observed. A possible role for methanethiol in biofilm formation and cytokine response with subsequent effects on oral malodor and periodontitis is suggested

Role of Porphyromonas gingivalis gingipains in multi-species biofilm formation

BackgroundPeriodontal diseases are polymicrobial diseases that cause the inflammatory destruction of the tooth-supporting (periodontal) tissues. Their initiation is attributed to the formation of subgingival biofilms that stimulate a cascade of chronic inflammatory reactions by the affected tissue. The Gram-negative anaerobes Porphyromonas gingivalis, Tannerella forsythia and Treponema denticola are commonly found as part of the microbiota of subgingival biofilms, and they are associated with the occurrence and severity of the disease. P. gingivalis expresses several virulence factors that may support its survival, regulate its communication with other species in the biofilm, or modulate the inflammatory response of the colonized host tissue. The most prominent of these virulence factors are the gingipains, which are a set of cysteine proteinases (either Arg-specific or Lys-specific). The role of gingipains in the biofilm-forming capacity of P. gingivalis is barely investigated. Hence, this in vitro study employed a biofilm model consisting of 10 ¿subgingival¿ bacterial species, incorporating either a wild-type P. gingivalis strain or its derivative Lys-gingipain and Arg-gingipan isogenic mutants, in order to evaluate quantitative and qualitative changes in biofilm composition.ResultsFollowing 64 h of biofilm growth, the levels of all 10 species were quantified by fluorescence in situ hybridization or immunofluorescence. The wild-type and the two gingipain-deficient P. gingivalis strains exhibited similar growth in their corresponding biofilms. Among the remaining nine species, only the numbers of T. forsythia were significantly reduced, and only when the Lys-gingipain mutant was present in the biofilm. When evaluating the structure of the biofilm by confocal laser scanning microscopy, the most prominent observation was a shift in the spatial arrangement of T. denticola, in the presence of P. gingivalis Arg-gingipain mutant.ConclusionsThe gingipains of P. gingivalis may qualitatively and quantitatively affect composition of polymicrobial biofilms. The present experimental model reveals interdependency between the gingipains of P. gingivalis and T. forsythia or T. denticola