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A Porphyromonas gingivalis haloacid dehalogenase family phosphatase interacts with human phosphoproteins and is important for invasion.

ABSTRACT: Haloacid dehalogenase (HAD) family phosphatases are widespread in prokaryotes and are generally involved in metabolic processes. Porphyromonas gingivalis, an invasive periodontal pathogen, secretes the HAD family phosphoserine phosphatase SerB653 when in contact with gingival epithelial cells. Here we characterize the structure and enzymatic activity of SerB653 and show that a SerB653 allelic replacement mutant of P. gingivalis is deficient in internalization and persistence in gingival epithelial cells. In contrast, mutation of a second HAD family serine phosphatase of P. gingivalis (SerB1170), or of a serine transporter, did not affect invasion. A pull-down assay identified GAPDH and heat-shock protein 90 as potential substrates for SerB653. Furthermore, exogenous phosphatase regulated microtubule dynamics in host cells. These data indicate that P. gingivalis has adapted a formerly metabolic enzyme to facilitate entry into host cells by modulating host cytoskeletal architecture. Our findings define a virulence-related role of a HAD family phosphatase and reveal an invasin of an important periodontal pathogen.

SUBMITTER: Tribble GD

PROVIDER: S-EPMC1544168 | biostudies-literature | 2006 Jul

REPOSITORIES: biostudies-literature

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A Porphyromonas gingivalis haloacid dehalogenase family phosphatase interacts with human phosphoproteins and is important for invasion.

Tribble Gena D GD Mao Song S James Chloe E CE Lamont Richard J RJ

Proceedings of the National Academy of Sciences of the United States of America 20060710 29

Haloacid dehalogenase (HAD) family phosphatases are widespread in prokaryotes and are generally involved in metabolic processes. Porphyromonas gingivalis, an invasive periodontal pathogen, secretes the HAD family phosphoserine phosphatase SerB653 when in contact with gingival epithelial cells. Here we characterize the structure and enzymatic activity of SerB653 and show that a SerB653 allelic replacement mutant of P. gingivalis is deficient in internalization and persistence in gingival epitheli ...[more]

PMID: 16832066

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Project description:Protein-tyrosine phosphorylation in bacteria plays a significant role in multiple cellular functions, including those related to community development and virulence. Metal-dependent protein tyrosine phosphatases that belong to the polymerase and histindinol phosphatase (PHP) family are widespread in Gram-positive bacteria. Here, we show that Porphyromonas gingivalis, a Gram-negative periodontal pathogen, expresses a PHP protein, Php1, with divalent metal ion-dependent tyrosine phosphatase activity. Php1 tyrosine phosphatase activity was attenuated by mutation of conserved histidine residues that are important for the coordination of metal ions and by mutation of a conserved arginine residue, a key residue for catalysis in other bacterial PHPs. The php1 gene is located immediately downstream of the gene encoding the bacterial tyrosine (BY) kinase Ptk1, which was a substrate for Php1 in vitro Php1 rapidly caused the conversion of Ptk1 to a state of low tyrosine phosphorylation in the absence of discernible intermediate phosphoforms. Active Php1 was required for P. gingivalis exopolysaccharide production and for community development with the antecedent oral biofilm constituent Streptococcus gordonii under nutrient-depleted conditions. In contrast, the absence of Php1 had no effect on the ability of P. gingivalis to form monospecies biofilms. In vitro, Php1 enzymatic activity was resistant to the effects of the streptococcal secreted metabolites pABA and H2O2, which inhibited Ltp1, an enzyme in the low-molecular-weight (LMW) phosphotyrosine phosphatase family. Ptk1 reciprocally phosphorylated Php1 on tyrosine residues 159 and 161, which independently impacted phosphatase activity. Loss of Php1 rendered P. gingivalis nonvirulent in an animal model of periodontal disease. Collectively, these results demonstrate that P. gingivalis possesses active PHP and LMW tyrosine phosphatases, a unique configuration in Gram-negatives which may allow P. gingivalis to maintain phosphorylation/dephosphorylation homeostasis in multispecies communities. Moreover, Php1 contributes to the pathogenic potential of the organism.IMPORTANCE Periodontal diseases are among the most common infections of humans and are also associated with systemic inflammatory conditions. Colonization and pathogenicity of P. gingivalis are regulated by signal transduction pathways based on protein tyrosine phosphorylation and dephosphorylation. Here, we identify and characterize a novel component of the tyrosine (de)phosphorylation axis: a polymerase and histindinol phosphatase (PHP) family enzyme. This tyrosine phosphatase, designated Php1, was required for P. gingivalis community development with other oral bacteria, and in the absence of Php1 activity P. gingivalis was unable to cause disease in a mouse model of periodontitis. This work provides significant insights into the protein tyrosine (de)phosphorylation network in P. gingivalis, its adaptation to heterotypic communities, and its contribution to colonization and virulence.

Project description: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 ΔPG0027 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 ΔPG0027 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 ΔPG0027 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 ΔPG0027 showed that both contained lipid A 1-phosphate, suggesting that the ΔPG0027 mutant strain lacked lipid A 1-phosphatase activity. The total phosphatase activities in the W50 and the ΔPG0027 mutant strains were similar, whereas the phosphatase activity in the periplasm of the ΔPG0027 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 ΔPG0027 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.

Dataset Information

A Porphyromonas gingivalis haloacid dehalogenase family phosphatase interacts with human phosphoproteins and is important for invasion.

Publications

A Porphyromonas gingivalis haloacid dehalogenase family phosphatase interacts with human phosphoproteins and is important for invasion.

Similar Datasets

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