RESUMO
Recombinant VimA protein can interact with the gingipains and several other proteins that may play a role in its biogenesis in Porphyromonas gingivalis. In silico analysis of PG2096, a hypothetical protein that was shown to interact with VimA, suggests that it may have environmental stress resistance properties. To further evaluate the role(s) of PG2096, the predicted open reading frame was PCR amplified from P. gingivalis W83 and insertionally inactivated using the ermF-ermAM antibiotic-resistance cassette. One randomly chosen PG2096-defective mutant created by allelic exchange and designated FLL205 was further characterized. Under normal growth conditions at 37 °C, Arg-X and Lys-X gingipain activities in FLL205 were reduced by approximately 35â% and 21â%, respectively, compared to the wild-type strain. However, during prolonged growth at an elevated temperature of 42 °C, Arg-X activity was increased by more than 40â% in FLL205 in comparison to the wild-type strain. In addition, the PG2096-defective mutant was more resistant to oxidative stress when treated with 0.25 mM hydrogen peroxide. Taken together these results suggest that the PG2096 gene, designated regT (regulator of gingipain activity at elevated temperatures), may be involved in regulating gingipain activity at elevated temperatures and be important in oxidative stress resistance in P. gingivalis.
Assuntos
Adesinas Bacterianas/metabolismo , Cisteína Endopeptidases/metabolismo , Estresse Oxidativo , Porphyromonas gingivalis/metabolismo , Adesinas Bacterianas/genética , Sequência de Aminoácidos , Cisteína Endopeptidases/genética , Cisteína Endopeptidases Gingipaínas , Temperatura Alta , Dados de Sequência Molecular , Mutagênese Insercional , Mutação , Peptídeo Hidrolases/metabolismo , Porphyromonas gingivalis/genética , Porphyromonas gingivalis/crescimento & desenvolvimento , Alinhamento de SequênciaRESUMO
The authors have shown previously that the vimA gene, which is part of the bcp-recA-vimA operon, plays an important role in protease activation in Porphyromonas gingivalis. The gingipain RgpB proenzyme is secreted in the vimA-defective mutant P. gingivalis FLL92. An important question that is raised is whether the vimA gene product could directly interact with the proteases for their activation or regulate a pathway responsible for protease activation. To further study the mechanism(s) of VimA-dependent protease activation, the vimA gene product was further characterized. A 39 kDa protein consistent with the size of the predicted VimA protein was purified. In protein-protein interaction studies, the VimA protein was shown to interact with gingipains RgpA, RgpB and Kgp. Immune sera from mice immunized with P. gingivalis immunoreacted with the purified VimA protein. Taken together, these data suggest an interaction of VimA with the gingipains and further confirm the role of this protein in their regulation or maturation.
Assuntos
Adesinas Bacterianas/metabolismo , Proteínas da Membrana Bacteriana Externa/fisiologia , Cisteína Endopeptidases/metabolismo , Precursores Enzimáticos/metabolismo , Genes Bacterianos , Hemaglutininas/metabolismo , Porphyromonas gingivalis/enzimologia , Virulência/genética , Adesinas Bacterianas/genética , Animais , Especificidade de Anticorpos , Apolipoproteínas E/deficiência , Apolipoproteínas E/genética , Proteínas da Membrana Bacteriana Externa/química , Infecções por Bacteroidaceae/imunologia , Cisteína Endopeptidases/genética , Ativação Enzimática , Precursores Enzimáticos/genética , Feminino , Regulação Bacteriana da Expressão Gênica , Cisteína Endopeptidases Gingipaínas , Hemaglutininas/genética , Soros Imunes/imunologia , Imunização , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Endogâmicos , Peso Molecular , Mutação , Óperon , Porphyromonas gingivalis/imunologia , Porphyromonas gingivalis/patogenicidade , Ligação ProteicaRESUMO
In several micro-organisms, HtrA, a serine periplasmic protease, is considered an important virulence factor that plays a regulatory role in oxidative and temperature stress. The authors have previously shown that the vimA gene product is an important virulence regulator in Porphyromonas gingivalis. Further, purified recombinant VimA physically interacted with the major gingipains and the HtrA from P. gingivalis. To further evaluate a role for HtrA in the pathogenicity of this organism, a 1.5 kb fragment containing the htrA gene was PCR-amplified from the chromosomal DNA of P. gingivalis W83. This gene was insertionally inactivated using the ermF-ermAM antibiotic-resistance cassette and used to create an htrA-deficient mutant by allelic exchange. In one randomly chosen isogenic mutant designated P. gingivalis FLL203, there was increased sensitivity to hydrogen peroxide. Growth of this mutant at an elevated temperature was more inhibited compared to the wild-type. Further, in contrast to the wild-type, there was a significant decrease in Arg-gingipain activity after heat shock in FLL203. However, the gingipain activity in the mutant returned to normal levels after a further 30 min incubation at room temperature. Collectively, these data suggest that HtrA may play a similar role in oxidative and temperature stress in P. gingivalis as observed in other organisms.
