Altered gingipain maturation in vimA- and vimE-defective isogenic mutants of Porphyromonas gingivalis.
We have previously shown that gingipain activity in Porphyromonas gingivalis is modulated by the unique vimA and vimE genes. To determine if these genes had a similar phenotypic effect on protease maturation and activation, isogenic mutants defective in those genes were further characterized. Western blot analyses with antigingipain antibodies showed RgpA-, RgpB-, and Kgp-immunoreactive bands in membrane fractions as well as the culture supernatant of both P. gingivalis W83 and FLL93, the vimE-defective mutant. In contrast, the membrane of P. gingivalis FLL92, the vimA-defective mutant, demonstrated immunoreactivity only with RgpB antibodies. With mass spectrometry or Western blots, full-length RgpA and RgpB were identified from extracellular fractions. In similar extracellular fractions from P. gingivalis FLL92 and FLL93, purified RgpB activated only arginine-specific activity. In addition, the lipopolysaccharide profiles of the vimA and vimE mutants were truncated in comparison to that of W83. While glycosylated proteins were detected in the membrane and extracellular fractions from the vimA- and vimE-defective mutants, a monoclonal antibody (1B5) that reacts with specific sugar moieties of the P. gingivalis cell surface polysaccharide and membrane-associated Rgp gingipain showed no immunoreactivity with these fractions. Taken together, these results indicate a possible defect in sugar biogenesis in both the vimA- and vimE-defective mutants. These modulating genes play a role in the secretion, processing, and/or anchorage of gingipains on the cell surface.
Vanterpool
Elaine
E
Department of Biochemistry and Microbiology, School of Medicine, Loma Linda University, Loma Linda, California 92350, USA. eguiness02x@som.llu.edu
Roy
Francis
F
Sandberg
Lawrence
L
Fletcher
Hansel M
HM
D018829:Adhesins, Bacterial; D001426:Bacterial Proteins / Q000235:genetics* / Q000378:metabolism; D002462:Cell Membrane / Q000201:enzymology; D003546:Cysteine Endopeptidases / Q000235:genetics / Q000378:metabolism*; D004789:Enzyme Activation; D004792:Enzyme Precursors / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism; D015964:Gene Expression Regulation, Bacterial; D000080867:Gingipain Cysteine Endopeptidases; D006031:Glycosylation; D006388:Hemagglutinins / Q000235:genetics / Q000378:metabolism*; D006801:Humans; D008565:Membrane Proteins / Q000235:genetics / Q000378:metabolism; D009154:Mutation; D016966:Porphyromonas gingivalis / Q000201:enzymology* / Q000235:genetics / Q000254:growth & development
D018829:Adhesins, Bacterial; D001426:Bacterial Proteins; D004792:Enzyme Precursors; D000080867:Gingipain Cysteine Endopeptidases; D006388:Hemagglutinins; D008565:Membrane Proteins; D003546:Cysteine Endopeptidases
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