Characterization of RagA and RagB in Porphyromonas gingivalis: study using gene-deletion mutants

Abstract

The major outer-membrane proteins RagA and RagB of Porphyromonas gingivalis are considered to form a receptor complex functionally linked to TonB. In this study, P. gingivalis mutants with ragA, ragB or both deleted were constructed from strain W83 as the parent to examine the physiological and pathological functions of RagA and RagB. The double-deletion mutant completely lacked both RagA and RagB, whereas the DragA mutant reduced RagB expression considerably and the DragB mutant produced degraded RagA. Growth of the three mutants in a nutrient-rich medium and synthetic media containing digested protein as a unique nutrient source was similar to that of the parental strain; however, both the DragA and DragAB mutants exhibited very slow growth in a synthetic medium containing undigested, native protein, and the two mutants tended to lose their viability during experiments, although gingipain (protease) activities were unchanged in the mutants. A mouse model showed that the DragB mutant had reduced virulence. Cell-surface labelling with biotin and dextran revealed that both RagA and RagB localized on the outermost cell surface. A cross-linking experiment using wild-type P. gingivalis showed that RagA and RagB were closely associated with each other. Furthermore, co-immunoprecipitation confirmed that RagA and RagB formed a protein-protein complex. These results suggest that physically associated RagA and RagB may stabilize themselves on the cell surface and function as active transporters of large degradation products of protein and in part as a virulence factor.

Selective Sorting of Cargo Proteins into Bacterial Membrane Vesicles

“…Certain forms of the gingipains are thought to exert a role in tissues distant from the site of the infection, and therefore it seems logical that these proteases are preferentially packed into OMV. In contrast, in agreement with their proposed roles in nutrient uptake or metabolite exchange, some porins (PG0694 and PG0695) and RagA/B (the remaining dominant OM proteins) are completely excluded from the OMV and remain in the OM (31,33,39). The specific protein packing process into OMV was aberrant in two LPS mutant strains.…”

“…RagA is an integral OM protein that exhibits all of the typical features of a TonB-linked OM receptor, whereas RagB is a lipoprotein (32). These two proteins are thought to form a complex in the OM that is responsible for nutrient uptake (31). The exact function of these proteins has yet to be determined.…”

“…3, B and D). RagA/B are immunodominant surface proteins of P. gingivalis (22,30,31). RagA is an integral OM protein that exhibits all of the typical features of a TonB-linked OM receptor, whereas RagB is a lipoprotein (32).…”

Role of Mfa5 in Expression of Mfa1 Fimbriae in Porphyromonas gingivalis

“…Genetic complementation strain FMFA5C was constructed using the expression vector pTCBex2 (Nagano et al 2007). In brief, the mfa5 region in ATCC 33277 was amplified by PCR using cMfa5FX and cMfa5RN with XbaI and NotI restriction sites, respectively (Appendix Table 2).…”

Inhibition of polysaccharide synthesis by the sinR orthologue PGN_0088 is indirectly associated with the penetration of Porphyromonas gingivalis biofilms by macrolide antibiotics

“…The amplified DNA fragment was cloned into a pGEM-T Easy vector (Promega). The sinR region obtained by NotI and BamHI digestion was inserted into NotI-BamHI-digested pTCB (Nagano et al, 2007) to yield pOD004 (sinR+). The pOD004 plasmid DNA was introduced into the sinR mutant by conjugation with E. coli S17-1 (Simon et al, 1983), harbouring pOD004, as a donor strain to generate strain ODP004 (sinR+-complemented strain; DsinR : : cat/ sinR+).…”