Involvement of an Skp-Like Protein, PGN_0300, in the Type IX Secretion System of Porphyromonas gingivalis

Abstract

The oral Gram-negative anaerobic bacterium Porphyromonas gingivalis is an important pathogen involved in chronic periodontitis. Among its virulence factors, the major extracellular proteinases, Arg-gingipain and Lys-gingipain, are of interest given their abilities to degrade host proteins and process other virulence factors. Gingipains possess C-terminal domains (CTDs) and are translocated to the cell surface or into the extracellular milieu by the type IX secretion system (T9SS). Gingipains contribute to the colonial pigmentation of the bacterium on blood agar. In this study, Omp17, the PGN_0300 gene product, was found in the outer membrane fraction. A mutant lacking Omp17 did not show pigmentation on blood agar and showed reduced proteolytic activity of the gingipains. CTD-containing proteins were released from bacterial cells without cleavage of the CTDs in the omp17 mutant. Although synthesis of the anionic polysaccharide (A-LPS) was not affected in the omp17 mutant, the processing of and A-LPS modification of CTD-containing proteins was defective. PorU, a C-terminal signal peptidase that cleaves the CTDs of other CTD-containing proteins, was not detected in any membrane fraction of the omp17 mutant, suggesting that the defective maturation of CTD-containing proteins by impairment of Omp17 is partly due to loss of function of PorU. In the mouse subcutaneous infection experiment, the omp17 mutant was less virulent than the wild type. These results suggested that Omp17 is involved in P. gingivalis virulence.

Structural and functional probing of PorZ, an essential bacterial surface component of the type-IX secretion system of human oral-microbiomic Porphyromonas gingivalis.

“…When the same proteins were detectable in the growth medium of the wild-type strain, they had much lower Mascot scores and no peptides corresponding to their respective CTDs. This “leaky” OM phenotype, which leads to release of non-cleaved CTDs from CTD-cargo proteins, is similar to that reported by Taguchi et al 36. These authors reported that the chaperone Skp-like protein (PGN_0300) is required for OM insertion of PorU sortase, which in turn is necessary for CTD cleavage from CTD-cargo proteins.…”

“…These authors reported that the chaperone Skp-like protein (PGN_0300) is required for OM insertion of PorU sortase, which in turn is necessary for CTD cleavage from CTD-cargo proteins. Consistently, deletion of PGN_0300 resulted in failure of PorU insertion and, thus, T9SS function36.…”

“…Due to the phenotypic similarity of the ΔPorZ mutant with the Skp-like PGN_0300 mutant, where PorU failed to insert into the OM36, we proceeded to investigate the surface exposure of PorU in the ΔPorZ and ΔPorN mutants as compared to wild type. Flow cytometry using an anti-PorU antibody revealed that PorU was absent from the cell surface of both ΔPorZ and ΔPorN, in stark contrast to the wild type (Fig.…”

Type IX secretion: the generation of bacterial cell surface coatings involved in virulence, gliding motility and the degradation of complex biopolymers

“…PG0534 is a predicted TonB-dependent receptor shown to be located in the outer membrane and required for T9SS function but it's specific role has not been determined (Saiki and Konishi, 2010a). A Skp-like protein, PGN_0300 has also been proposed to be involved in the T9SS (Taguchi et al, 2015). Since Skp is known in other species to be involved in the assembly of outer membrane proteins (Noinaj et al, 2017), it remains to be demonstrated that PGN_0300 is specific to the T9SS, rather than simply performing its expected role of assembling certain outer membrane proteins including one or more components of the T9SS.…”

The role of kinases and phosphatases in the pathobiology of porphyromonas gingivalis.

“…A-LPS is a phosphorylated branched mannan [67], that is attached to many proteins translocated through the type IX secretion system (T9SS), thus anchoring them to the bacterial surface [68]. A mutant unable to incorporate A-LPS into T9SS substrates is less virulent in a mouse subcutaneous infection model [69] Gingipains are yet another important aspect of P. gingivalis virulence. These enzymes play important roles in bacterial housekeeping and infection.…”