Porphyromonas gingivalis and Tannerella forsythia are two of the primary pathogens that are associated in the etiology and progression of chronic periodontitis. In T. forsythia, KLIKK proteases are the recently identified group of proteolytic enzymes that are secreted through Type IX secretion system (T9SS). Among, these KLIKK proteases a synergistic relationship was observed between karilysin and mirolysin in invading the host complement system for the survival of the bacteria. Since, karilysin has been already characterized, in this study we propose to study about mirolysin through structural, biochemical and biological characterization. The obtained results from the experiments has shown the propensity o... More
Porphyromonas gingivalis and Tannerella forsythia are two of the primary pathogens that are associated in the etiology and progression of chronic periodontitis. In T. forsythia, KLIKK proteases are the recently identified group of proteolytic enzymes that are secreted through Type IX secretion system (T9SS). Among, these KLIKK proteases a synergistic relationship was observed between karilysin and mirolysin in invading the host complement system for the survival of the bacteria. Since, karilysin has been already characterized, in this study we propose to study about mirolysin through structural, biochemical and biological characterization. The obtained results from the experiments has shown the propensity of the proenzyme (mirolysin) to autocatalytically process itself at Xaa-Arg peptide bonds. Also, the catalytic enzyme has shown the capablity to degrade an array of physiological substrates like fibrinogen, fibronectin, insulin and LL-37 antimicrobial peptide. In P. gingivalis, gingipains are the major proteolytic enzymes that are also secreted through T9SS and possess a conserved CTD similar to KLIKK proteases. The conserved C-Terminal Domain (CTD) of the T9SS cargo proteins is found essential for the export of these proteins from the periplasm across the outer membrane. Although the mechanism of CTD as an export signal is not well understood so far, taking previous literature into consideration a phenomenon of CTD dimerization has been hypothesized as a possible mechanism in translocation of the cargo proteins through T9SS. Hence, based on the structure of an already crystallized rCTD of RgpB [95] and PorZ (PG1604), in which the CTD is not cleaved off during the translocation of PorZ across outer membrane, four mutations (A637R, G699R, A719R, and R721E) were incorporated into the structure of the RgpB CTD. These mutants were later characterized for their secretory phenotype by quantification of the gingipain activity and by analyzing the processing of the protein through western blots. Out of the four, G699R and A719R mutations have completely occluded the export of protein, while the proteins bearing A637R and R721E mutations were partially processed and secreted. The full-length ProRgpB (native) and its recombinant Ig-CTD tandem proteins, with and without insertion of the factor Xa cleavage site (inserted between Ig and CTD), were purified to investigate the effect of A719R and R721E mutations on CTD dimerization. Preliminary results with the glutaraldehyde crosslinking on recombinant Ig-CTD proteins with A719R and R721E mutations revealed the lack of dimer formation in contrast to the wild-type recombinant Ig-CTD protein without any mutations. In future, these studies will certainly help in understanding the mechanism of T9SS in Gram-negative bacteria.
