Type IX secretion system : characterization of an effector protein and an insight into the role of c-terminal domain dimeration in outer membrane translocation.

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

The outer-membrane export signal of Porphyromonas gingivalis type IX secretion system (T9SS) is a conserved C-terminal \beta-sandwich domain

In the recently characterized Type IX Secretion System (T9SS), the conserved C-terminal domain (CTD) in secreted proteins functions as an outer membrane translocation signal for export of virulence factors to the cell surface in the Gram-negative Bacteroidetes phylum. In the periodontal pathogen Porphyromonas gingivalis, the CTD is cleaved off by PorU sortase in a sequence-independent manner, and anionic lipopolysaccharide (A-LPS) is attached to many translocated proteins, thus anchoring them to the bacterial surface. Here, we solved the atomic structure of the CTD of gingipain B (RgpB) from P. gingivalis, alone and together with a preceding immunoglobulin-superfamily domain (IgSF). The CTD was found to possess a typical Ig-like fold encompassing seven antiparallel β-strands organized in two β-sheets, packed into a β-sandwich structure that can spontaneously dimerise through C-terminal strand swapping. Small angle X-ray scattering (SAXS) revealed no fixed orientation of the CTD with respect to the IgSF. By introducing insertion or substitution of residues within the inter-domain linker in the native protein, we were able to show that despite the region being unstructured, it nevertheless is resistant to general proteolysis. These data suggest structural motifs located in the two adjacent Ig-like domains dictate the processing of CTDs by the T9SS secretion pathway

Purification and characterisation of recombinant His-tagged RgpB gingipain from Porphymonas gingivalis

Gingipain proteases are important virulence factors from the periodontal pathogen Porphyromonas gingivalis and are the target of many in vitro studies. Due to their close biochemical properties, purification of individual gingipains is difficult and requires multiple chromatographic steps. In this study, we demonstrate that insertion of a hexahistidine affinity tag upstream of a C-terminal outer membrane translocation signal in RgpB gingipain leads to the secretion of a soluble, mature form of RgpB bearing the affinity tag which can easily be purified by nickel-chelating affinity chromatography. The final product obtained in high yielding and high purity is biochemically indistinguishable from the native RgpB enzyme