Abstract
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.
Highlights
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
Translocation of gingipains from the periplasm across the OM is dependent on a conserved C-terminal domain (CTD), which appears to be important for secretion of the proteins[13] and in particular, truncation of the last few C-terminal residues of this domain leads to accumulation of gingipains in the periplasm[14]
Our previous report described the strange behavior of non-glycosylated RgpB production in the native organism when the junction between the immunoglobulin-superfamily domain (IgSF) and CTD was lengthened by insertion of a hexapeptide motif[18], to investigate this phenomenon, we produced recombinant IgSF-CTD with insertion or substitution of hexapeptide motifs in the linker region for analysis
Summary
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. 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 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. With the exception of T1SS and T2SS, which are widely distributed among prokaryotes, all other types of secretion systems are limited to proteobacteria with scattered occurrence in a few evolutionary lineages of bacteria[2] This is the case for Porphyromonas gingivalis, a bacterium belonging to the Bacteroidetes phylum recognized as a major periodontal pathogen[3]. In the case of RgpB, covalent attachment of A-LPS depends on the length of a linker sequence between the CTD and the preceding immunoglobulin-superfamily domain (IgSF), and occurs concurrently with the proteolytic cleavage of the CTD18
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