Porphyromonas gingivalis secretes strong proteases called gingipains that are implicated in periodontal pathogenesis. Protein secretion systems common to other Gram-negative bacteria are lacking in P. gingivalis, but several proteins, including PorT, have been linked to gingipain secretion. Comparative genome analysis and genetic experiments revealed 11 additional proteins involved in gingipain secretion. Six of these (PorK, PorL, PorM, PorN, PorW, and Sov) were similar in sequence to Flavobacterium johnsoniae gliding motility proteins, and two others (PorX and PorY) were putative two-component system regulatory proteins. Real-time RT-PCR analysis revealed that porK, porL, porM, porN, porP, porT, and sov were down-regulated in P. gingivalis porX and porY mutants. Disruption of the F. johnsoniae porT ortholog resulted in defects in motility, chitinase secretion, and translocation of a gliding motility protein, SprB adhesin, to the cell surface, providing a link between a unique protein translocation system and a motility apparatus in members of the Bacteroidetes phylum.gingipain | Porphyromonas gingivalis | Flavobacterium | chitinase

Dentatorubral and pallidoluysian atrophy (DRPLA) is an autosomal dominant neurodegenerative disorder characterized by combined systemic degeneration of the dentatofugal and pallidofugal pathways. We investigated a candidate gene and found that DRPLA patients had an expanded CAG trinucleotide repeat in a gene on the short arm of chromosome 12. The repeat size varied from 7-23 in normal individuals. In patients one allele was expanded to between 49-75 repeats or occasionally even more. Expansion was usually associated with paternal transmission and only occasionally with maternal transmission. Repeat size showed a close correlation with age of onset of symptoms and disease severity. We conclude that DRPLA is the seventh genetic disorder known to be associated with expansion of an unstable trinucleotide repeat.

The anaerobic Gram-negative bacterium Porphyromonas gingivalis is a major pathogen in severe forms of periodontal disease and refractory periapical perodontitis. We have recently found that P. gingivalis has a novel secretion system named the Por secretion system (PorSS), which is responsible for secretion of major extracellular proteinases, Arg-gingipains (Rgps) and Lys-gingipain. These proteinases contain conserved C-terminal domains (CTDs) in their C-termini. Hemin-binding protein 35 (HBP35), which is one of the outer membrane proteins of P. gingivalis and contributes to its haem utilization, also contains a CTD, suggesting that HBP35 is translocated to the cell surface via the PorSS. In this study, immunoblot analysis of P. gingivalis mutants deficient in the PorSS or in the biosynthesis of anionic polysaccharide-lipopolysaccharide (A-LPS) revealed that HBP35 is translocated to the cell surface via the PorSS and is glycosylated with A-LPS. From deletion analysis with a GFP-CTD[HBP35] green fluorescent protein fusion, the C-terminal 22 amino acid residues of CTD[HBP35] were found to be required for cell surface translocation and glycosylation. The GFP-CTD fusion study also revealed that the CTDs of CPG70, peptidylarginine deiminase, P27 and RgpB play roles in PorSS-dependent translocation and glycosylation. However, CTD-region peptides were not found in samples of glycosylated HBP35 protein by peptide map fingerprinting analysis, and antibodies against CTD-regions peptides did not react with glycosylated HBP35 protein. These results suggest both that the CTD region functions as a recognition signal for the PorSS and that glycosylation of CTD proteins occurs after removal of the CTD region. Rabbits were used for making antisera against bacterial proteins in this study.

The dual membrane envelopes of Gram-negative bacteria provide two barriers of unlike nature that regulate the transport of molecules into and out of organisms. Organisms have developed several systems for transport across the inner and outer membranes. The Gramnegative periodontopathogenic bacterium Porphyromonas gingivalis produces proteinase and adhesin complexes, gingipains/adhesins, on the cell surface and in the extracellular milieu as one of the major virulence factors. Gingipains and/or adhesins are encoded by kgp, rgpA, rgpB, and hagA on the chromosome. In this study, we isolated a P. gingivalis mutant (porT), which showed very weak activities of gingipains in the cell lysates and culture supernatants. Subcellular fractionation and immunoblot analysis demonstrated that precursor forms of gingipains and adhesins were accumulated in the periplasmic space of the porT mutant cells. Peptide mass fingerprinting and N-terminal amino acid sequencing of the precursor proteins and the kgp-rgpB chimera gene product in the porT mutant indicated that these proteins lacked the signal peptide regions, consistent with their accumulation in the periplasm. The PorT protein seemed to be membrane-associated and exposed to the periplasmic space, as revealed by subcellular fractionation and immunoblot analysis using antiPorT antiserum. These results suggest that the membrane-associated protein PorT is essential for transport of the kgp, rgpA, rgpB, and hagA gene products across the outer membrane from the periplasm to the cell surface, where they are processed and matured.

Cells of Flavobacterium johnsoniae and of many other members of the phylum Bacteroidetes exhibit rapid gliding motility over surfaces by a unique mechanism. These cells do not have flagella or pili; instead, they rely on a novel motility apparatus composed of Gld and Spr proteins. SprB, a 669-kDa cell-surface adhesin, is required for efficient gliding. SprB was visualized by electron microscopy as thin 150-nm-long filaments extending from the cell surface. Fluorescence microscopy revealed movement of SprB proteins toward the poles of the cell at ∼2 μm/s. The fluorescent signals appeared to migrate around the pole and continue at the same speed toward the opposite pole along an apparent left-handed helical closed loop. Movement of SprB, and of cells, was rapidly and reversibly blocked by the addition of carbonyl cyanide m-chlorophenylhydrazone, which dissipates the proton gradient across the cytoplasmic membrane. In a gliding cell, some of the SprB protein appeared to attach to the substratum. The cell body moved forward and rotated with respect to this point of attachment. Upon reaching the rear of the cell, the attached SprB often was released from the substratum, and apparently recirculated to the front of the cell along a helical path. The results suggest a model for Flavobacterium gliding, supported by mathematical analysis, in which adhesins such as SprB are propelled along a closed helical loop track, generating rotation and translation of the cell body.cell motility | proton motive force | immunofluorescence microscopy | continuous track | left-handed helix C ells of Flavobacterium johnsoniae, and of many other members of the phylum Bacteroidetes, move rapidly over surfaces at speeds of 1-3 μm/s by gliding motility (1). These cells lack flagella and pili, and the mechanism of cell movement is poorly understood. Flavobacterium gliding is thought to rely on motors embedded in the cell envelope that propel large cell-surface adhesins such as SprB and related proteins (2). Deletion of sprB results in dramatic reduction in motility. Twelve Gld proteins also are required for gliding (3