Por Secretion System of Porphyromonas gingivalis

The virulence factors of pathogenic bacteria are major secretory proteins that are directly linked to their pathogenicity. These secretory proteins are translocated across the membranes of bacterial cells by translocase nanomachines, which consists of integral membrane proteins. The periodontal pathogen, Porphyromonas gingivalis, secretes trypsin-like proteases(gingipains)either as a large complex on the bacterial cell surface or into the extracellular milieu. Gingipains are important virulence factors, because they degrade host proteins. They are responsible for the processing/maturation of other P. gingivalis virulence factors. At least six types of translocase nanomachines have been found in Gram-negative bacteria;however, P. gingivalis does not have genes homologous to those coding these secretion systems in the bacterial genome and not much is known about the mechanism of gingipain secretion. In this study, the proteins responsible for gingipain secretion, i.e., PorK, PorL, PorM, PorN, and PorW, were identified by comparative genome analysis and genetic experiments. We named the gingipain secretion system the Por secretion system(PorSS). Genes encoding PorSS proteins are conserved among a group of bacteria including periodontal pathogens such as Tannellera forsythia and Prevotella intermedia in the phylum Bacteroidetes. In addition, homologous genes are involved in gliding motility and chitinase secretion in Flavobacterium johnsoniae, another member of the phylum Bacteroidetes. Two other genes, porX and porY, encoding the regulatory factors of PorSS gene expression were identified at the same time. The expression of the porT, porK, porL, porM, and porN genes was downregulated in PorXor PorY-defective mutants. PorSS and its regulatory system appear to be associated with the pathogenicity of various bacteria in the phylum Bacteroidetes. § Corresponding author E-mail:[email protected] secreted and inner membrane proteins in an unfolded conformation. On the other hand, the Tat system is involved in the transport of proteins in a folded conformation. After cleavage of a signal peptide by signal peptidase, the secreted protein is released into the periplasm and then translocated across the outer membrane to the extracellular environment through one of the secretion systems. In the typeII secretion system, the precursor secreted proteins are translocated across the inner membrane by the Sec translocon or the Tat pathway. After cleavage of signal peptide by a signal peptidase, the secreted proteins are translocated across the outer membrane by the type II secretion system, which consists of multicomponent machines spanning inner and outer membranes. The typeII secretion system is involved in the secretion of virulence factors in many Gram-negative bacteria, such as aerolysin in Aeromonas hydrophila, the heat-labile toxin in enterotoxigenic Escherichia coli, and cholera toxin in Vibrio cholerae. The type V secretion system:The autotransporter and the twopartner secretion systems are grouped within the type V secretion system due to the similarities between these systems. The translocator and passenger domains are encoded by a single gene in the autotransporter system, but are encoded by two separate genes in the two-partner secretion system. After these precursor proteins have been transported across the inner membrane through the Sec translocon, the translocator domains are transported across the outer membrane using their passenger domain. In the one-step system, the typeI, III, IV, and VI secretion systems form complexes across the two membranes and allow both direct export of the virulence factors from the cytoplasm to the extracellular milieu or injection of effector proteins into the cytoplasm of the host cells(Fig. 1). The typeI secretion system of Gram-negative bacteria consists of three proteins:the ABC transporter protein, the oligomeric membrane fusion protein, and the outer membrane protein. This simple system is associated with, for example, the secretion of HlyA(hemolysin A)from E. coli, CyaA(Pore forming/adenylate cyclase)from Bordetella pertussis, and HasA(Haemophore)from Serratia maecescens. The type III secretion system, which consists of a membrane-embedded basal body, needle, and tip structure, are conserved in both flagellar and virulence-associated secretion systems. 188 K. Sato:Por Secretion System of Porphyromonas gingivalis Fig. 1 Schematic models of typeI―VI secretion systems in Gram-negative bacteria In the typeI secretion system, secreted proteins are transported from the cytoplasm to the extracellular milieu in a single step. In the typeII secretion system, secreted proteins are transported from the cytoplasm to the extracellular milieu in a two-step process. Initially, proteins are transported through the inner membrane by the Sec machinery and are then translocated through the outer membrane by the typeII secretion system. In the type III secretion system, secreted proteins are transported from the cytoplasm to the host cell. Pathogenic bacteria including Salmonell typhimurium, enteropathogenic, and enterohaemorrhagic E. coli, inject effector proteins directly into their host cells using the type III secretion system. The type IV secretion system is found in both Gram-positive and Gram-negative bacteria and is associated with the conjugative transfer of plasmid DNA or transposons, DNA uptake from and release into the extracellular milieu, and the transport of effector molecules into target cells. Conjugative transfer contributes to the emergence of antibiotic-resistant bacteria. Pathogenic bacteria such as Helicobacter pylori and Legionella pneumophila use the type IV secretion system. The type VI secretion system was recently identified and its mechanism has not yet been elucidated. The type VI secretion system is usually encoded by a cluster of 12―25 genes within a pathogenicity island in Gramnegative bacteria, such as Pseudomonas aeruginosa and Salmonella typhimurium. The function and characterization of the components of the type VI system remain to be elucidated. These secretion systems are not, however, present in periodontal pathogens of the Bacteroidetes phylum, such as Porphyromonas gingivalis, Tannerella forsythia, and Prevotella intermedia. P. gingivals, a principal pathogen of periodontal disease, has many virulence factors, including proteinases, fimbriae, and hemagglutinins. Most of the proteolytic activities of the extracellular and cell surface proteinases are derived from a group of cysteine proteinases named gingipains. In addition to the degradation of host proteins, gingipains are responsible for the processing/ maturation of P. gingivalis virulence factors, including hemagglutinins and fimbriae. Gingipains are encoded by three separate genes in the P. gingivalis genome, kgp for the Lys-specific cysteine proteinase, and rgpA and rgpB for two Arg-specific cysteine proteinases. They are synthesized in the cytoplasm as precursor proteins with a signal peptide, a pro-region, a proteinase domain, hemagglutinin domain, and a C-terminal domain. These molecules are then translocated across the inner membrane, periplasm, and outer membrane. The transported gingipains are then located on the bacterial cell surface as large complexes or secreted into the extracellular milieu(Fig. 2A). Because P. gingivalis has no homology with the gene clusters of known secretion systems, the mechanism of gingipain secretion is not well understood. A porT mutant, which was constructed by transposon mutagenesis in a previous study, accumulates gingipain precursor forms in the periplasmic space and shows no gingipain activity. In the porT mutant, gingipain, TapA protein, and Hbp35 are not secreted at the cell surface. These proteins contain the C-terminal domain, which has conserved DxxG and GxY motifs as well as charged residues at the Cterminal end. In addition, these proteins immunoreact with a monoclonal antibody, 1B5, which is immunoreactive to a cell surface anionic polysaccharide(APS) and to the glycan addition of gingipains. It is possible that these outer membrane proteins, which contain the conserved C-terminal domain, are secreted by a previously unknown secretion system. P. gingivalis Genes Involved in Colonial Black Pigmentation on Blood Agar P. gingivalis forms characteristic black-pigmented colonies on blood agar plates due to the accumulation of μ-oxo heme dimers on the cell surface(Fig. 2B). Rgp activity is crucial for converting oxyhaemoglobin to methaemoglobin, which is more susceptible to Kgp degradation for the eventual release of iron(III)protoporphyrin IX and the production of μ-oxo heme dimers;therefore, the kgp and rgpA rgpB kgp mutants form less and non-pigmented colonies, respectively, caused by defects in the biosynthesis of μ-oxo heme dimers. Because the black pigmentation on blood agar is associated with gingipain activity at the cell surface, the pigmentation-related genes characterized so far can be classified as three types of genes:those which are involved in gene expression, surface attachment of gingipain-adhesin complexes, and membrane transportation. Gene expression:The kgp and rgpA rgpB kgp mutants form less and non-pigmented colonies. Surface attachment of gingipain-adhesin complexes:Kgp and Rgp are present as cell surface gingipain-adhesin complexes consisting of Rgp and 189