Yuichiro Kikuchi

A two-component system regulates gene expression of the type IX secretion component proteins via an ECF sigma factor

The periodontopathogen Porphyromonas gingivalis secretes potent pathogenic proteases, gingipains, via the type IX secretion system (T9SS). This system comprises at least 11 components; however, the regulatory mechanism of their expression has not yet been elucidated. Here, we found that the PorY (PGN_2001)-PorX (PGN_1019)-SigP (PGN_0274) cascade is involved in the regulation of T9SS. Surface plasmon resonance (SPR) analysis revealed a direct interaction between a recombinant PorY (rPorY) and a recombinant PorX (rPorX). rPorY autophosphorylated and transferred a phosphoryl group to rPorX in the presence of Mn2+. These results demonstrate that PorX and PorY act as a response regulator and a histidine kinase, respectively, of a two component system (TCS), although they are separately encoded on the chromosome. T9SS component-encoding genes were down-regulated in a mutant deficient in a putative extracytoplasmic function (ECF) sigma factor, PGN_0274 (SigP), similar to the porX mutant. Electrophoretic gel shift assays showed that rSigP bound to the putative promoter regions of T9SS component-encoding genes. The SigP protein was lacking in the porX mutant. Co-immunoprecipitation and SPR analysis revealed the direct interaction between SigP and PorX. Together, these results indicate that the PorXY TCS regulates T9SS-mediated protein secretion via the SigP ECF sigma factor.

Involvement of the Type IX Secretion System in Capnocytophaga ochracea Gliding Motility and Biofilm Formation

ABSTRACT Capnocytophaga ochracea is a Gram-negative, rod-shaped bacterium that demonstrates gliding motility when cultured on solid agar surfaces. C. ochracea possesses the ability to form biofilms; however, factors involved in biofilm formation by this bacterium are unclear. A type IX secretion system (T9SS) in Flavobacterium johnsoniae was shown to be involved in the transport of proteins (e.g., several adhesins) to the cell surface. Genes orthologous to those encoding T9SS proteins in F. johnsoniae have been identified in the genome of C. ochracea; therefore, the T9SS may be involved in biofilm formation by C. ochracea. Here we constructed three ortholog-deficient C. ochracea mutants lacking sprB (which encodes a gliding motility adhesin) or gldK or sprT (which encode T9SS proteins in F. johnsoniae). Gliding motility was lost in each mutant, suggesting that, in C. ochracea, the proteins encoded by sprB, gldK, and sprT are necessary for gliding motility, and SprB is transported to the cell surface by the T9SS. For the ΔgldK, ΔsprT, and ΔsprB strains, the amounts of crystal violet-associated biofilm, relative to wild-type values, were 49%, 34%, and 65%, respectively, at 48 h. Confocal laser scanning and scanning electron microscopy revealed that the biofilms formed by wild-type C. ochracea were denser and bacterial cells were closer together than in those formed by the mutant strains. Together, these results indicate that proteins exported by the T9SS are key elements of the gliding motility and biofilm formation of C. ochracea.

Role of extracytoplasmic function sigma factors in biofilm formation of Porphyromonas gingivalis

BackgroundPorphyromonas gingivalis has been implicated as a major pathogen in the development and progression of chronic periodontitis. P. gingivalis biofilm formation in the subgingival crevice plays an important role in the ability of the bacteria to tolerate stress signals outside the cytoplasmic membrane. Some bacteria use a distinct subfamily of sigma factors to regulate their extracytoplasmic functions (the ECF subfamily). The objective of this study was to determine if P. gingivalis ECF sigma factors affect P. gingivalis biofilm formation.MethodsTo elucidate the role of ECF sigma factors in P. gingivalis, chromosomal mutants carrying a disruption of each ECF sigma factor-encoding gene were constructed. Bacterial growth curves were measured by determining the turbidity of bacterial cultures. The quantity of biofilm growing on plates was evaluated by crystal violet staining.ResultsComparison of the growth curves of wild-type P. gingivalis strain 33277 and the ECF mutants indicated that the growth rate of the mutants was slightly lower than that of the wild-type strain. The PGN_0274- and PGN_1740-defective mutants had increased biofilm formation compared with the wild-type (p < 0.001); however, the other ECF sigma factor mutants or the complemented strains did not enhance biofilm formation.ConclusionThese results suggest that PGN_0274 and PGN_1740 play a key role in biofilm formation by P. gingivalis.

Point-of-care detection of Tannerella forsythia using an antigen–antibody assisted dielectrophoretic impedance measurement method

UNLABELLED The importance of periodontal treatment planning based on diagnosis with clinical detection of periodontal pathogens has been well recognized. However, reliable detection and quantification methods that can be conveniently used at chair-side have yet to be developed. This study aimed to evaluate the clinical use of a novel apparatus which uses an antigen-antibody reaction assisted dielectrophoretic impedance measurement (AA-DEPIM) for the detection of a prominent periodontal pathogen, Tannerella forsythia. A total of 15 patients with a clinical diagnosis of chronic periodontitis, three periodontally healthy volunteers and two with gingivitis were subjected to clinical and microbiological examinations. Saliva samples were analyzed for the presence of T. forsythia using AA-DEPIM, PCR-Invader and real-time PCR methods. The measurement values for total bacteria and T. forsythia using the prototype AA-DEPIM apparatus were significantly greater in periodontitis group than those in healthy/gingivitis group. Using the AA-DEPIM apparatus with tentative cut-off values, T. forsythia was detected for 14 (12 with periodontitis and 2 either healthy or with gingivitis) out of 20 individuals. The measurement for the detection of T. forsythia by the AA-DEPIM method showed a significant positive correlation with the detection by PCR-Invader (r = 0.541, p = 0.01) and the real-time PCR method (r = 0.834, p = 0.01). When the PCR-Invader method was used as a reference, the sensitivity and specificity of the AA-DEPIM method were 76.5% and 100%, respectively. The results suggested that the AA-DEPIM method has potential to be used for clinically evaluating salivary presence of T. forsythia at chair-side. TRIAL REGISTRATION UMIN Clinical Trials Registry (UMIN-CTR) UMIN000012181.

Treponema denticola Induces Epithelial Barrier Dysfunction in Polarized Epithelial Cells

Treponema denticola, an anaerobic spirochete found mainly in the oral cavity, is associated with periodontal disease and has a variety of virulence factors. Although in vitro studies have shown that T. denticola is able to penetrate epithelial cell monolayers, its effect on the epithelial barrier junction is not known. Human gingival epithelial cells are closely associated with adjacent membranes, forming barriers in the presence of tight junction proteins, including zonula occludens-1 (ZO-1), claudin-1, and occludin. Tight junction proteins are also expressed by Madin-Darby canine kidney (MDCK) cells in culture. In this study, the MDCK cell profile was investigated following infection with T. denticola (ATCC 35405) wild-type, as well as with its dentilisin-deficient mutant, K1. Basolateral exposure of MDCK cell monolayers to T. denticola at a multiplicity of infection (MOI) of 104 resulted in a decrease in transepithelial electrical resistance (TER). Transepithelial electrical resistance in MDCK cell monolayers also decreased following apical exposure to T. denticola (MOI=104), although this took longer with basolateral exposure. The effect on the TER was time-dependent and required the presence of live bacteria. Meanwhile, MDCK cell viability showed a decrease with either basolateral or apical exposure. Immunofluorescence analysis demonstrated decreases in the amounts of immunoreactive ZO-1 and claudin-1 in association with disruption of cell-cell junctions in MDCK cells exposed apically or basolaterally to T. denticola. Western blot analysis demonstrated degradation of ZO-1 and claudin-1 in culture lysates derived from T. denticola-exposed MDCK cells, suggesting a bacteria-induced protease capable of cleaving these tight junction proteins.

Characterization of a novel potential peptide import system in Treponema denticola

Treponema denticola is a major etiologic agent of chronic periodontitis. On the outer sheath of T. denticola, several proteins, such as the major outer sheath protein and dentilisin were detected, and among them, a 95 kDa protein which has not yet been characterized. The aim of this study was to characterize the function of this 95 kDa protein containing gene cluster. A gene encoding this 95 kDa protein (TDE_1072) of T. denticola was inactivated by homologous recombination. We compared growth curves between the TDE_1072 mutant and wild-type strains as well as differences in gene expression by DNA microarray analysis. Differential expression of genes identified by microarray analysis was confirmed by quantitative reverse transcription-polymerase chain reaction. The proteins encoded by TDE_1072, TDE_1073, TDE_1074, TDE_1075, and TDE_1076 shared respective similarities to the substrate-binding domain (DppA) of an ABC-type dipeptide/oligopeptide/nickel transport system, and to the permease components (DppB and DppC) and ATPase components (DppD and DppF) of an ABC-type dipeptide/oligopeptide/nickel transport system. Inactivation of dppA attenuated the growth of T. denticola and dppA-dppF were co-transcribed. In contrast, expression of oppB-oppF was up-regulated in the mutant. Our findings indicate that TDE_1072 may be a potential periplasmic solute binding protein encoded by dppA that is involved in the organization of a peptide uptake system with dppB-dppF.

Treponema denticola transcriptional profiles in serum-restricted conditions

Treponema denticola is a major pathogen in periodontal disease and is frequently isolated from the lesions of patients with chronic periodontitis. Treponema denticola utilizes serum components as nutrient sources so as to colonize and proliferate in the gingival crevice. However, the mechanisms of serum utilization remain unclear. Therefore, the aim of the present study was to identify T. denticola serum utilization genes. Precultured T. denticola cells were suspended in a tryptone-yeast extract-gelatin-volatile fatty acids medium containing 0, 1% and 10% serum, respectively, and incubated anaerobically for 17 h. Total RNA was isolated, and T. denticola gene expression was compared by microarray and reverse transcription-polymerase chain reaction. In serum-depleted conditions, the expression levels of a potential hydroxylamine reductase, several ABC transporters, and phosphoenolpyruvate synthase were increased, while those of genes encoding methyl-accepting chemotaxis proteins and a transcriptional regulator were decreased. These results suggest that T. denticola may uptake serum components mainly through the action of ABC transporters. In particular, the decrease in the dmcA expression level with decreasing serum concentration suggests its involvement in chemotaxis toward serum-rich environments.

Identification of a specific domain of Porphyromonas gingivalis Hgp44 responsible for adhesion to Treponema denticola

Interaction between two periodontal pathogens, Porphyromonas gingivalis and Treponema denticola, contributes to plaque biofilm formation. Porphyromonas gingivalis forms aggregates with T. denticola through its adhesion/hemagglutinin domain (Hgp44). In this study, we investigated the specific domain of P. gingivalis Hgp44 responsible for adhesion to T. denticola using expression vectors harboring P. gingivalis Hgp44 DNA sequences encoding amino acid residues 1-419. Six plasmids harboring fragments in this region were generated by PCR amplification and self-ligation, and recombinant proteins r-Hgp44 (residues 1-419), r-Hgp441 (residues 1-124), r-Hgp442 (1-199), r-Hgp443 (1-316), r-Hgp444 (199-419), r-Hgp445 (124-198) and r-Hgp446 (199-316) were produced, as confirmed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and immunoblotting. r-Hgp44, r-Hgp443 and r-Hgp446 showed greater adhesion to T. denticola sonicates than the control, as determined by enzyme-linked immunosorbent assay. r-Hgp446 reduced the coaggregation of P. gingivalis and T. denticola. Scanning electron and confocal laser scanning microscopy analyses revealed that r-Hgp446 reduced dual-species biofilm formation. Our results indicate that residues 199-316 of P. gingivalis Hgp44 are mainly responsible for adhesion to T. denticola; inhibiting this domain could potentially disrupt periodontopathic biofilm formation and maturation.

Effect of extracytoplasmic function sigma factors on autoaggregation, hemagglutination, and cell surface properties of Porphyromonas gingivalis

Porphyromonas gingivalis is a bacterium frequently isolated from chronic periodontal lesions and is involved in the development of chronic periodontitis. To colonize the gingival crevice, P. gingivalis has to adapt to environmental stresses. Microbial gene expression is regulated by transcription factors such as those in two-component systems and extracytoplasmic function (ECF) sigma factors. ECF sigma factors are involved in the regulation of environmental stress response genes; however, the roles of individual ECF sigma factors are largely unknown. The purpose of this study was to investigate the functions, including autoaggregation, hemagglutination, gingipain activity, susceptibility to antimicrobial agents, and surface structure formation, of P. gingivalis ECF sigma factors encoded by SigP (PGN_0274), SigCH (PGN_0319), PGN_0450, PGN_0970, and SigH (PGN_1740). Various physiological aspects of the sigP mutant were affected; autoaggregation was significantly decreased at 60 min (p < 0.001), hemagglutination activity was markedly reduced, and enzymatic activities of Kgp and Rgps were significantly decreased (p < 0.001). The other mutants also showed approximately 50% reduction in Rgps activity. Kgp activity was significantly reduced in the sigH mutant (p < 0.001). No significant differences in susceptibilities to tetracycline and ofloxacin were observed in the mutants compared to those of the wild-type strain. However, the sigP mutant displayed an increased susceptibility to ampicillin, whereas the PGN_0450 and sigH mutants showed reduced susceptibility. Transmission electron microscopy images revealed increased levels of outer membrane vesicles formed at the cell surfaces of the sigP mutant. These results indicate that SigP is important for bacterial surface-associated activities, including gingipain activity, autoaggregation, hemagglutination, vesicle formation, and antimicrobial susceptibility.

Possible Involvement of Surface Antigen Protein 2 in the Morphological Transition and Biofilm Formation of Candida albicans

Surface antigen protein 2 (Csa2) is a member of the Candida albicans Common in Fungal Extracellular Membranes (CFEM) protein superfamily. We previously established its role in iron acquisition in C. albicans. However, the other roles of Csa2 remain unknown. Here, we compared growth, morphological transition, and biofilm formation among wild-type, Csa2-mutant, and complemented strains of C. albicans. Deletion of the Csa2 gene resulted in smaller and reduced colony growth, significant attenuation of the dimorphic transition under serum-inducing conditions, and reduced biofilm formation; complementation restored these levels to those of the wild-type. Our findings demonstrated that Csa2 participated in yeast-to-hyphae morphological switching under serum-inducing conditions and contributed to the biofilm formation of C. albicans. This work, therefore, provides novel insights into the potential roles of Csa2 in virulence of C. albicans.