Masaru Ohara

Identification of the Staphylococcus aureus etd pathogenicity island which encodes a novel exfoliative toxin, ETD, and EDIN-B

ABSTRACT We identified a novel pathogenicity island in Staphylococcus aureus which contains open reading frames (ORFs) similar to the exfoliative toxin (ET) gene, glutamyl endopeptidase gene, and edin-B gene in tandem and the phage resistance gene, flanked by hsdM, hsdS (restriction and modification system), and IS256. The protein encoded by the ET-like gene showed 40, 59, and 68% amino acid sequence identities with exfoliative toxin A (ETA), exfoliative toxin B (ETB), and Staphylococcus hyicus ETB (ShETB), respectively. When injected into neonatal mice, the recombinant protein derived from the ET-like gene induced exfoliation of the skin with loss of cell-to-cell adhesion in the upper part of the epidermis as observed in histological examinations, just as was found in neonatal mice injected with ETA or ETB. Western blot analysis indicated that the recombinant protein is serologically distinct from ETA and ETB. Therefore, the product encoded by this new ORF is a new ET member produced by S. aureus and is termed ETD. ETD did not induce blisters in 1-day-old chickens. In the skins of mice injected with ETD, cell surface staining of desmoglein 1 (Dsg1), a cadherin type cell-to-cell adhesion molecule in desmosomes, was abolished without affecting that of desmoglein 3 (Dsg3). Furthermore, in vitro incubation of the recombinant extracellular domains of Dsg1 and Dsg3 with the recombinant protein demonstrated that both mouse and human Dsg1, but not Dsg3, were directly cleaved in a dose-dependent manner. These results demonstrate that ETD and ETA induce blister formation by identical pathophysiological mechanisms. Clinical strains positive for edin-B were suggested to be clonally associated, and all edin-B-positive strains tested were positive for etd. Among 18 etd-positive strains, 12 produced ETD extracellularly. Interestingly, these strains are mainly isolated from other sources of infections and not from patients with bullous impetigo or staphylococcal scalded-skin syndrome. This strongly suggests that ETD might play a pathogenic role in a broader spectrum of bacterial infections than previously considered.

Enteropathogenic and enterohaemorrhagic Escherichia coli deliver a novel effector called Cif, which blocks cell cycle G2/M transition.

Enteropathogenic Escherichia coli (EPEC) and enterohaemorrhagic E. coli (EHEC) are closely related pathogens. Both use a type III secretion system (TTSS) encoded by the ‘locus of enterocyte effacement’ (LEE) to subvert and attach to epithelial cells through the injection of a repertoire of effector molecules. Here, we report the identification of a new TTSS translocated effector molecule called Cif, which blocks cell cycle G2/M transition and induces the formation of stress fibres through the recruitment of focal adhesions. Cif is not encoded by the LEE but by a lambdoid prophage present in EPEC and EHEC. A cif mutant causes localized effacement of microvilli and intimately attaches to the host cell surface, but is defective in the ability to block mitosis. When expressed in TTSS competent LEE‐positive pathogens, Cif is injected into the infected epithelial cells. These cells arrested at the G2/M phase displayed accumulation of inactive phosphorylated Cdk1. In conclusion, Cif is a new member of a growing family of bacterial cyclomodulins that subvert the host eukaryotic cell cycle.

Complete Nucleotide Sequence of a Staphylococcus aureus Exfoliative Toxin B Plasmid and Identification of a Novel ADP-Ribosyltransferase, EDIN-C

ABSTRACT The complete nucleotide sequence of pETB, a 38.2-kbStaphylococcus aureus plasmid encoding the exfoliative toxin B (ETB), was determined. A total of 50 open reading frames were identified on the plasmid genome and, among these, 32 showed sequence similarity to known proteins. pETB contains three copies of IS257, which divide the pETB genome into three regions: (i) a cadmium resistance operon-containing region, (ii) a lantibiotic production gene-containing region, and (iii) the remaining part where genes for plasmid replication and/or maintenance are dispersed. In the third region, genes of various kinds of functions are present among the replication- and maintenance-related genes. They include two virulence-related genes, the etb gene and a gene encoding a novel ADP-ribosyltransferase closely related to EDIN, which belongs to the C3 family of ADP-ribosyltransferases modifying Rho GTPases. They also include genes for a cell wall-anchoring surface protein and a phage resistance protein. Based on the determined sequence of pETB, the genome structures of etb-bearing plasmids (ETB plasmids) from various clinical isolates were analyzed by the PCR scanning method. The data indicate that, although the ETB plasmids are highly heterogeneous in genome size, the fundamental genome organization is well conserved. The size variation of the plasmid is mainly attributed to defined regions which may be hot spots for gene shuffling.

An N-terminal Segment of the Active Component of the Bacterial Genotoxin Cytolethal Distending Toxin B (CDTB) Directs CDTB into the Nucleus

Cytolethal distending toxin (CDT), produced by Actinobacillus actinomycetemcomitans, is a putative virulence factor in the pathogenesis of periodontal diseases. It is a cell cycle specific inhibitor at the G2/M transition. CDTB, one of the subunits of the CDT holotoxin, is implicated in a genotoxic role after entering the target cells, whereby chromosomal damage induces checkpoint phosphorylation cascades. CDTB microinjected into the cytoplasm was shown to localize in the nucleus and induce chromatin collapse. To investigate the molecular mechanism involved in nuclear transport of CDTB, we used transient expression and microinjection of a CDTB-green fluorescent protein (GFP) fusion protein. After microinjection, His-tagged CDTB-GFP entered the nucleus in 3–4 h. Leptomycin B did not increase the speed of entry of the fusion protein, suggesting that the relatively slow entry of the fusion protein is not due to the CRM1-dependent nuclear export of the protein. Nuclear localization of the CDTBGFP was temperature-dependent. An in vitro transport assay demonstrated that the nuclear localization of CDTB is mediated by active transport. An assay using transient expression of a series of truncated CDTB-GFP fusion proteins revealed that residues 48–124 constitute the minimum region involved in nuclear transport of CDTB. A domain swapping experiment of the region involved in nuclear transport of CDTB with an SV40 T nuclear localization signal indicated that CDTB is composed of two domains, an N-terminal domain for nuclear transport and a C-terminal active domain. Our results strongly suggest that nuclear localization of CDTB is required for the holotoxin to induce cytodistension and cell cycle block. This is the first demonstration that a bacterial toxin possessing a unique domain for nuclear transport is transferred to the animal cell nucleus by active transport.

Clonal association of Staphylococcus aureus causing bullous impetigo and the emergence of new methicillin-resistant clonal groups in Kansai district in Japan

A molecular epidemiological analysis was performed to reveal the clonal association of Staphylococcus aureus strains isolated from patients with bullous impetigo. Pulsed-field gel electrophoresis with cluster analysis, genetic and phenotypic characterizations, and antimicrobial susceptibility profiling of 88 S. aureus strains isolated from outpatients at 4 hospitals in the Kansai district in Japan were undertaken. Three distinct clonal groups were identified: 2 of them carried the exfoliative toxin (ET) A gene (eta), and the other carried the ETB gene (etb). The former groups represent 2 eta-positive clonal groups that have not been described previously. All the strains in the more dominant eta-positive clonal group and some of the strains in the etb-positive clonal group were methicillin-resistant S. aureus (MRSA) showing borderline-to-moderate resistance to beta-lactams. These MRSA strains appear to be emerging clonal groups that have not been considered in previous epidemiological studies of ET-producing S. aureus in Japan and thus pose a significant threat for future treatment of patients with bullous impetigo and/or staphylococcal scalded-skin syndrome.

Increased prevalence and clonal dissemination of multidrug-resistant Pseudomonas aeruginosa with the blaIMP-1 gene cassette in Hiroshima

OBJECTIVES The aim of this study was to evaluate the dissemination of metallo-beta-lactamase (MBL)-encoding genes among multidrug-resistant (MDR) Pseudomonas aeruginosa isolates recovered from major hospitals in the Hiroshima region. METHODS During July to December from 2004 to 2006, a surveillance of eight major hospitals in the Hiroshima region identified 387 non-duplicate isolates resistant to imipenem (MIC >or= 16 mg/L). They were screened for resistance to amikacin (MIC >or= 64 mg/L) and ciprofloxacin (MIC >or= 4 mg/L) and MBL-encoding genes. The structure of the variable regions of the integrons was determined using PCR mapping. Clonality was assessed using PFGE and multilocus sequence typing (MLST). RESULTS The frequency of MBL-positive isolates in MDR P. aeruginosa isolates significantly increased from 42.3% in 2004 to 81.4% in 2006. Most of the MBL-positive isolates produced IMP-1 followed by VIM-2. The bla(IMP-1) and bla(VIM-2) genes were present in class 1 integrons. Characterization of the variable regions of the integron showed the presence of six different gene cassette arrays in bla(IMP-1) cassettes and a single array in bla(VIM-2) cassettes. The IMP-1 producers belonged to two clonal lineages using PFGE and MLST analyses and the integron variations correlated well with the clonal complexes. Among them, strains positive for a newly identified In113-derived bla(IMP-1) gene cassette array were most widely distributed in Hiroshima. CONCLUSIONS This study shows a dramatic increase in MBL genes, primarily bla(IMP-1), in MDR P. aeruginosa isolates in Hiroshima during these 3 years. In addition, MDR P. aeruginosa with the newly discovered In113-derived bla(IMP-1) gene cassette array appears to be clonally expanding.

The gate controlling cell wall synthesis in Staphylococcus aureus

Glucosamine‐6‐P occupies a central position between cell wall synthesis and glycolysis. In the initial steps leading to peptidoglycan precursor formation glucosamine‐6‐P is processed sequentially to UDP‐N‐acetylglucosamine, while to enter the glycolysis pathway, glucosamine‐6‐P is isomerized by NagB to fructose‐6‐P. Although we could not demonstrate NagB activity, nagB inactivation significantly reduced growth. Mutational analysis showed that NagA was involved in glucosamine‐6‐P formation from N‐acetylglucosamine‐6‐P, and GlmS in that from fructose‐6‐P. Inactivation of glmS prevented growth on glucose as sole carbon source, which resumed after complementation with N‐acetylglucosamine. Transcription of glmS as well as the amount of GlmS was reduced in the presence of N‐acetylglucosamine. This and the preferential incorporation of N‐acetylglucosamine over glucose into cell wall material showed that N‐acetylglucosamine was used exclusively for cell wall synthesis, while glucose served both cell wall synthesis and glycolysis. These observations suggest furthermore GlmS to be the key and only enzyme leading from glucose to cell wall synthesis in Staphylococcus aureus, and show that there exists a tight regulation and hierarchy in sugar utilization. Inactivation of nagA, nagB or glmS affected the susceptibility of S. aureus to cell wall synthesis inhibitors, suggesting an interdependence between efficiency of cell wall precursor formation and resistance levels.

Molecular Characterization of an atl Null Mutant of Staphylococcus aureus

atl is a gene encoding a bifunctional peptidoglycan hydrolase of Staphylococcus aureus. The gene product of atl is a 138 kDa protein that has an amidase domain and a glucosaminidase domain, and undergoes processing to generate two major peptidoglycan hydrolases, a 51 kDa glucosaminidase and a 62 kDa amidase in culture supernatant. An atl null mutant was isolated by allelic replacement and characterized. The mutant grew in clusters and sedimented when grown in broth culture. Analysis of peptidoglycan prepared from the wild type and the mutant revealed that there were no differences in muropeptide composition or in glycan chain length distribution. On the other hand, the atl mutation resulted in pleiotropic effects on cell surface nature. The mutant cells showed complete inhibition of metabolic turnover of cell wall peptidoglycan and revealed a rough outer cell wall surface. The mutation also decreased the amount of protein non‐covalently bound to the cell surface and altered the protein profile, but did not affect proteins covalently associated with the cell wall. Lysis of growing cells treated with otherwise lytic concentration of penicillin G was completely inhibited in the mutant, but that of non‐growing cells was not affected by the mutation. The atl mutation did not significantly affect the ability of S. aureus to provoke an acute infection when inoculated intraperitoneally in a mouse sepsis model. These results further support the supposition that atl gene products are involved in cell separation, cell wall turnover and penicillin‐induced lysis of the cells.

Caspase-2 and caspase-7 are involved in cytolethal distending toxin-induced apoptosis in Jurkat and MOLT-4 T-cell lines

ABSTRACT Cytolethal distending toxin (CDT) from Actinobacillus actinomycetemcomitans is a G2/M cell-cycle-specific growth-inhibitory toxin that leads to target cell distension followed by cell death. To determine the mechanisms by which A. actinomycetemcomitans CDT acts as an immunosuppressive factor, we examined the effects of highly purified CDT holotoxin on human T lymphocytes. Purified CDT was cytolethal toward normal peripheral T lymphocytes that were activated by in vitro stimulation with phytohemagglutinin. In addition, purified CDT showed cytolethal activity against Jurkat and MOLT-4 cells, which are known to be sensitive and resistant, respectively, to Fas-mediated apoptosis. Death in these cell lines was accompanied by the biochemical features of apoptosis, including membrane conformational changes, intranucleosomal DNA cleavage, and an increase in caspase activity in the cells. Pretreatment of Jurkat cells with the general caspase inhibitor z-VAD-fmk mostly suppressed CDT-induced apoptosis. Furthermore, specific inhibitors of caspase-2 and -7 showed significant inhibitory effects on CDT-induced apoptosis in Jurkat cells, and these inhibitory effects were fully associated with reduced activity of caspase-2 or -7 in the CDT-treated Jurkat cells. These results strongly suggest that CDT possesses the ability to induce human T-cell apoptosis through activation of caspase-2 and -7.

Identification of six major outer membrane proteins from Actinobacillus actinomycetemcomitans

We have identified six major sarcosyl-insoluble outer membrane proteins (Omp) of Actinobacillus actinomycetemcomitans Y4, and designated them as Omp100, Omp64, Omp39, Omp29, Omp18 and Omp16 according to the molecular mass. A similar N-terminal sequence was found in the first 15 amino acid residues of Omp16 and Omp18. The N-terminal sequence of Omp29 matched perfectly with the sequence previously identified. We cloned and determined the DNA sequences of three complete genes encoding Omp100, Omp64 and Omp18/16, and one incomplete gene encoding Omp39. Each Omp revealed homologies with some bacterial virulence factors responsible for adhesion, invasion, serum resistance, or protein antigenicity. Serum from patients with periodontitis suspected to be related to A. actinomycetemcomintans infection strongly reacted with Omp100, Omp29 and Omp16 as did serum from mice immunized with A. actinomycetemcomitans Y4 whole bacteria. These findings suggest that Omps of A. actinomycetemcomitans can be associated with periodontal disease.