Type III secretion machinery (TTSM), composed of a needle, a basal body, and a C-ring compartment, delivers a subset of effectors into host cells. Here, we show that Shigella Spa33 is an essential component of the C-ring compartment involved in mediating the transit of various TTSM-associated translocated proteins. Electron microscopic analysis and pull-down assay revealed Spa33 to be localized beneath the TTSM via interaction with MxiG and MxiJ (basal body components). Spa33 is also capable of interacting with Spa47 (TTSM ATPase), MxiK, MxiN (required for the transit of MxiH, the needle component), Spa32 (required for determining needle length), and several effectors. Genetic and functional analyses of the Spa33 C-terminal region, which is highly conserved in the SpaO-YscQ-HrcQ B -FliN family, indicate that some of the conserved residues are crucial for needle formation via interactions with MxiN. Thus, Spa33 plays a central role as the C-ring component in recruiting/exporting TTSM-associated proteins.Type III secretion machinery (TTSM) 2 is found in many Gram-negative bacteria and plays a central role in mediating the translocation of virulence proteins (effectors) into host cells. TTSM consists of three major components: a needle portion, a basal body and a C-ring compartment, and the entire complex is needed to provide a conduit for direct translocation of effectors from the bacterial cytoplasm into host cells (1-3). Structural studies of the TTSM of Salmonella and Shigella indicate that the basal body resembles the flagella basal body, which consists of two upper and two lower rings (4 -8). The compartment called the C-ring, assembled beneath the flagella basal body, is also present beneath the TTSM basal body. However, its relevant component and exact role in protein translocation via the TTSM remain largely speculative (9 -12). The needle portion of the Shigella TTSM is composed of MxiH and MxiI, and the basal body is composed of MxiD, MxiG, and MxiJ (8,13). Genetic and functional analyses indicate that some of the TTSM-associated proteins such as MxiK, MxiN, Spa32, and Spa47 are involved in forming the functional needle, where MxiN is thought to be required for translocation of MxiH, the major needle component, through the basal body (14 -19). The C-ring compartment of TTSM is assumed to be involved in recognition of the secreted signals carried by effectors and to be responsible for the promiscuous character of the TTSM in regards to heterologous secreted proteins. However, the mechanisms underlying the recognition and export of proteins via the TTSM components remain essentially unknown.Previous studies have indicated that Pseudomonas syringae HrcQ B is located on the cytoplasmic side of the TTSM. HrcQ B shares partial amino acid sequence similarity with FliN (a flagella C-ring component), Shigella Spa33, Salmonella SpaO, and Yersinia YscQ (12,20,21). We therefore sought to characterize the role of Spa33 in mediating the translocation of Shigella effectors via the TTSM. Our results provide the first...

Each set contained six to nine primer pairs that amplify products of markedly different sizes. This genetic methodology (E. coli O-genotyping PCR) allowed for comprehensive, rapid, and low-cost typing. Validation of the PCR system using O-serogroup references and wild strains showed that the correct O serogroups were specifically and accurately identified for 100% (182/182) and 90.8% (522/575) of references and wild strains, respectively. The PCR-based system reported here might be a promising tool for the subtyping of E. coli strains for epidemiological studies as well as for the surveillance of pathogenic E. coli during outbreaks.

To analyze the co-occurrence of virulence genes among bovine and human commensal E. coli strains and visualize it in the network interface, we constructed a pairwise co-occurrence matrix for each gene (Supplemental Table S8). Only one co-occurrence

EHEC O157:H7 clade 6 strains harboring stx2a and/or stx2c and clade 8 strains harboring stx2a or stx2a/stx2c were frequently associated with childhood HUS cases in Japan. Rapid and specific detection of such lineages are required for infection control measures.

In 2009, the first high-level ceftriaxone-resistant Neisseria gonorrhoeae strain (H041) was isolated in Kyoto, Japan. The present study describes an intensified surveillance (antimicrobial resistance and molecular typing) of Neisseria gonorrhoeae isolates in Kyoto and its neighboring prefecture Osaka, Japan, in 2010 to 2012, which was initiated after the identification of H041. From April 2010 to March 2012, 193 N. gonorrhoeae isolates were collected and the MICs (g/ml) to six antimicrobials, including ceftriaxone, were determined. All isolates showed susceptibility to ceftriaxone and cefixime (MIC values, <0.5 g/ml), and spectinomycin. The rates of resistance (intermediate susceptibility) to azithromycin, penicillin G, and ciprofloxacin were 3.6% (19.7%), 24.4% (71.0%), and 78.2% (0.5%), respectively. Multilocus sequence typing (MLST) showed that 40.9%, 19.2%, and 17.1% of isolates belonged to ST1901, ST7359, and ST7363, respectively. Furthermore, N. gonorrhoeae multiantigen sequence typing (NG-MAST) revealed that 12 (63%) of the 19 isolates with decreased susceptibility to ceftriaxone (MIC > 0.064 g/ml) were of ST1407. NG-MAST ST1407 was also the most prevalent ST (16.1%; 31 of 193 isolates). In those NG-MAST ST1407 strains, several mosaic type penA alleles were found, including SF-A type (penicillin binding protein 2 allele XXXIV) and its derivatives. These were confirmed using transformation of the penA mosaic alleles as critical determinants for enhanced cefixime and ceftriaxone MICs. The intensified surveillance in Kyoto and Osaka, Japan, did not identify any dissemination of the highlevel ceftriaxone-resistant N. gonorrhoeae strain H041, suggesting that H041 might have caused only a sporadic case and has not spread further.