Hiroki Iwai

A Bacterial Effector Targets Mad2L2, an APC Inhibitor, to Modulate Host Cell Cycling

The gut epithelium self-renews every several days, providing an important innate defense system that limits bacterial colonization. Nevertheless, many bacterial pathogens, including Shigella, efficiently colonize the intestinal epithelium. Here, we show that the Shigella effector IpaB, when delivered into epithelial cells, causes cell-cycle arrest by targeting Mad2L2, an anaphase-promoting complex/cyclosome (APC) inhibitor. Cyclin B1 ubiquitination assays revealed that APC undergoes unscheduled activation due to IpaB interaction with the APC inhibitor Mad2L2. Synchronized HeLa cells infected with Shigella failed to accumulate Cyclin B1, Cdc20, and Plk1, causing cell-cycle arrest at the G2/M phase in an IpaB/Mad2L2-dependent manner. IpaB/Mad2L2-dependent cell-cycle arrest by Shigella infection was also demonstrated in rabbit intestinal crypt progenitors, and the IpaB-mediated arrest contributed to efficient colonization of the host cells. These results strongly indicate that Shigella employ special tactics to influence epithelial renewal in order to promote bacterial colonization of intestinal epithelium.

A novel caspase-1/Toll-like receptor 4-independent pathway of cell death induced by cytosolic Shigella in infected macrophages

Shigella-induced macrophage cell death is an important step in the induction of acute inflammatory responses that ultimately lead to bacillary dysentery. Cell death was previously reported to be dependent upon the activation of caspase-1 via interaction with IpaB secreted by intracellular Shigella, but in this study, we show that Shigella infection of macrophages can also induce cell death independent of caspase-1 or IpaB activity. Time-lapse imaging and electron microscopic analyses indicated that caspase-1-dependent and -independent cell death is morphologically indistinguishable and that both resemble necrosis. Analyses of Shigella mutants or Escherichia coli using co-infection with Listeria suggested that a component common to Gram-negative bacteria is involved in inducing caspase-1-independent cell death. Further studies revealed that translocation of bacterial lipid A into the cytosol of macrophages potentially mediates cell death. Notably, cell death induced by cytosolic bacteria was TLR4-independent. These results identify a novel cell death pathway induced by intracellular Gram-negative bacteria that may play a role in microbial-host interactions and inflammatory responses.

High Vaccine Efficacy against Shigellosis of Recombinant Noninvasive Shigella Mutant That Expresses Yersinia Invasin

Live attenuated Shigella vaccines elicit protective immune responses, but involve a potential risk of inducing a strong inflammatory reaction. The bacterial invasiveness that is crucial for Ag delivery causes inflammatory destruction of infected epithelial cells and proinflammatory cell death of infected macrophages. In this study, the noninvasive Shigella mutant ΔipaB was equipped with Yersinia invasin protein, which has been shown to mediate bacterial invasion and targeting to M cells located in follicle-associated epithelium. Invasin-expressing ΔipaB (ΔipaB/inv) was internalized into epithelial cells and retained in the intraphagosomal space. ΔipaB/inv did not induce necrotic cell death of infected macrophages nor cause symptomatic damage after intranasal vaccination of mice. ΔipaB/inv was safer and more effective than the conventional live vaccine, ΔvirG. Infection by ΔipaB/inv caused polymorphonuclear neutrophil infiltration in the lung, but did not induce production of large amounts of proinflammatory cytokines. We concluded that the low experimental morbidity and high vaccine efficacy of ΔipaB/inv are primarily based on high protective immune responses, which may be enhanced by the polymorphonuclear neutrophil infiltration unaccompanied by tissue injury.

Complete Annotated Genome Sequence of Mycobacterium tuberculosis Erdman

We report the completely annotated genome sequence of Mycobacterium tuberculosis Erdman (TMC 107; ATCC 35801), which is a well-known laboratory strain of M. tuberculosis.

Complete annotated genome sequence of Mycobacterium tuberculosis (Zopf) Lehmann and Neumann (ATCC35812) (Kurono)

We report the completely annotated genome sequence of Mycobacterium tuberculosis (Zopf) Lehmann and Neumann (ATCC35812) (Kurono), which is a used for virulence and/or immunization studies. The complete genome sequence of M. tuberculosis Kurono was determined with a length of 4,415,078 bp and a G+C content of 65.60%. The chromosome was shown to contain a total of 4,340 protein-coding genes, 53 tRNA genes, one transfer messenger RNA for all amino acids, and 1 rrn operon. Lineage analysis based on large sequence polymorphisms indicated that M. tuberculosis Kurono belongs to the Euro-American lineage (lineage 4). Phylogenetic analysis using whole genome sequences of M. tuberculosis Kurono in addition to 22 M. tuberculosis complex strains indicated that H37Rv is the closest relative of Kurono based on the results of phylogenetic analysis. These findings provide a basis for research using M. tuberculosis Kurono, especially in animal models.

Peroxiredoxin 1 Contributes to Host Defenses against Mycobacterium tuberculosis

Peroxiredoxin (PRDX)1 is an antioxidant that detoxifies hydrogen peroxide and peroxinitrite. Compared with wild-type (WT) mice, Prdx1-deficient (Prdx1−/−) mice showed increased susceptibility to Mycobacterium tuberculosis and lower levels of IFN-γ and IFN-γ–producing CD4+ T cells in the lungs after M. tuberculosis infection. IL-12 production, c-Rel induction, and p38 MAPK activation levels were lower in Prdx1−/− than in WT bone marrow–derived macrophages (BMDMs). IFN-γ–activated Prdx1−/− BMDMs did not kill M. tubercuosis effectively. NO production levels were lower, and arginase activity and arginase 1 (Arg1) expression levels were higher, in IFN-γ–activated Prdx1−/− than in WT BMDMs after M. tuberculosis infection. An arginase inhibitor, Nω-hydroxy-nor-arginine, restored antimicrobial activity and NO production in IFN-γ–activated Prdx1−/− BMDMs after M. tuberculosis infection. These results suggest that PRDX1 contributes to host defenses against M. tuberculosis. PRDX1 positively regulates IL-12 production by inducing c-Rel and activating p38 MAPK, and it positively regulates NO production by suppressing Arg1 expression in macrophages infected with M. tuberculosis.

A Mutation in the 16S rRNA Decoding Region Attenuates the Virulence of Mycobacterium tuberculosis

ABSTRACT Mycobacterium tuberculosis contains a single rRNA operon that encodes targets for antituberculosis agents, including kanamycin. To date, only four mutations in the kanamycin binding sites of 16S rRNA have been reported in kanamycin-resistant clinical isolates. We hypothesized that another mutation(s) in the region may dramatically decrease M. tuberculosis viability and virulence. Here, we describe an rRNA mutation, U1406A, which was generated in vitro and confers resistance to kanamycin while highly attenuating M. tuberculosis virulence. The mutant showed decreased expression of 20% (n = 361) of mycobacterial proteins, including central metabolic enzymes, mycolic acid biosynthesis enzymes, and virulence factors such as antigen 85 complexes and ESAT-6. The mutation also induced three proteins, including KsgA (Rv1010; 16S rRNA adenine dimethyltransferase), which closely bind to the U1406A mutation site on the ribosome; these proteins were associated with ribosome maturation and translation initiation processes. The mutant showed an increase in 17S rRNA (precursor 16S rRNA) and a decrease in the ratio of 30S subunits to the 70S ribosomes, suggesting that the U1406A mutation in 16S rRNA attenuated M. tuberculosis virulence by affecting these processes.

LPS-dependent changes in the expression of 57 kDa and 53 kDa cell membrane proteins without participation of CD14

It is widely presumed that in addition to CD14, other molecules are necessary for lipopolysaccharide (LPS)-induced cell activation. In order to shed light on some of the biological and biochemical properties of these molecules, we examined the LPS responsiveness of CD14-negative, ST2 cells. Although ST2 cells do not express CD14 mRNA, they, nonetheless, expressed IL-6 mRNA and synthesized IL-6 protein when incubated with LPS in serum-free medium (i.e. without soluble CD14). Paxlitacel (Taxol™) also induced IL-6 mRNA expression in ST2 cells, while Rhodobacter sphaeroides diphoshoryl lipid A (RsDPLA) inhibited both LPS- and Taxol-induced expression of IL-6 mRNA. Collectively, these data suggest that LPS, RsDPLA, and Taxol all recognize the same receptor complex on ST2 cells and do not require the participation of CD14. In addition, using antibody raised against the ST2 cell membrane fraction, we detected a set of LPS-specific membrane antigens in murine peritoneal macrophages, including two designated p57 (57 kDa) and p53 (53 kDa). There was no qualitative difference in the expression of p57 and p53 in LPS-responsive, C3H/HeN and LPS-hyporesponsive, C3H/HeJ macrophages. However, after stimulating the macrophages with LPS or Taxol, expression of p57 and p53 was diminished in C3H/HeN macrophages, but not in C3H/HeJ macrophages. Phorbol ester (PMA) and A23187 calcium ionophore did not suppress p57 or p53 expression, and the lipid A precursor, PE406, did not bind to either protein. Thus, p57 and p53 may play important roles in LPS-evoked responses, but they do not appear to serve as LPS receptors.

Genetic diversity of Mycobacterium tuberculosis isolates from Tochigi prefecture, a local region of Japan

BackgroundForeign-born patients with tuberculosis (TB) may introduce globally disseminated isolates of Mycobacterium tuberculosis into large cities in Japan. The risk of dissemination of these isolates into local regions, however, has not been determined. This study analyzed the molecular epidemiology of M. tuberculosis isolates obtained from TB patients living in a local region of Japan.MethodsWhole genome sequences of 169 M. tuberculosis isolates, obtained from 148 Japanese-born and 21 foreign-born patients living in Tochigi, Japan, were analyzed using the Comprehensive analysis server for the Mycobacteriumtuberculosis complex (CASTB).ResultsThe 169 isolates were clustered into four clades; Lineage 2 (111 isolates 65.7%), Lineage 4 (43 isolates, 25.4%), Lineage 1 (13 isolates, 7.7%), and Lineage 3 (2 isolates, 1.2%). Of the 111 isolates belonging to Lineage 2, 79 (71.2%) were of the atypical Beijing sub-genotype. Of the 13 Lineage 1 isolates, nine (69.2%) were from foreign-born patients. The isolates belonging to Lineage 4 were further clustered into three clades, two containing isolates shared by both Japanese- and foreign-born patients. The two isolates belonging to Lineage 3 were obtained from foreign-born patients.ConclusionsThe genotypic diversity of M. tuberculosis in a local region of Japan is increased primarily by the presence of isolates obtained from foreign-born patients.

Protective effect of OK-432 on mice against endotoxemia and infection with Pseudomonas aeruginosa and Salmonella enteritidis

OK‐432 has been used clinically as a biological response modifier for cancer therapy. We investigated here the protective effects of OK‐432 against endotoxic shock and infectious death caused by Pseudomonas aeruginosa and Salmonella enteritidis in mice and proposed a possible mechanism. Pre‐treatment of OK‐432 reduced the lethality of lipopolysaccharide (LPS)‐induced endotoxic shock in d‐(+)‐galactosamine‐sensitized C3H/HeN mice. OK‐432 did not affect the TNFα production in blood, but it did decrease the susceptibility to TNFα. Furthermore, an acceleration of LPS clearance from blood was detected. The pretreatment of OK‐432 also decreased the lethality of mice in bacterial infection caused by P. aeruginosa and S. enteritidis. The rapid decrease of the viable bacteria from the circulating blood and in spleen and liver in mice was observed in a manner similar to LPS clearance. These findings indicate that the protective effect of OK‐432 against the endotoxemia and bacteremia may depend on an up‐regulation of clearance of LPS and bacteria and the augmented resistance to TNFα.