Aki Kaneko

PROTECTION AGAINST SHIGA TOXIN 1 CHALLENGE BY IMMUNIZATION OF MICE WITH PURIFIED MUTANT SHIGA TOXIN 1

ABSTRACT Shiga toxin 1 (Stx1) of enterohemorrhagic Escherichia coli O157:H7 was cloned, and four mutant Stx1s were constructed by site-directed mutagenesis with PCR. The wild-type and mutant Stx1s with amino acid replacements at positions 167 and 170 of the A subunit were purified by one-step affinity chromatography with commercially available Globotriose Fractogel, and the mutant Stxs were used for the immunization of mice. The mutant toxins were nontoxic to Vero cells in vitro and to mice in vivo and induced the immunoglobulin G antibody against the wild-type Stx1, which neutralized the cytotoxicity of Stx1. The induced antibody titers depended on the mutation at position 170 of the A subunit. The mice immunized with the mutant Stx1s were protected against a challenge of approximately 100 times the 50% lethal dose of the wild-type Stx1, suggesting that the mutant toxins are good candidates for toxoid vaccines for infection by Stx1-producing E. coli.

Deletion of the CaBIG1 Gene Reduces β-1,6-Glucan Synthesis, Filamentation, Adhesion, and Virulence in Candida albicans

ABSTRACT The human fungal pathogen Candida albicans is able to change its shape in response to various environmental signals. We analyzed the C. albicans BIG1 homolog, which might be involved in β-1,6-glucan biosynthesis in Saccharomyces cerevisiae. C. albicans BIG1 is a functional homolog of an S. cerevisiae BIG1 gene, because the slow growth of an S. cerevisiae big1 mutant was restored by introduction of C. albicans BIG1. CaBig1p was expressed constitutively in both the yeast and hyphal forms. A specific localization of CaBig1p at the endoplasmic reticulum or plasma membrane similar to the subcellular localization of S. cerevisiae Big1p was observed in yeast form. The content of β-1,6-glucan in the cell wall was decreased in the Cabig1Δ strain in comparison with the wild-type or reconstituted strain. The C. albicans BIG1 disruptant showed reduced filamentation on a solid agar medium and in a liquid medium. The Cabig1Δ mutant showed markedly attenuated virulence in a mouse model of systemic candidiasis. Adherence to human epithelial HeLa cells and fungal burden in kidneys of infected mice were reduced in the Cabig1Δ mutant. Deletion of CaBIG1 abolished hyphal growth and invasiveness in the kidneys of infected mice. Our results indicate that adhesion failure and morphological abnormality contribute to the attenuated virulence of the Cabig1Δ mutant.

Association of a Regulatory Gene, slyA with a Mouse Virulence of Salmonella Serovar Choleraesuis

The influence of slyA gene, originally found in Salmonella serovar Typhimurium as a regulatory gene for the expression of virulence genes, on a mouse virulence of S. serovar Choleraesuis was investigated by using an slyA‐defective mutant. The defective mutant was constructed by the insertion of a kanamycin‐resistance gene (aph) into the cloned slyA gene, and the homologous recombination with the intact slyA gene on the chromosome. The mutant strain showed the LD50 value for BALB/c mouse approximately 105 higher than that of the parent strain. The increase of the LD50 value was the same order as that shown by the mutation of the slyA gene of S. serovar Typhimurium, although LD50 of the wild‐type strain of S. serovar Choleraesuis was 40‐fold higher than that of S. serovar Typhimurium. The time course of infection observed in the mice organs also proved the clear difference of the virulence between the parent and the mutant strains. These results suggested that the slyA gene product functions as a virulence‐associated regulator also in S. serovar Choleraesuis.

TTG as the Initiation Codon of Salmonella slyA, a Gene Required for Survival within Macrophages

The slyA gene, which has been implicated in the virulence of Salmonella serovar Typhimurium and its survival in macrophages, is widely distributed among different Salmonella serovars. In this study, we cloned and sequenced the translational initiation region of the slyA gene from nine different serovars and found sequence differences in the previously proposed ATG initiation codon but not in a TTG triplet, another putative initiation codon in the slyA gene. Therefore, we determined the actual translational initiation site of the slyA gene by analyzing slyA genes with defined mutation in either the ATG or TTG sequences in an in vitro translation assay and a quantitative hemolytic assay in Escherichia coli. The replacement of TTG by TTC in the slyA gene significantly reduced both the amount of protein synthesized and the hemolytic activity of a transformed strain of E. coli, while replacement of ATG by ATC had no effect in these assays. In addition, the amino acid sequence analysis of the His‐tagged SlyA protein showed that it was identical with the amino acid sequence deduced from the 5′ end of the slyA gene with a TTG initiation codon. Our results suggest that TTG serves as the translational initiation codon for the slyA gene of Salmonella.

Molecular heterogeneity of heat-stable toxin-producing and non-producing Escherichia coli O25 strains isolated in Japan.

Escherichia coli O25 strains that produce heat‐stable toxin (ST) have been recently isolated in Japan, and epidemiological study of this type of enterotoxigenic E. coli is required. In this study the heterogeneity of 16 ST‐producing and non‐producing strains of E. coli O25 was investigated. All eight ST‐producing strains were shown to have STIb gene, and seven of them had similar profiles of plasmids, ladder‐banding of LPS in SDS‐polyacrylamide gel electrophoresis, and chromosomal DNA digestions in pulsed‐field gel electrophoresis (PFGE). In contrast, ST‐non‐producing strains were more heterogeneous in all parameters examined. PFGE of the digested chromosomal DNA with several restriction enzymes was proved to be an effective procedure to compare the closely related strains of E. coli O25.

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.