Phil Youl Ryu

Regulation of Vibrio vulnificus virulence by the LuxS quorum-sensing system

Vibrio vulnificus is a halophilic estuarine bacterium that causes fatal septicaemia and necrotizing wound infections. We tested whether V. vulnificus produces signalling molecules (autoinducer 1 and/or 2) stimulating Vibrio harveyi quorum‐sensing system 1 and/or 2. Although there was no evidence for signalling system 1, we found that V. vulnificus produced a signalling activity in the culture supernatant that induced luminescence expression in V. harveyi through signalling system 2. Maximal autoinducer 2 (AI‐2) activity was observed during mid‐exponential to early stationary phase and disappeared in the late stationary phase when V. vulnificus was grown in heart infusion broth containing 2.5% NaCl. V. vulnificus showed increased signalling activity when it was cultured in the presence of glucose (0.5%) and at low pH (pH 6.0). From a cosmid library of V. vulnificus type strain ATCC 29307, we have identified the AI‐2 synthase gene (luxSVv) showing 80% identity with that of V. harveyi (luxSVh) at the amino acid level. To investigate the pathogenic role of luxSVv, a deletion mutant of the clinical isolate V. vulnificus MO6‐24/O was constructed. The luxSVv mutant showed a significant delay in protease production and an increase in haemolysin production. The decreased protease and increased haemolysin activities were restored to the isogenic wild‐type level by complementation with the wild‐type luxSVv allele. The change in phenotypes was also complemented by logarithmic phase spent media produced by the wild‐type bacteria. Transcriptional activities of the haemolysin gene (vvhA) and protease gene (vvpE) were also observed in the mutant using chromosomal PvvhA::lacZ and PvvpE::lacZ transcriptional reporter constructs: transcription of vvhA was increased and of vvpE decreased by the mutation. The mutation resulted in an attenuation of lethality to mice. Intraperitoneal LD50 of the luxSVv mutant increased by 10‐ and 750‐fold in ferric ammonium citrate‐non‐overloaded and ferric ammonium citrate‐overloaded mice respectively. The time required for the death of mice was also significantly delayed in the luxSVv mutant. Cytotoxic activity of the organism against HeLa cells, measured by lactate dehydrogenase (LDH) release assay, was also decreased significantly by the mutation. Taken together, the V. vulnificus LuxS quorum‐sensing system seems to play an important role in co‐ordinating the expression of virulence factors.

Immunological responses induced by asd and wzy/asd mutant strains of Salmonella enterica serovar Typhimurium in BALB/c mice

Attenuated bacteria have long been developed as vaccine candidates but can have some disadvantages, such as the potential for damage to immune organs due to insufficient clearance. To minimize these disadvantages, we generated Salmonella enterica serovar Typhimurium mutants SHJ2104 (asd::cm) and HTSaYA (wzy::km, asd::cm). The wzy gene codes for the O-antigen polymerase, which is involved in lipopolysaccharide (LPS) biosynthesis, and asd codes for aspartate β-semialdehyde dehydrogenase, which participates in cell wall formation. The strains synthesized LPS with a short-chain length, and showed lower cytotoxicity and reduced intracellular proliferation in animal cells compared to wild-type bacteria. After oral infection, the mutants were cleared in immune tissues, including the Peyer’s patch, mesenteric lymph node, and spleen, within 5 days. The LD50 of the mutants in Balb/c mice was estimated to be 106 higher than wild-type bacteria when administered either via an oral or i.p. route, indicating that the two strains are highly attenuated. To compare the immune response to and protective effects of the mutants against wild-type bacterial infection, we inoculated the mutants into mice via an oral (1×1010CFU) or i.p. (1×107 CFU) route once or twice at a two week interval. All immune responses, such as serum IgG and secretory IgA levels, cytokine production, and delayed hypersensitivity, were highly induced by two rounds of immunization. HTSaYA and SHJ2104 induced similar immune responses, and mice immunized with HTSaYA or SHJ2104 via an i.p. route were protected against wild-type Salmonella infection even at 100-fold of the LD50 (5×106 CFU). Taken together, these data indicate that HTSaYA and SHJ2104 could be developed as live attenuated Salmonella vaccine candidates.

Molecular cloning and characterization of clyA genes in various serotypes of Salmonella enterica

Cytolysin A (ClyA) is a pore-forming hemolytic protein encoded by the clyA gene. It has been identified in Salmonella enterica serovars Typhi and Paratyphi A. To identify and characterize the clyA genes in various Salmonella enterica strains, 21 different serotypes of strains isolated from clinical specimens were presently examined. Full-length clyA genes were found in S. enterica serovar Brandenburg, Indiana, Panama, and Schwarzengrund strains by polymerase chain reaction amplification. The ClyA proteins from these four strains showed >97% amino acid identity to that of S. enterica serovar Typhi. Although all four serovars expressed detectable levels of ClyA as determined by Western blot analysis, they did not show a strong hemolytic effect on blood agar, indicating that ClyA may not be efficiently expressed or secreted. Escherichia coli transformed with clyA genes from the four serovars enhanced production of ClyA proteins and hemolytic activities to a level similar to S. enterica serovar Typhi ClyA. The present results suggest that ClyA may play a role in the pathogenesis of S. enterica serovar Brandenburg, Indiana, Panama and Schwarzengrund.

In vivo monitoring of CD44+ cancer stem-like cells by γ-irradiation in breast cancer

There is increasing evidence that cancer contains cancer stem cells (CSCs) that are capable of regenerating a tumor following chemotherapy or radiotherapy. CD44 and CD133 are used to identify CSCs. This study investigated non-invasive in vivo monitoring of CD44-positive cancer stem-like cells in breast cancer by γ-irradiation using molecular image by fusing the firefly luciferase (fLuc) gene with the CD44 promoter. We generated a breast cancer cell line stably expressing fLuc gene by use of recombinant lentiviral vector controlled by CD44 promoter (MCF7-CL). Irradiated MCF7-CL spheres showed upregulated expression of CD44 and CD133, by immunofluorescence and flow cytometry. Also, gene expression levels of CSCs markers in irradiated spheres were clearly increased. CD44+ CSCs increased fLuc expression and tumor growth in vivo and in vitro. When MCF7-CL was treated with siCD44 and irradiated, CD44 expression was inhibited and cell survival ratio was decreased. MCF7-CL subsets were injected into the mice and irradiated by using a cobalt-60 source. Then, in vivo monitoring was performed to observe the bioluminescence imaging (BLI). When breast cancer was irradiated, relative BLI signal was increased, but tumor volume was decreased compared to non-irradiated tumor. These results indicate that increased CD44 expression, caused by general feature of CSCs by irradiation and sphere formation, can be monitored by using bioluminescence imaging. This system could be useful to evaluate CD44-expressed CSCs in breast cancer by BLI in vivo as well as in vitro for radiotherapy.

Effect of iron on cytolysin a expression in Salmonella enterica serovar Typhi.

Previously, a novel protein ClyA (Cytolysin A) has been identified in Escherichia coli K-12, Salmonella enterica serovars Typhi and Paratyphi A and Shigella. Salmonella spp. synthesize substantial amounts of ClyA upon infection of the human host, although the mechanism by which ClyA is induced in vivo is unclear. Since environmental signals could control the expression of virulence determinants, ClyA expression in S. Typhi Ty2 was tested by Western blotting in the presence of normal pooled human serum (NPS). The level of ClyA expression increased in the presence of NPS in a concentration-dependent manner. RPMI 1640 medium similarly induced ClyA expression. ClyA expression was inversely proportional to the concentration of iron in RPMI medium. Therefore, we speculated that iron inhibited the expression of ClyA in S. Typhi Ty2, and free iron depletion may be one of the causes of S. Typhi-mediated induction of ClyA in vivo. Transcription from a clyA-lacZ fusion gene decreased as iron concentration increased, but not as significantly as the ClyA protein expression. It is concluded that the regulatory effect of iron on clyA expression is mainly at translational level.

Salicylic Acid Reduces OmpF Expression, Rendering Salmonella enterica Serovar Typhimurium More Resistant to Cephalosporin Antibiotics

Salmonella enterica serovar Typhimurium is one of the most important bacterial pathogens causing diarrhea. The resistance of S. typhimurium to antimicrobial agents, which has recently been isolated from patients, is causing serious problems. We investigated the effects of salicylic acid (Sal) and acetyl salicylate (AcSal) on the susceptibility of S. typhimurium to cephalosporin antibiotics, which are known to increase resistance to cephalosporin and quinolone antibiotics. The MIC of cephalosporin antibiotics was higher than that of the media without Sal. The rate of accumulation of ethidium bromide (EtBr) in the bacteria by the outer membrane protein (Omp) was not different from that of the bacteria cultured in the medium containing Sal. However, Carbonyl cyanide-m-chlorophenylhydrazone (CCCP), an inhibitor of bacterial efflux pumps, significantly reduced the rate of accumulation of EtBr in bacteria cultured on Sal containing medium. In the medium containing CCCP, the MIC of the antimicrobial agent tended to decrease as compared with the control. In addition, the MIC of the bacteria treated with CCCP and Sal was higher than that of the antimicrobial agent against the CCCP treated experimental bacteria. These results suggest that Sal decreases the expression of OmpF in the Omp of S. typhimurium and reduces the permeability of cephalosporin antibiotics to bacteria, which may induce tolerance to cephalosporin antibiotics.

Effect of promoter-upstream sequence on σ38-dependent stationary phase gene transcription

Abstractσ38 in Escherichia coli is required for expression of a subset of stationary phase genes. However, the promoter elements for σ38-dependent genes are virtually indistinguishable from that for σ70-dependent house-keeping genes. hdeABp is a σ38-dependent promoter and LEE5p is a σ70-dependent promoter, but both are repressed by H-NS, a bacterial histone-like protein, which acts at promoter upstream sequence. We swapped the promoter upstream sequences of the two promoters and found that the σ dependency was switched. This was further verified using lacUV5 core promoter. The results suggested that the determinant for σ38-dependent promoter lies in the promoter upstream sequence.