IVET-based identification of virulence factors in Vibrio vulnificus MO6-24/O.
Vibrio vulnificus is an opportunistic pathogen that causes septicemia in humans. To identify the genes associated with its pathogenicity, in vivo expression technology (IVET) was used to select genes specifically expressed in a host, yet not significantly in vitro. Random lacZ-fusions in the genome of V. vulnificus strain MO6-24/O were constructed using an IVET vector, pSG3, which is a suicide vector containing promoterless-aph and -lacZ as reporter genes. A total of approximately 18,000 resulting library clones were then intraperitoneally injected into BALB/c mice using a colony forming unit (CFU) of 1.6 x 10(6). Two hours after infection, kanamycin was administered at 200 microg per gram of mouse weight. After two selection cycles, 11 genes were eventually isolated, which were expressed only in the host. Among these genes, VV20781 and VV21007 exhibiting a homology to a hemagglutinin gene and tolC, respectively, were selected based on having the highest frequency. When compared to wild-type cells, mutants with lesions in these genes showed no difference in the rate of growth rate, yet a significant decrease in cytotoxicity and the capability to form a biofilm.
Lee
Ko-Eun
KE
Department of Life Science and Interdisciplinary Program of Integrated Biotechnology, Sogang University, Seoul 121-742, Korea.
Bang
Ji-Sun
JS
Baek
Chang-Ho
CH
Park
Dae-Kyun
DK
Hwang
Won
W
Choi
Sang Ho
SH
Kim
Kun-Soo
KS
D000595:Amino Acid Sequence; D000818:Animals; D019677:Artificial Gene Fusion / Q000379:methods*; D018441:Biofilms; D005260:Female; D015723:Gene Library; D016680:Genome, Bacterial; D006367:HeLa Cells; D006801:Humans; D051379:Mice; D008807:Mice, Inbred BALB C; D008969:Molecular Sequence Data; D041261:Vibrio vulnificus / Q000235:genetics / Q000472:pathogenicity*; D037521:Virulence Factors / Q000032:analysis* / Q000737:chemistry / Q000235:genetics / Q000633:toxicity
D037521:Virulence Factors