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Infection and Immunity, October 2003, p. 5461-5471, Vol. 71, No. 10
0019-9567/03/$08.00+0     DOI: 10.1128/IAI.71.10.5461-5471.2003
Copyright © 2003, American Society for Microbiology. All Rights Reserved.

Characterization and Pathogenic Significance of Vibrio vulnificus Antigens Preferentially Expressed in Septicemic Patients

Young Ran Kim,1 Shee Eun Lee,1,{dagger} Choon Mee Kim,1 Soo Young Kim,1 Eun Kyoung Shin,1 Dong Hyeon Shin,2 Sun Sik Chung,2 Hyon E. Choy,2 Ann Progulske-Fox,3 Jeffrey D. Hillman,3 Martin Handfield,3 and Joon Haeng Rhee1,2*

National Research Laboratory of Molecular Microbial Pathogenesis, Research Institute of Vibrio Infection and Genome Research Center for Enteropathogenic Bacteria,1 Department of Microbiology, Chonnam National University Medical School, Kwangju 501-746, South Korea,2 Center for Molecular Microbiology and Department of Oral Biology, College of Dentistry, University of Florida, Gainesville, Florida 32610-04243

Received 5 February 2003/ Returned for modification 28 April 2003/ Accepted 14 July 2003

Many important virulence genes of pathogenic bacteria are preferentially expressed in vivo. We used the recently developed in vivo-induced antigen technology (IVIAT) to identify Vibrio vulnificus genes induced in vivo. An expression library of V. vulnificus was screened by colony blot analysis by using pooled convalescent-phase serum that had been thoroughly adsorbed with in vitro-expressed V. vulnificus whole cells and lysates. Twelve clones were selected, and the sequences of the insert DNAs were analyzed. The DNA sequences showed homologies with genes encoding proteins of diverse functions: these functions included chemotaxis (a methyl-accepting chemotaxis protein), signaling (a GGDEF-containing protein and a putative serine/threonine kinase), biosynthesis and metabolism (PyrH, PurH, and IlvC), secretion (TatB and plasmid Achromobacter secretion [PAS] factor), transcriptional activation (IlvY and HlyU), and the activity of a putative lipoprotein (YaeC). In addition, one identified open reading frame encoded a hypothetical protein. Isogenic mutants of the 12 in vivo-expressed (ive) genes were constructed and tested for cytotoxicity. Cytotoxic activity of the mutant strains, as measured by lactate dehydrogenase release from HeLa cells, was nearly abolished in pyrH, purH, and hlyU mutants. The intraperitoneal 50% lethal dose in mice increased by ca. 10- to 50-fold in these three mutants. PyrH and PurH seem to be essential for in vivo growth. HlyU appears to be one of the master regulators of in vivo virulence expression. The successful identification of ive genes responsible for the in vivo bacterial virulence, as done in the present study, demonstrates the usefulness of IVIAT for the detection of new virulence genes.

* Corresponding author. Mailing address: National Research Laboratory of Molecular Microbial Pathogenesis and Department of Microbiology, Chonnam National University Medical School, 5 Hak-Dong, Dong-Ku, Kwangju 501-746, South Korea. Phone: 82-62-220-4136. Fax: 82-62-228-7294. E-mail: address: jhrhee{at}chonnam.chonnam.ac.kr.

Editor: V. J. DiRita

{dagger} Present address: Department of Dental Pharmacology, Chonnam National University Dental College, Kwangju, Korea.

Infection and Immunity, October 2003, p. 5461-5471, Vol. 71, No. 10
0019-9567/03/$08.00+0     DOI: 10.1128/IAI.71.10.5461-5471.2003
Copyright © 2003, American Society for Microbiology. All Rights Reserved.



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