Journal of Basic Microbiology 2011, 51, 243 – 252 243 © 2011 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim www.jbm-journal.com Research Paper Establishment of a Caenorhabditis elegans infection model for Vibrio alginolyticus Sellegounder Durai, Shunmugiah Karutha Pandian and Krishnaswamy Balamurugan Department of Biotechnology, Alagappa University, Karaikudi – 630 003, India Vibrio alginolyticus, a common bacterium in the marine environment, is a threat to marine ani- mals and humans by causing serious infections. The present study reveals the establishment of a Caenorhabditis elegans infection model for Vibrio alginolyticus. The infection and colonization was localized in the animal by tagging V. alginolyticus with GFP and using Confocal Laser Scanning Microscopy. Chemotactic response of C. elegans to V. alginolyticus, pharyngeal disten- tion and blockage of vulval region leading to internal hatching were analyzed. The time required for causing infection, and the bacterial loads in the intestine of C. elegans were deter- mined. Regulation of innate immune related genes, lys-7, clec-60 and clec-87, were also analyzed using real time PCR. The pathogen infected animals appeared to ward-off infection by up-regul- ating the candidate antimicrobial gene(s) for few hours, before succumbing to the pathogen. For the first time, the pathogenicity of V. alginolyticus at both physiological and molecular level has been studied in detail using the model organism C. elegans. Abbreviations: PAMPs – Pathogen-Associated Molecular Patterns; TDH – Thermostable direct hemolysin; CTLD – C-type lectin domain; NGM – Nematode growth medium; TCBS agar – Thiosulfate Citrate Bile salts Sucrose agar; CFU – Colony Forming Unit; RT-PCR – Reverse transcriptase-Polymerase chain reaction; PCR – Polymerase Chain Reaction; GFP – Green fluorescent protein; ATCC – American Type Culture Collection Keywords: Vibrio alginolyticus / Caenorhabditis elegans / Host-pathogen interactions / Innate immunity Received: August 04, 2010; accepted: October 07, 2010 DOI 10.1002/jobm.201000303 Introduction * The nematode C. elegans represents a good model for studying various aspects of bacterial pathogenesis. Re- cently V. cholerae virulent factors haemolysin [1] and protease [2] were proved to be necessary for killing C. elegans. The virulence factors of V. vulnificus, necessary for causing infection in mammals, play an important role in causing infection in C. elegans [3] making it a suitable model for host pathogen interaction studies. Most of the bacteria lethal to C. elegans primarily cause intestinal infection [4 – 6], bacteria reach the intestine by escaping the pharynx thereby manage to fake the host in one or many ways. On reaching the gut, bacte- Correspondence: Dr. K. Balamurugan, Department of Biotechnology, Alagappa University, Karaikudi 630 003, India E-mail: bsuryar@yahoo.com Phone: +91 4565 225215 Fax: +91 4565 225202 ria multiply and cause distension and in some cases damage the intestinal epithelium which leads to death [4, 7]. Vibrionaceae genus is of high threat to human be- ings. Of them, V. cholerae, V. parahaemolyticus and V. alginolyticus are highly pathogenic to humans and marine animals. V. alginolyticus is a common inhabitant of the marine environment in both temperate and tropical waters [8, 9]. V. alginolyticus is one of the most common and important pathogens that cause vibriosis in human and marine species and infection in humans was first recognized in 1973. Infection of fish by V. alginolyticus results in biofilm formation in their in- testine, which leads to fish mortality and potentially significant economic losses. V. alginolyticus has been described to cause human infections including wound infection (71%), gastroenteritis (12%), ear infection and septicemia [10]. Other clinical syndromes reported in association with V. alginolyticus infection include