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Applied and Environmental Microbiology, April 2009, p. 2166-2175, Vol. 75, No. 7
0099-2240/09/$08.00+0     doi:10.1128/AEM.02449-08
Copyright © 2009, American Society for Microbiology. All Rights Reserved.

High-Throughput Bioluminescence-Based Mutant Screening Strategy for Identification of Bacterial Virulence Genes{triangledown}

Attila Karsi,1,2* Nagihan Gülsoy,3 Erin Corb,1 Pradeep R. Dumpala,1 and Mark L. Lawrence1,2*

Department of Basic Sciences, College of Veterinary Medicine,1 Institute for Digital Biology, Mississippi State University, Mississippi State, Mississippi 39762,2 Department of Biology, Faculty of Art and Science, Marmara University, Göztepe, Istanbul 34722, Turkey3

Received 24 October 2008/ Accepted 29 January 2009

A high-throughput bioluminescence screening procedure for identification of virulence genes in bacteria was developed and applied to the fish pathogen Edwardsiella ictaluri. A random transposon mutant library expressing bioluminescence was constructed and robotically arrayed on 384-well plates. Mutants were cultivated and mixed with catfish serum and neutrophils in 96-well plates, and bioluminescence was used to detect mutants that are more susceptible to killing by these host factors. The virulence and vaccine efficacy of selected mutants were determined in channel catfish. Transposon insertion sites in 13 mutants attenuated in the natural host were mapped to the E. ictaluri genome. Ten unique genes were mutated, including genes encoding a negative regulator of sigmaE activity, a glycine cleavage system protein, tricarboxylic acid cycle enzymes, an O polysaccharide biosynthesis enzyme, proteins encoded on the native plasmid pEI1, and a fimbrial chaperon protein. Three of these mutants were found to have potential as live attenuated vaccines. This study demonstrates a novel application of bioluminescence to identify bacterial genes required for host resistance; as a result, efficacious and genetically defined live attenuated vaccine candidates were developed.

* Corresponding author. Mailing address: College of Veterinary Medicine, 240 Wise Center Dr., Mississippi State, MS 39762-6100. Phone: (662) 325-1195. Fax: (662) 325-1031. E-mail for M. Lawrence: lawrence{at}cvm.msstate.edu. E-mail for A. Karsi: karsi{at}cvm.msstate.edu

{triangledown} Published ahead of print on 5 February 2009.

Applied and Environmental Microbiology, April 2009, p. 2166-2175, Vol. 75, No. 7
0099-2240/09/$08.00+0     doi:10.1128/AEM.02449-08
Copyright © 2009, American Society for Microbiology. All Rights Reserved.





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