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Infection and Immunity, September 2001, p. 5689-5697, Vol. 69, No. 9
0019-9567/01/$04.00+0   DOI: 10.1128/IAI.69.9.5689-5697.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.

Opsonized Virulent Edwardsiella tarda Strains Are Able To Adhere to and Survive and Replicate within Fish Phagocytes but Fail To Stimulate Reactive Oxygen Intermediates

Putanae S. Srinivasa Rao,1 Tit Meng Lim,1 and Ka Yin Leung1,2,*

Department of Biological Sciences, Faculty of Science,1 and Tropical Marine Science Institute,2 National University of Singapore, Singapore 119260

Received 2 January 2001/Returned for modification 12 March 2001/Accepted 7 June 2001

Edwardsiella tarda is responsible for hemorrhagic septicemia (edwardsiellosis) in fish and also causes diseases in higher vertebrates such as birds, reptiles, and mammals, including humans. Interactions of E. tarda with blue gourami phagocytes were studied by light microscopy as well as by adherence, intracellular replication, and superoxide anion assays. Both nonopsonized virulent (PPD130/91 and AL9379) and avirulent (PPD125/87 and PPD76/87) bacteria could adhere to and survive and replicate within phagocytes, while only opsonized virulent strains replicated within the phagocytes. Furthermore, only avirulent E. tarda elicited a higher rate of production of reactive oxygen intermediates (ROIs) by phagocytes, indicating that they were unable to avoid and/or resist reactive oxygen radical-based killing by the fish phagocytes. TnphoA transposon mutagenesis was used to construct a library of 200 alkaline phosphatase (PhoA+) fusion mutants from a total of 182,000 transconjugants derived from E. tarda PPD130/91. Five of these mutants induced more ROI production in phagocytes than the wild-type strain. Two mutants had lower replication ability inside phagocytes and moderately higher 50% lethal dose values than the wild-type strain. Sequence analysis revealed that three of these mutants had insertions at sequences having homology to PhoS, dipeptidase, and a surface polymer ligase of lipid A core proteins of other pathogens. These three independent mutations might have changed the cell surface characteristics of the bacteria, which in turn induced phagocytes to produce increased ROIs. Sequences from two other mutants had no homology to known genes, indicating that they may be novel genes for antiphagocytic killing. The present study showed that there are differences in the interactions of virulent and avirulent E. tarda organisms with fish phagocytes and PhoA+ fusion mutants that could be used successfully to identify virulence genes. The information elucidated here would help in the development of suitable strategies to combat the disease caused by E. tarda.

* Corresponding author. Mailing address: Department of Biological Sciences, Faculty of Science, The National University of Singapore, Science Drive 4, Singapore 117543, Singapore. Phone: (65) 874 7835. Fax: (65) 779 2486. E-mail: dbslky{at}nus.edu.sg.

Infection and Immunity, September 2001, p. 5689-5697, Vol. 69, No. 9
0019-9567/01/$04.00+0   DOI: 10.1128/IAI.69.9.5689-5697.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.


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