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Identification of Burkholderia pseudomallei Genes Required for the Intracellular Life Cycle and In Vivo Virulence

Institute of Medical Microbiology, Hannover Medical School, Carl-Neuberg-Str. 1, 30625 Hannover, Germany,¹ Friedrich Loeffler Institute for Medical Microbiology, Ernst Moritz Arndt University Greifswald, Martin-Luther-Strasse 6, 17487 Greifswald, Germany,² Department of Microbiology, Institute of Plant Biology, University of Zürich, Zollikerstrasse 107, 8008 Zürich, Switzerland³

Received 4 August 2005/ Returned for modification 19 October 2005/ Accepted 27 February 2006

The bacterial pathogen Burkholderia pseudomallei invades host cells, escapes from endocytic vesicles, multiplies intracellularly, and induces the formation of actin tails and membrane protrusions, leading to direct cell-to-cell spreading. This study was aimed at the identification of B. pseudomallei genes responsible for the different steps of this intracellular life cycle. B. pseudomallei transposon mutants were screened for a reduced ability to form plaques on PtK2 cell monolayers as a result of reduced intercellular spreading. Nine plaque assay mutants with insertions in different open reading frames were selected for further studies. One mutant defective in a hypothetical protein encoded within the Bsa type III secretion system gene cluster was found to be unable to escape from endocytic vesicles after invasion but still multiplied within the vacuoles. Another mutant with a defect in a putative exported protein reached the cytoplasm but exhibited impaired actin tail formation in addition to a severe intracellular growth defect. In four mutants, the transposon had inserted into genes involved in either purine, histidine, or p-aminobenzoate biosynthesis, suggesting that these pathways are essential for intracellular growth. Three mutants with reduced plaque formation were shown to have gene defects in a putative cytidyltransferase, a putative lipoate-protein ligase B, and a hypothetical protein. All nine mutants proved to be significantly attenuated in a murine model of infection, with some mutants being essentially avirulent. In conclusion, we have identified a number of novel major B. pseudomallei virulence genes which are essential for the intracellular life cycle of this pathogen.

Editor: J. T. Barbieri

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