1887

Abstract

is a bacterial parasite of freshwater amoebae which also grows in alveolar macrophages and thus causes the potentially fatal pneumonia Legionnaires' disease. Intracellular growth within amoebae and macrophages is mechanistically similar and requires the Icm/Dot type IV secretion system. This paper reports the development of an assay, the amoebae plate test (APT), to analyse growth of wild-type and / mutant strains spotted on agar plates in the presence of . In the APT, wild-type formed robust colonies even at high dilutions, , -, - or mutants failed to grow, and or - mutants were partially growth defective. The or mutant strains were used to screen an chromosomal library for genes that suppress the growth defect in the presence of the amoebae. An suppressor plasmid was isolated that harboured the and flanking genes, indicating that this plasmid complements the intracellular growth defect of the mutant. In contrast, different suppressor plasmids rendered the mutant more cytotoxic for without enhancing intracellular multiplication in amoebae or RAW264.7 macrophages. Deletion of individual genes in the suppressor plasmids inserts identified (egionella ytotoxic uppressor) -, -, - and - as being required for enhanced cytotoxicity of an mutant strain. The corresponding proteins show sequence similarity to hydrolases, NlpD-related metalloproteases, lipid A disaccharide synthases and ABC transporters, respectively. Overexpression of LcsC, a putative paralogue of the lipid A disaccharide synthase LpxB, increased cytotoxicity of an mutant but not that of other / or mutant strains against . Based on sequence comparison and chromosomal location, and probably encode enzymes involved in cell wall maintenance and peptidoglycan metabolism. The APT established here may prove useful to identify other bacterial factors relevant for interactions with amoeba hosts.

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2005-01-01
2019-11-13
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