The amoebae plate test implicates a paralogue of lpxB in the interaction of Legionella pneumophila with Acanthamoeba castellanii

Abstract

Legionella pneumophilais 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 ofL. pneumophilawild-type andicm/dotmutant strains spotted on agar plates in the presence ofAcanthamoeba castellanii. In the APT, wild-typeL. pneumophilaformed robust colonies even at high dilutions,icmT, -R, -PordotBmutants failed to grow, andicmSor -Gmutants were partially growth defective. TheicmSoricmGmutant strains were used to screen anL. pneumophilachromosomal library for genes that suppress the growth defect in the presence of the amoebae. AnicmSsuppressor plasmid was isolated that harboured theicmSand flankingicmgenes, indicating that this plasmid complements the intracellular growth defect of the mutant. In contrast, differenticmGsuppressor plasmids rendered theicmGmutant more cytotoxic forA. castellaniiwithout enhancing intracellular multiplication in amoebae or RAW264.7 macrophages. Deletion of individual genes in the suppressor plasmids inserts identifiedlcs(Legionellacytotoxicsuppressor) -A, -B, -Cand -Das being required for enhanced cytotoxicity of anicmGmutant 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 anicmGmutant but not that of othericm/dotorrpoSmutant strains againstA. castellanii. Based on sequence comparison and chromosomal location,lcsBandlcsCprobably 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.