A significant role for ladC in the initiation of Legionella pneumophila infection

Australian Bacterial Pathogenesis Program, Department of Microbiology, Monash University, Victoria 3800, Australia; Unité de Génomique des Microorganismes Pathogènes, Institut Pasteur, Paris Cedex 15 and CNRS URA 2171, France; Department of Microbiology and Immunology, Northwestern University Medical School, Chicago, Illinois 60611, USA; Department of Microbiology and Immunology, University of Melbourne, Victoria 3010, Australia

^* To whom correspondence should be addressed. Email: Hartland{at}unimelb.edu.au.

	Abstract

Previously, we identified ladC in a cohort of genes that were present in L. pneumophila but absent in other Legionella species. Here we constructed a ladC mutant of L. pneumophila and assessed its ability to replicate in mammalian cell lines and Acanthamoeba castellanii. The ladC mutant was recovered in significantly lower numbers than wild type L. pneumophila at early time points, which was reversed upon transcomplementation with ladC but not ladC_N430A/R434A, encoding a putative catalytically inactive derivative of the protein. In fact complementation of ladC::km with ladC_N430A/R434A resulted in a severe replication defect within human and amoeba cell models of infection that did not follow a typical dominant negative phenotype. Using differential immunofluorescence staining to distinguish adherent from intracellular bacteria, we found that the ladC mutant exhibited a 10 fold reduction in adherence to THP-1 macrophages but no difference in uptake by THP-1 cells. When tested in vivo in A/J mice, the competitive index of the ladC mutant dropped 5-fold over 72 h indicating a significant attenuation compared to wild type L. pneumophila. Although localization of LadC to the bacterial inner membrane suggested that the protein may be involved in signaling pathways that regulate virulence gene expression, microarray analysis indicated that ladC does not influence the transcriptional profile of L. pneumophila in vitro or during A. castellanii infection. Although the mechanism by which LadC modulates the initial interaction between the bacterium and host cell remains unclear, we have established that LadC plays an important role in L. pneumophila infection.