Significant Role for ladC in Initiation of Legionella pneumophila Infection

Abstract

Previously, we identified ladC in a cohort of genes that were present in Legionella 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, which 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 fivefold 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.

Life Stage-specific Proteomes of Legionella pneumophila Reveal a Highly Differential Abundance of Virulence-associated Dot/Icm effectors

“…A study by Hoffmann et al characterized the bacterial LCV proteome at 1h after infection (29). Hoffmann et al found some of the here described PE-up proteins, including RavZ (124), RalF (119), SidC (111,162), SidM/DrrA (113,115,163) but also some E-up proteins, such as LegS2 (105), LepA (108), and those with equal abundance, such as Lem3 (164), Ceg28/ RidL (165), SdhA (166), SidD (110,167). This shows that a broad variety of Dot/Icm effectors is present in LCV proteomes.…”

Intracellular Growth and Morphological Characteristics of <i>Legionella pneumophila</i> during Invasion and Proliferation in Different Cells

“…Although it is not very clear how the bacteria are internalized by eukaryotic cells, many reports indicated that different mechanisms of uptake were used by macrophage-like cell and non-phagocytic cell. 5,[19][20][21] Our results indicated that internalization of L. pneumophila by macrophage-like cell lines were easier than that by non-phagocytic cell lines. Correlational analyses from our results showed positive correlation between adherence and invasiveness (Fig.…”

The PmrA/PmrB Two-Component System ofLegionella pneumophilaIs a Global Regulator Required for Intracellular Replication within Macrophages and Protozoa

“…The four sequenced genomes of L. pneumophila contain each an average of 3,000 genes with 200 to 300 genes specific to each genome, but all contain the pmrAB locus. It is important to note that some of the genes present in the clinical strain AA100/130b may be absent in the Philadelphia-1 strain and vice versa and that gene variation among bacterial strains of the same species usually represents a small proportion of the genome (53). Despite potential minor differences between the Philadelphia-1 strain genome and our clinical strain AA100/a30b, ϳ10% of the core genome appears to be under the regulation of PmrA.…”