Legionella pneumophilaEnhC is required for efficient replication in tumour necrosis factor α-stimulated macrophages

Abstract

Summary Legionella pneumophila enhC-mutants were originally identified as being defective for uptake into host cells. In this work, we found that the absence of EnhC resulted in defective intracellular growth when dissemination of intracellular bacteria to neighbouring cells was expected to occur. No such defect was observed during growth within the amoeba Dictyostelium discoideum. Culture supernatants containing the secreted products of infected macrophages added to host cells restricted the growth of the DenhC strain, while tumour necrosis factor a (TNF-a), at concentrations similar to those found in macrophage culture supernatants, could reproduce the growth restriction exerted by culture supernatants on L. pneumophila DenhC. The absence of EnhC also caused defective trafficking of the Legionellacontaining vacuole in TNF-a-treated macrophages. EnhC was shown to be an envelope-associated protein largely localized to the periplasm, with its expression induced in post-exponential phase, as is true for many virulence-associated proteins. Furthermore, the absence of EnhC appeared to affect survival under stress conditions, as the DenhC mutant was more susceptible to H 2O2 treatment than the wildtype strain. EnhC therefore is a unique virulence factor that is required for growth specifically when macrophages have heightened potential to restrict microbial replication.

Type II Secretion Is Necessary for Optimal Association of the Legionella-Containing Vacuole with Macrophage Rab1B but Enhances Intracellular Replication Mainly by Rab1B-Independent Mechanisms

“…2A). The observation that 1 to 3% of the inoculum entered into the macrophages is similar to past observations made with L. pneumophila and this type of gentamicin protection assay (53). To confirm our results, we utilized a trypan blue quenching method that was recently adapted to assess L. pneumophila entry into host cells (54)(55)(56).…”

“…To assess the extent of L. pneumophila entry into macrophages, a gentamicin protection assay was done (36,53). Briefly, the bacterial strains were added to the BMD macrophages at an MOI equal to 1 or 50, and then the infection was synchronized as described above.…”

Implication of Proteins Containing Tetratricopeptide Repeats in Conditional Virulence Phenotypes of Legionella pneumophila

“…1F). In contrast, a ⌬enhC mutant of strain Lp02 was not defective for entry into HEp-2 cells or macrophages derived from bone marrow of A/J mice (30). Differences in entry phenotypes between L. pneumophila strains AA100, JR32, and Lp02 may be attributable to genomic differences, including the absence of an Lvh T4SS in strain Lp02.…”

The Type II Secretion System of Legionella pneumophila Dampens the MyD88 and Toll-Like Receptor 2 Signaling Pathway in Infected Human Macrophages

“…The noninflammasome NLRs NOD1 and NOD2 are stimulated during L. pneumophila infection. This has been shown in the context of lung infection of knockout mice and infected BMD macrophages obtained from knockout mice as well as a human epithelial cell line (33,(40)(41)(42)(43). The activation of the MAPK and NF-B pathways is in part dependent upon NOD signaling, which is contingent upon RIP2 (44,45).…”