Legionella pneumophila EnhC is required for efficient replication in tumour necrosis factor α‐stimulated macrophages

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 Δ enhC strain, while tumour necrosis factor α (TNF‐α), at concentrations similar to those found in macrophage culture supernatants, could reproduce the growth restriction exerted by culture supernatants on L. pneumophila Δ enhC . The absence of EnhC also caused defective trafficking of the Legionella‐ containing vacuole in TNF‐α‐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 Δ enhC mutant was more susceptible to H 2 O 2 treatment than the wild‐type strain. EnhC therefore is a unique virulence factor that is required for growth specifically when macrophages have heightened potential to restrict microbial replication.