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Legionella pneumophila is a Gram-negative bacterium found in natural and man-made water systems where it replicates within amoebas and ciliates. In humans, once inside the lungs, L. pneumophila replicates in alveolar macrophages and causes Legionnaires’ disease, a severe pneumonia. The Icm/Dot type IVb secretion system is a major virulence factor required for intracellular multiplication. The Icm/Dot system allows the secretion of effectors into the cytoplasm of the host cell. These effectors modify host cell vesicular trafficking and prevent maturation of the phagosome. The innate immune response is crucial in restricting L. pneumophila proliferation. TNF-α is one of the major cytokines involved in this process as it renders macrophages more resistant to L. pneumophila infection and induces apoptosis of L. pneumophila-infected macrophages. Tail-specific proteases (Tsp) are involved in tolerating thermal stress and in virulence. We have previously characterized the Tsp encoded by L. pneumophila, showing that it is important for surviving thermal stress and for infection of amoeba when a temperature change occurs during infection. Here, we demonstrated that Tsp is required for intracellular multiplication in macrophages. Absence of tsp is associated with higher production of TNF-α by macrophages in response to L. pneumophila infection. This effect is independent of the Icm/Dot secretion system.

Legionella pneumophila (Lp) is an inhabitant of natural and man-made water systems where it replicates within amoebae and ciliates and survives within biofilms. When Lp-contaminated aerosols are breathed in, Lp will enter the lungs and infect human alveolar macrophages, causing a severe pneumonia known as Legionnaires Disease. Lp is often found in hot water distribution systems (HWDS), which are linked to nosocomial outbreaks. Heat treatment is used to disinfect HWDS and reduce the concentration of Lp. However, Lp is often able to recolonize these water systems, indicating an efficient heat-shock response. Tail-specific proteases (Tsp) are typically periplasmic proteases implicated in degrading aberrant proteins in the periplasm and important for surviving thermal stress. In this paper, we show that Tsp, encoded by the lpg0499 gene in Lp Philadelphia-1, is important for surviving thermal stress in water and for optimal infection of amoeba when a shift in temperature occurs during intracellular growth. Tsp is expressed in the post-exponential phase but repressed in the exponential phase. The cis-encoded small regulatory RNA Lpr17 shows opposite expression, suggesting that it represses translation of tsp. In addition, tsp is regulated by CpxR, a major regulator in Lp, in a Lpr17-independent manner. Deletion of CpxR also reduced the ability of Lp to survive heat shock. In conclusion, this study shows that Tsp is an important factor for the survival and growth of Lp in water systems.IMPORTANCELegionella pneumophila (Lp) is a major cause of nosocomial and community-acquired pneumonia. Lp is found in water systems including hot water distribution systems. Heat treatment is a method of disinfection often used to limit Lp’s presence in such systems; however, the benefit is usually short term as Lp is able to quickly recolonize these systems. Presumably, Lp respond efficiently to thermal stress, but so far not much is known about the genes involved. In this paper, we show that the Tail-specific protease (Tsp) and the two-component system CpxRA are required for resistance to thermal stress, when Lp is free in water and when it is inside host cells. Our study identifies critical systems for the survival of Lp in its natural environment under thermal stress.

We describe a strain of Legionella quinlivanii isolated from a bronchoalveolar lavage specimen from an 83-year-old patient in the province of Québec. Identification was done using 16S rRNA sequencing. The strain could replicate efficiently in human THP-1 macrophages and maintained a low level of cytotoxicity. Upon analyzing the whole genome sequencing data, the icm/dot secretion system was present, but the strain lacked some effector genes known to express proteins toxic to cells. The pathogenicity of this Legionella species should be investigated further.

Surviving the nutrient-poor aquatic environment for extended periods of time is important for the transmission of various water-borne pathogens, including Legionella pneumophila (Lp). Previous work concluded that the stringent response and the sigma factor RpoS are essential for the survival of Lp in water. In the present study, we investigated the role of the LetA/S two-component signal transduction system in the successful survival of Lp in water. In addition to cell size reduction in the post-exponential phase, LetS also contributes to cell size reduction when Lp is exposed to water. Importantly, absence of the sensor kinase results in a significantly lower survival as measured by CFUs in water at various temperatures and an increased sensitivity to heat shock. According to the transcriptomic analysis, LetA/S orchestrates a general transcriptomic downshift of major metabolic pathways upon exposure to water leading to better culturability, and likely survival, suggesting a potential link with the stringent response. However, the expression of the LetA/S regulated small regulatory RNAs, RsmY and RsmZ, is not changed in a relAspoT mutant, which indicates that the stringent response and the LetA/S response are two distinct regulatory systems contributing to the survival of Lp in water.