The Tail-Specific Protease Is Important for Legionella pneumophila To Survive Thermal Stress in Water and inside Amoebae

Abstract

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 can enter the lungs and may 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 Lp Philadelphia-1, Tsp is encoded by the lpg0499 gene. In this paper, we show that Tsp is important for surviving thermal stress in water and for optimal infection of amoeba when a shift in temperature occurs during intracellular growth. We also demonstrate that Tsp is expressed in the post-exponential phase but repressed in the exponential phase and that the cis-encoded small regulatory RNA Lpr17 shows opposite expression, suggesting that it represses translation of tsp. In addition, our results show that 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, our study shows that Tsp is likely an important factor for the survival and growth of Lp in water systems. IMPORTANCE Legionella 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 the presence of Lp in such systems; however, the benefit is usually short term as Lp is able to quickly recolonize these systems. Presumably, Lp responds 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.

A tail-specific protease is required for Legionella pneumophila intracellular multiplication

“…Early study did not find a function associated with Tsp (Lawrence et al 2014). We have recently shown that Tsp is involved in surviving a heat treatment at 55 • C and replicating inside the amoeba Vermamoeba vermiformis when the temperature during the infection is increased (Saoud et al 2021). In addition, Tsp appears to be negatively regulated by Lpr17, a cis-encoded small regulatory RNA (sRNA), in the exponential (E) phase (Saoud et al 2021).…”

“…We have recently shown that Tsp is involved in surviving a heat treatment at 55 • C and replicating inside the amoeba Vermamoeba vermiformis when the temperature during the infection is increased (Saoud et al 2021). In addition, Tsp appears to be negatively regulated by Lpr17, a cis-encoded small regulatory RNA (sRNA), in the exponential (E) phase (Saoud et al 2021). Tsp is expressed in the post-exponential phase (PE) and during thermal stress, conditions in which Lpr17 is not expressed (Saoud et al 2021).…”

Legionella pneumophila—Virulence Factors and the Possibility of Infection in Dental Practice

“…The registered case number of infections is increasing despite numerous Legionella can be found in water distribution cooling towers, where it can replicate within protozoa. It is often Vermamoeba vermiformis that protects Legionella pneumophila from the effects of heat and disinfectants, which can result in nosocomial infections [28]. Coevolution with multiple protozoan species has resulted in the development of mechanisms that allow L. pneumophila to occupy different hosts, and the possibility of human cell infection [2,29,30].…”

Targeted Protein Degradation: The New Frontier of Antimicrobial Discovery?

“…HtrA is also located in the periplasm. 83,93 In spite of this, ClpXP is the principle degrader of SsrA-tagged proteins in E. coli. 92 The delivery of SsrA tagged proteins to the ClpXP protease complex is facilitated by the adaptor protein SspB in conjunction with its unfoldase partner ClpX.…”

“…In E. coli , the proteases ClpXP, ClpAP, Lon, FtsH, and Tsp recognize and degrade proteins with an SsrA tag, thus providing full coverage of the three bacterial compartments (cytoplasm, membrane, and periplasm) since ClpXP, ClpAP, and Lon are expressed in the cytoplasm, FtsH is membrane-bound and Tsp is located in the periplasm. HtrA is also located in the periplasm. , In spite of this, ClpXP is the principle degrader of SsrA-tagged proteins in E. coli . The delivery of SsrA tagged proteins to the ClpXP protease complex is facilitated by the adaptor protein SspB in conjunction with its unfoldase partner ClpX. , …”