The CpxRA two‐component system contributes to Legionella pneumophila virulence

Abstract

The bacterium Legionella pneumophila is capable of intracellular replication within freshwater protozoa as well as human macrophages, the latter of which results in the serious pneumonia Legionnaires' disease. A primary factor involved in these host cell interactions is the Dot/Icm Type IV secretion system responsible for translocating effector proteins needed to establish and maintain the bacterial replicative niche. Several regulatory factors have been identified to control the expression of the Dot/Icm system and effectors, one of which is the CpxRA two-component system, suggesting essentiality for virulence. In this study, we generated cpxR, cpxA and cpxRA in-frame null mutant strains to further delineate the role of the CpxRA system in bacterial survival and virulence. We found that cpxR is essential for intracellular replication within Acanthamoeba castellanii, but not in U937-derived macrophages. Transcriptome analysis revealed that CpxRA regulates a large number of virulence-associated proteins including Dot/Icm effectors as well as Type II secreted substrates. Furthermore, the cpxR and cpxRA mutant strains were more sodium resistant than the parental strain Lp02, and cpxRA expression reaches maximal levels during postexponential phase. Taken together, our findings suggest the CpxRA system is a key contributor to L. pneumophila virulence in protozoa via virulence factor regulation.

Legionella pneumophila OxyR Is a Redundant Transcriptional Regulator That Contributes to Expression Control of the Two-Component CpxRA System

“…The aforementioned cascade is quite interesting, as it draws connections to different aspects and observations in this study, as detailed in the following: (i) negative regulation of the CpxRA system may in part explain the limited growth phenotype of the OxyR Lp overexpression strain Lp02 OE ( Fig. 2A), as we recently identified the CpxRA system to be absolutely required by L. pneumophila for intracellular replication in A. castellanii (25); (ii) transcriptomic analysis has determined that the CpxRA system regulates a significant number of genes in L. pneumophila (25), implicating a wider regulatory influence for oxyR Lp expression and a rationale for limiting OxyR Lp levels at a threshold throughout the progressive growth phases, as observed (Fig. 1), potentially avoiding perturbations to multiple regulatory networks; (iii) limiting OxyR Lp expression also correlates with the observation that its repression of icmR and cpxRA expression is secondary to the activation elicited by CpxR (Fig.…”

“…Several mutants have been successfully generated in previous studies using this method (34,40); however, the success rate is fairly low, and thus, the use of an alternate suicide vector may be more effective. To test this, an in-frame null mutant strategy that has efficiently generated single and multigene deletion mutants recently in our laboratory, as well as in previous studies elsewhere, was selected (25,41,42). This approach employs generating an in-frame knockout fragment via specific primer design (Table 2) and the suicide vector pSR47s, which has sacB as the counterselectable marker (see Materials and Methods for details) ( Table 1) (43).…”

“…The site within the P lpg1441-cpxA region, containing seven mismatches, is located 103 bp upstream of the Lpg1441 translational start site and overlaps the CpxR binding motif identified in our recent work that examined this region (Fig. 8B) (25). Each of these sites has comparable mismatches to the functional OxyR Lp binding site determined elsewhere for the ahpC2 promoter region and therefore may be functional within these promoter regions as well (36).…”

“…To investigate this potential interaction, the reporter construct pJT730 containing the promoter region that drives the expression of the cpxRA two-component system (P lpg1441-cpxA ) was electroporated into the ΔoxyR Lp mutant strain; the promoter activity was compared to the activities in parental Lp02 and Δcpx mutant strains. In the ΔcpxA mutant strain, P lpg1441-cpxA activity is elevated due to the direct positive autoregulation exhibited by CpxR and elevated in the ΔcpxR and ΔcpxRA mutant strains due to indirect negative autoregulation (Table 1) (25). Similarly to what is observed in ΔcpxA, P lpg1441-cpxA activity is initially slightly elevated in the ΔoxyR Lp mutant strain but decreases with time to levels just below those observed in Lp02; after 10 h, it increases at an accelerated rate in comparison to Lp02 (Fig.…”

“…These factors include a group of Ͼ300 effector molecules delivered into the host cell cytoplasm by a Dot/Icm type IVB secretion system that enables L. pneumophila to evade the lysosome-phagosome pathway and subsequently establish a replicative niche (17)(18)(19). Expression of the Dot/Icm system and associated effectors is under the control of several regulatory factors that include LetAS, PmrAB, CpxRA, and LqsRS two-component systems, as well as the stationary-phase sigma factor RpoS; each is able to positively and negatively impact the expression of this virulence system (20)(21)(22)(23)(24)(25)(26).…”