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

Abstract

Nominally an environmental organism, Legionella pneumophila is an intracellular parasite of protozoa but is also the causative agent of the pneumonia termed Legionnaires' disease, which results from inhalation of aerosolized bacteria by susceptible humans. Coordination of gene expression by a number of identified regulatory factors, including OxyR, assists L. pneumophila in adapting to the stresses of changing environments. L. pneumophila OxyR (OxyR_Lp) is an ortholog of Escherichia coli OxyR; however, OxyR_Lp was shown elsewhere to be functionally divergent, such that it acts as a transcription regulator independently of the oxidative stress response. In this study, the use of improved gene deletion methods has enabled us to generate an unmarked in-frame deletion of oxyR in L. pneumophila Lack of OxyR_Lp did not affect in vitro growth or intracellular growth in Acanthamoeba castellanii protozoa and U937-derived macrophages. The expression of OxyR_Lp does not appear to be regulated by CpxR, even though purified recombinant CpxR bound a DNA sequence similar to that reported for CpxR elsewhere. Surprisingly, a lack of OxyR_Lp resulted in elevated activity of the promoters located upstream of icmR and the lpg1441-cpxA operon, and OxyR_Lp directly bound to these promoter regions, suggesting that OxyR_Lp is a direct repressor. Interestingly, a strain overexpressing OxyR_Lp demonstrated reduced intracellular growth in A. castellanii but not in U937-derived macrophages, suggesting that balanced expression control of the two-component CpxRA system is necessary for survival in protozoa. Taken together, this study suggests that OxyR_Lp is a functionally redundant transcriptional regulator in L. pneumophila under the conditions evaluated herein.IMPORTANCELegionella pneumophila is an environmental pathogen, with its transmission to the human host dependent upon its ability to replicate in protozoa and survive within its aquatic niche. Understanding the genetic factors that contribute to L. pneumophila survival within each of these unique environments will be key to limiting future point-source outbreaks of Legionnaires' disease. The transcriptional regulator L. pneumophila OxyR (OxyR_Lp) has been previously identified as a potential regulator of virulence traits warranting further investigation. This study demonstrated that oxyR is nonessential for L. pneumophila survival in vitro and in vivo via mutational analysis. While the mechanisms of how OxyR_Lp expression is regulated remain elusive, this study shows that OxyR_Lp negatively regulates the expression of the cpxRA two-component system necessary for intracellular survival in protozoa.

Keywords: CpxRA; Legionella pneumophila; OxyR; gene expression; intracellular pathogen; microbial genetics; protozoa; transcriptional regulation.

FIG 1

OxyR_Lp does not exhibit growth-phase-dependent changes in cellular levels. Immunoblot (A) and quantitative densitometry (B) analyses of OxyR_Lp and isocitrate dehydrogenase (ICDH) in early exponential (EE), exponential (E), and postexponential (PE) growth phases in L. pneumophila Lp02. (A) Lp02 harboring the oxyR_Lp complementation construct, pJT396, during postexponential phase (overexpressing [OE-PE]) and the ΔoxyR_Lp mutant strain grown to postexponential phase (ΔO) were also included. Results represent one of two independent experiments. (B) Means of OxyR_Lp/ICDH ratios obtained for each growth phase. For the OxyR_Lp OE strain, the 4-fold dilution factor of the sample was incorporated into the densitometric value and the OxyR_Lp/ICDH ratio was subsequently determined and plotted. An asterisk indicates statistical significance, with a P value of <0.05; ns indicates no significant differences in OxyR_Lp protein levels by Student's t test with Welch's correction. Error bars represent the standard errors of the means (SEM) of the results from two independent experiments.

FIG 2

OxyR_Lp is not required for intracellular growth. Intracellular growth kinetics of L. pneumophila Lp02, the ΔoxyR_Lp mutant, the ΔoxyR_Lp mutant harboring oxyR_Lp complementation plasmid pJT396 (ΔoxyR_Lp Comp), Lp02 harboring oxyR_Lp complementation plasmid pJT396 (overexpressing OxyR_Lp [Lp02 OE]), the ΔoxyR_Lp mutant containing pJB908 (vector control [ΔoxyR_Lp VC]), and the ΔdotA mutant in Acanthamoeba castellanii (A) or U937-derived macrophages (B). (A) Lp02-pThyA, ΔoxyR_Lp-pThyA, and ΔdotA-pThyA strains harbor pBH6119 (pThyA). Infections were conducted at 25°C and 37°C within A. castellanii and U937-derived macrophages, respectively. Data are means from duplicate infections plated in triplicate and represent the results from one of three independent experiments. Errors bars represent standard errors of the means (SEM).

FIG 3

Growth profiles of L. pneumophila strains in BYE broth are not affected when OxyR_Lp is deleted, complemented, or overexpressed. Examined were L. pneumophila Lp02 (Lp02-pThyA), the ΔoxyR_Lp mutant (ΔoxyR_Lp-pThyA), the ΔoxyR_Lp mutant harboring oxyR_Lp complementation construct pJT396 (ΔoxyR_Lp Comp), Lp02 harboring oxyR_Lp complementation construct pJT396 (Lp02 OE), and Lp02 and ΔoxyR_Lp mutant strains harboring empty vector pJB908 (Lp02 VC and ΔoxyR_Lp VC, respectively). Lp02-pThyA and ΔoxyR_Lp-pThyA strains harbor pBH6119 (pThyA). Data represent the results from one of three independent experiments conducted in triplicate, with error bars representing standard errors of the means (SEM) of three technical replicates.

FIG 4

In vitro expression of oxyR_Lp is not regulated by OxyR_Lp and/or the CpxRA two-component system. (A, C, and D) Each strain examined harbored the pJT277 reporter construct in which the P_oxyR region drives the expression of GFP. All data are presented as normalized fluorescence values (relative fluorescence units/optical density at 600 nm). (A) P_oxyR activity was assessed in L. pneumophila Lp02 and ΔoxyR_Lp. (B) Schematic diagram of the P_oxyR region annotated with a potential CpxR binding site (BR) (underlined), with boldface font indicating the sequence likely recognized by CpxR and nonboldface font indicating the nucleotide spacer region. The transcriptional start site determined by Sahr et al. (71) is in boldface font and marked with an asterisk. The oxyR_Lp coding sequence is in italicized boldface font with the first nucleotide of the start codon designated +1. Numbering for the promoter region begins at the first nucleotide upstream of the oxyR_Lp start codon. (C and D) P_oxyR activity was assessed in L. pneumophila Lp02, ΔcpxR, ΔcpxA, and ΔcpxRA (C), as well as L. pneumophila Lp02, ΔcpxR ΔoxyR_Lp, ΔcpxA ΔoxyR_Lp, and ΔcpxRA ΔoxyR_Lp (D). Results in panels A, C, and D represent one of three independent experiments, with error bars representing standard errors of the means (SEM) of three technical replicates and ns indicating no significant differences in P_oxyR activity between the strains examined for the time period within the line segment, as determined by Student's t test with Welch's correction.

FIG 5

Purified recombinant CpxR is functional as it binds to the icmR promoter region (P_icmR). (A) Mobility shift assay performed with purified His₁₀-CpxR and ³²P-radiolabeled P_icmR region. The amount of His₁₀-CpxR used in each binding reaction is indicated above each lane. (B) DNase I protection assay with footprint denoted by the shaded bar. ³²P-radiolabeled P_icmR region was incubated with (+) or without (−) His₁₀-CpxR prior to treatment with DNase I. A Sanger sequencing ladder of the P_icmR region is indicated by G, A, T, and C. (C) Schematic of the P_icmR region. The CpxR binding region deduced by DNase I protection assay is boxed, with the sequence recognized by CpxR in boldface font with dashed underlining. Nucleotide numbering for the bound region corresponds to the distance from the translational start site. The −10 RNA polymerase binding site and transcriptional start site determined by Gal-Mor et al. (58) are designated by boldface font with underlining and boldface font with an asterisk, respectively. Arrows indicate primers used in promoter fragment generation, with the icmR coding sequence in boldface font and italicized.

FIG 6

CpxR binds to the oxyR_Lp promoter region (P_oxyR). (A) Mobility shift assay performed with purified His₁₀-CpxR and ³²P-radiolabeled P_oxyR region. The amount of His₁₀-CpxR used in each binding reaction is indicated above each lane. (B) DNase I protection assay with footprint denoted by the shaded bar. ³²P-radiolabeled P_oxyR region was incubated with (+) or without (−) His₁₀-CpxR prior to treatment with DNase I. A Sanger sequencing ladder of the P_oxyR region is indicated by G, A, T, and C. (C) Schematic of the P_oxyR region. The CpxR binding region deduced by DNase I protection assay is within the open box, with the sequence recognized by CpxR in boldface font with dashed underlining. The solid line of the box indicates readable sequence associated with the sequencing ladder, and the dashed line indicates continued footprint without corresponding readable sequencing ladder. Nucleotide numbering for the bound region corresponds to the distance from the translational start site. The transcriptional start site determined by Sahr et al. (71) is in boldface font and marked with an asterisk. Arrows indicate primers used in promoter fragment generation, with the oxyR_Lp coding sequence in boldface font and italicized.

FIG 7

OxyR_Lp negatively regulates the expression of icmR and the cpxRA two-component system. GFP reporter constructs were expressed in L. pneumophila Lp02 strains, and normalized fluorescence values (relative fluorescence units/optical density at 600 nm) were determined. L. pneumophila Lp02, ΔcpxR, ΔcpxA, ΔcpxRA, and ΔoxyR_Lp strains harbored the pJT502 reporter construct in which the P_icmR region drives the expression of GFP (A) or the pJT730 reporter construct in which the P_lpg1441-cpxA region drives the expression of GFP (B). Results represent one of three independent experiments conducted in triplicate. Error bars represent standard errors of the means (SEM) of three technical replicates.

FIG 8

OxyR_Lp directly interacts with the icmR (P_icmR) and lpg1441-cpxA (P_lpg1441-cpxA) promoter regions. (A and B) Schematic diagrams of the P_icmR region (A) and P_lpg1441-cpxA region (B) annotated with a potential OxyR_Lp binding site outlined within a box (BR), with the gray-highlighted font indicating the motifs likely recognized by OxyR_Lp and nonhighlighted font indicating the nucleotide spacer region. The transcriptional start site determined by Gal-Mor et al. (58) for P_icmR or by Sahr et al. (71) for P_lpg1441-cpxA is in boldface font and marked with an asterisk. The CpxR binding site identified within P_icmR by Gal-Mor and Segal (22) or identified within P_lpg1441-cpxA by Tanner et al. (25) is designated by italicized font, with the boldface font indicating the sequence bound by CpxR and the nonbold font indicating the nucleotide spacer. icmR and lpg1441 coding sequences are in boldface font, with the first nucleotide of the start codon designated +1 and the −10 RNA polymerase binding site of P_icmR determined by Gal-Mor et al. (58) indicated by underlining. Numbering for the promoter region begins at the first nucleotide upstream of the respective start codons for icmR and lpg1441. Arrows indicate primers used in promoter fragment generation. (C and D) Mobility shift assay performed with purified OxyR_Lp-His₆ and ³²P-radiolabeled P_icmR region (C) or ³²P-radiolabeled P_lpg1441-cpxA region (D). The amount of OxyR_Lp-His₆ used in each binding reaction is indicated above each lane.

FIG 9

OxyR_Lp regulation of icmR and the cpxRA two-component system is secondary to the regulation elicited by CpxR. GFP reporter constructs were expressed in L. pneumophila Lp02 strains, and normalized fluorescence values (relative fluorescence units/optical density at 600 nm) were determined. L. pneumophila Lp02, ΔcpxR ΔoxyR_Lp, ΔcpxA ΔoxyR_Lp, and ΔcpxRA ΔoxyR_Lp harbored the pJT502 reporter construct in which the P_icmR region drives the expression of GFP (A) or the pJT730 reporter construct in which the P_lpg1441-cpxA region drives the expression of GFP (B). Results represent one of three independent experiments conducted in triplicate. Error bars represent standard errors of the means (SEM) of the results from three technical replicates.

FIG 10

Summary of OxyR_Lp and CpxR regulatory interactions. OxyR_Lp negatively (T-bar) regulates the expression of the cpxRA system and icmR by directly interacting with their respective promoters. This interaction, however, is secondary to the regulatory effects elicited by CpxR, where CpxR positively (solid arrows) regulates itself (autoregulation) (25), substrates of the type II secretion system (25), and Dot/Icm system components and chaperones (21, 22), as well as Dot/Icm system effectors, in which CpxR also elicits negative regulation (21, 22, 25, 51). oxyR_Lp expression is likely enhanced or repressed (dotted arrow or T-bar, respectively) by the direct binding of CpxR to its promoter region under conditions still to be determined (question mark) (Feldheim et al. [51] and this work). The direct binding of CpxR to the oxyR_Lp promoter region and OxyR_Lp's direct repression of the promoter of cpxRA generate a regulatory loop, where CpxR has potential to indirectly enhance or suppress its expression through the direct control of oxyR_Lp expression. Open arrowheads depict promoter regions of the indicated downstream genes.