A conserved OmpA-like protein in Legionella pneumophila required for efficient intracellular replication

Abstract

The OmpA-like protein domain has been associated with peptidoglycan-binding proteins, and is often found in virulence factors of bacterial pathogens. The intracellular pathogen Legionella pneumophila encodes for six proteins that contain the OmpA-like domain, among them the highly conserved uncharacterized protein we named CmpA. Here we set out to characterize the CmpA protein and determine its contribution to intracellular survival of L. pneumophila Secondary structure analysis suggests that CmpA is an inner membrane protein with a peptidoglycan-binding domain at the C-teminus. A cmpA mutant was able to replicate normally in broth, but failed to compete with an isogenic wild-type strain in an intracellular growth competition assay. The cmpA mutant also displayed significant intracellular growth defects in both the protozoan host Acanthamoeba castellanii and in primary bone marrow-derived macrophages, where uptake into the cells was also impaired. The cmpA phenotypes were completely restored upon expression of CmpA in trans The data presented here establish CmpA as a novel virulence factor of L. pneumophila that is required for efficient intracellular replication in both mammalian and protozoan hosts.

Keywords: Dot/Icm; Legionella; OmpA-Like; intracellular pathogen; secretion system; vacuolar pathogen.

Figure 1.

CmpA belongs to the C-OmpA-like family of L. pneumophila proteins. (A) Schematic representation of predicted protein domains found in the CmpA protein sequence. The cmpA gene encodes for a protein 249 amino acids in length. Two putative transmembrane domains are depicted as gray rectangles marked TM1 and TM2, predicted using both TMpred (Hofmann and Stoffel 1993) and the SMART database (Letunic, Doerks and Bork 2012). TM1 ranges between amino acids 29 and 47, and TM2 ranges between amino acids 76 and 98. The SMART database also predicts the presence of two additional domains, defined by the Pfam database (Finn et al. 2013). A glycine zipper domain, Pfam PF13488, is predicted at amino acid range 61–103 and portrayed by a white rectangle encompassing TM2. The OmpA-like domain, Pfam PF00691, ranges between amino acids 132 and 228 and is represented by a black rectangle. The length and location of the different domains are shown to scale, with the ruler at the bottom marking residue numbers. (B) Multiple sequence alignment of all six C-OmpA-like proteins found in L. pneumophila. Sequence alignment was performed using the T-Coffee server (Notredame, Higgins and Heringa ; Wallace et al. 2006) and color coded for degree of homology with pink denoting strong homology, as shown in the key. The C-OmpA-like region is highly conserved among all six proteins.

Figure 2.

Genotypic characterization of the cmpA mutant. The cmpA mutant was analyzed to verify the location of the transposon insertion within the cmpA gene. (A) A schematic representation of the Lpg0657 locus encoding the cmpA gene. The location of the transposon insertion at nucleotide 262 is marked with a large triangle, and the primers used to characterize the strain are marked by arrows, FWD indicating primer SN242, and REV indicating primer SN243. The two primers span the transposon insertion site, and are designed to generate a 400 base pair (bp) fragment in a PCR with the LP01 wild-type strain CR39, and a 1800 bp fragment in the isogenic cmpA transposon insertion mutant SN272. (B) Gel electrophoresis of genotyping PCRs using genomic DNA as template with primer pair SN242 and SN243. Lanes are marked on the top to indicate the strain tested, WT refers to the LP01strain CR39, and cmpA indicates the cmpA transposon mutant SN272. (C) Image showing an immunoblot probed with a monoclonal anti-Flag antibody for detection of the Flag-CmpA fusion protein expressed off the pIG1 plasmid. Whole cell lysates from L. pneumophila cmpA mutant harboring the pCmpA plasmid pIG1 (strain SN316) were analyzed. A specific band corresponding to a protein with a molecular weight slightly above 36 kDa was detected in whole-cell lysates from cells that have been induced with IPTG (+IPTG). The anti-Flag-reactive product was not detected in lysates isolated from cells that were not induced with IPTG (no IPTG). The positions of molecular weight standards (kDa) are indicated.

Figure 3.

The CmpA protein is required for efficient intracellular replication in both protozoan and mammalian hosts. L. pneumophila growth rates were determined in the protozoan host A. castellanii (A), and in mouse bone marrow-derived macrophages (B). Intracellular growth kinetics of L. pneumophila strain SN272 (cmpA, triangles) was compared to that of wild-type L. pneumophila strain CR39 (WT, closed circles), and ΔdotA mutant strain CR58 (ΔdotA, squares). Growth was significantly attenuated in the cmpA mutant, and this defect was fully complemented in the cmpA mutant expressing CmpA off a plasmid (cmpA pCmpA, inverted triangles). No difference in growth rate was detected in the wild-type strain overexpressing CmpA (WT pCmpA, open circles). Each time point represents the mean number of viable bacteria recovered from triplicate wells ± standard error of the mean. Error bars are obscured by data symbols in data points with very low error. Statistical significance was determined using a Student's t-test (***P ≤ 0.001, ****P ≤ 0.0001 vs WT). (C) Wild-type strain CR39 (WT, circles) and the isogenic mutant strain SN272 (cmpA, triangles) were mixed at a 1:1 ratio and used at an MOI of 0.25 in an intracellular competition assay in the protozoan host A. castellanii, as outlined in Materials and Methods. All experiments were repeated three times, and a representative experiment of each is shown.

Figure 4.

The cmpA mutant has an intermediate salt resistance phenotype. The ability of the different strains to grow in the presence of 100 mM NaCl was assayed by spotting bacteria on plain CYE plates (control) or CYE plates supplemented with 100 mM NaCl. Plate grown cells were scraped, resuspended in H₂O and adjusted for OD_{600 nm}. Ten-fold serial dilutions of the wild-type strain CR39 (WT), mutant strain SN272 (cmpA) and dotA-deficient strain CR58 (ΔdotA) were plated and allowed to grow at 37°C for 3 days. A representative experiment out of three repeats is shown.

Figure 5.

Uptake into mouse macrophages is reduced in the cmpA mutant. The efficiency of uptake into bone marrow-derived A/J mouse macrophages was tested using a gentamicin protection assay. L. pneumophila strains were used to infect macrophages at an MOI of 100, uptake was allowed to proceed for 10 min before the cells were treated with gentamicin to eliminate extracellular bacteria and cell lysates plated for CFU. CFU recovered from the cells infected with wild-type strain CR39 (WT), mutant strain SN272 (cmpA), dotA-deficient strain CR58 (ΔdotA) and the wild type and cmpA strains harboring plasmid pIG1 (WT pCmpA and cmpA pCmpA, respectively) are plotted. Uptake of the cmpA strain is reduced by roughly 50%, and is restored to wild-type levels in the complemented strain. Values represent the mean number of viable bacteria recovered from triplicate wells ± standard error of the mean. Statistical significance was determined using a Student's t-test (**P ≤ 0.01, ***P ≤ 0.001).