Characterization of the role of the pathogenicity island and vapG in the virulence of the intracellular actinomycete pathogen Rhodococcus equi

Abstract

Rhodococcus equi, a facultative intracellular pathogen of macrophages, causes severe, life-threatening pneumonia in young foals and in people with underlying immune deficiencies. R. equi virulence is dependent on the presence of a large virulence plasmid that houses a pathogenicity island (PAI) encoding a novel family of surface-localized and secreted proteins of largely unknown function termed the virulence-associated proteins (VapACDEFGHI). To date, vapA and its positive regulators virR and orf8 are the only experimentally established virulence genes residing on the virulence plasmid. In this study, a PAI deletion mutant was constructed and, as anticipated, was attenuated for growth both in macrophages and in mice due to the absence of vapA expression. Expression of vapA in the PAI mutant from a constitutive promoter, thereby eliminating the requirement for the PAI-encoded vapA regulators, resulted in delayed bacterial clearance in vivo, yet full virulence was not restored, indicating that additional virulence genes are indeed located within the deleted pathogenicity island region. Based on previous reports demonstrating that the PAI-carried gene vapG is highly upregulated in macrophages and in the lungs of R. equi-infected foals, we hypothesized that vapG could be an important virulence factor. However, analysis of a marked vapG deletion mutant determined the gene to be dispensable for growth in macrophages and in vivo in mice.

FIG. 1.

Confirmation of the PAI deletion. (A) Schematic of the pathogenicity island and locations of the PCR primers (arrows) used in analysis of the PAI mutant. (B) Results of PCR analysis of the PAI mutant using primers annealing within the deleted region. Primers specific for either vapA, -C, -D, -F, -G, or -H were used to confirm the deletion in the mutant. PCR amplicons for these primer pairs are shown. Total DNA from R. equi 103− (lanes −), R. equi 103+ (lane +), and the PAI mutant (lanes Δ) was used as the template in PCRs. DNA standards (lanes M) are shown to the left of each group of lanes. (C) Results of PCR analysis of the virulence plasmid backbone in the PAI mutant. Primer pairs annealing to different regions spanning the virulence plasmid outside the deleted pathogenicity island region were used in PCRs using the same template DNAs as for panel A. Annealing site coordinates are shown in parentheses under the respective primer pair.

FIG. 2.

Expression of VapA in the PAI mutant. (A) Western blot analysis. Whole-cell extracts were prepared from R. equi 103+ (lane 1), virulence plasmid-cured 103− (lane 2), 103−/vapA (lane 3), 103+ ΔvapA (lane 4), 103+ ΔvapA/vapA (lane 5), ΔPAI (lane 6), and ΔPAI/vapA (lane 7), all cultured at 37°C as described in Materials and Methods. The presence of VapA protein was detected with rabbit polyclonal antiserum to VapA. Sizes of protein molecular mass standards are indicated on the left. (B) Flow cytometry profile showing the expression of VapA on the surface of R. equi. Bacteria were grown in liquid broth at 37°C, washed, and stained with a polyclonal antiserum to VapA. The relative median fluorescence values of wild-type isolate 103+, the PAI mutant strain (ΔPAI), and the recombinant pMV261-vapA-transformed PAI mutant strain (ΔPAI/vapA) were compared. Analyses included a comparison to bacteria stained with an irrelevant antibody (control; preimmune serum). The number in the upper left corner of each histogram represents the median fluorescence intensity for each strain. The values above the gate reflect the percentage of VapA-positive cells within the population analyzed.

FIG. 3.

Expression of vapA fails to restore the intracellular growth defect of the PAI mutant. (A) RAW264.7 macrophage monolayers were infected with R. equi strains 103+, 103−, ΔPAI, and ΔPAI/vapA at an MOI of 10:1. Following uptake and repeated washing to remove unbound bacteria and the addition of antibiotic to kill any remaining extracellular bacteria, triplicate macrophage monolayers were lysed at 1 h, 24 h, and 48 h postinfection. Lysates were plated onto BHI agar plates to determine the associated CFU. The data are expressed as means ± standard deviations. The graphs shown here are representative of three independent experiments. (B) Fold change in the CFU of intracellular bacteria at 24 h and 48 h postinfection relative to 1 h postinfection (hpi). A positive ratio reflects an increase in bacterial CFU over time resulting from bacterial replication in macrophages, whereas a negative ratio reflects a decrease in bacterial number over time. Values shown are the means ± standard deviations for triplicate monolayers from an individual experiment. The data shown are representative of three independent experiments.

FIG. 4.

Expression of vapA fails to rescue the PAI mutant of R. equi from clearance in SCID mice. SCID mice were injected intravenously with 5 × 10⁶ CFU of R. equi 103+, 103−, 103−/vapA, 103+ ΔvapA, 103+ ΔvapA/vapA, ΔPAI, and ΔPAI/vapA. At 0, 2, 7, and 14 days postinfection, mice were humanely sacrificed and organs removed aseptically. The total numbers of bacteria in the spleens (A), lungs (B), and livers (C) were determined by dilution plating of organ homogenates. Each point on the graph represents the mean ± standard deviation of bacterial counts for four mice.

FIG. 5.

Confirmation of deletion of vapG. (A) PCR analysis of the vapG mutant. Primer pairs that specifically amplify regions across the deletion site were used in PCRs in which total DNA from R. equi 103+ (+) and the vapG mutant (Δ) served as the template. Standard DNA markers are indicated on the left (M). A no-template control (−) is also shown. (B) RT-PCR analysis of vapG expression. Total RNA was extracted from R. equi strains 103+ (+), the vapG mutant (Δ), and the complemented vapG mutant (Δ/G). cDNA was synthesized using equivalent concentrations of total RNA as the template. The presence of vapG and gyrB (internal control) was assessed by PCR using primer pairs specific for internal regions of each of the genes. Standard DNA markers are indicated on the left (M). No-template controls (−) and no-RT controls (NRT) are shown.

FIG. 6.

vapG is dispensable for growth in murine macrophages. (A) Intracellular replication of R. equi strains in RAW264.7 macrophages. Macrophage monolayers were infected with R. equi 103+, 103−, 103+ ΔvapG, and 103+ ΔvapG/vapG, and bacterial intracellular replication was followed as described in the legend to Fig. 3. (B) Fold change in intracellular bacterial CFU among the various strains at 24 h and 48 h postinfection (hpi). Values shown are the means ± standard deviations for monolayers assessed in triplicate. The data shown here are representative of three independent experiments.

FIG. 7.

vapG is dispensable for growth in equine alveolar macrophages. Intracellular replication of R. equi strains in equine alveolar macrophages is shown. (A) The number of bacteria associated with 200 macrophages was determined at 1 h, 24 h, and 48 h postinfection by fluorescence microscopy. (B) Number of macrophages with 10 or more intracellular bacteria at the indicated time points postinfection. Values shown are the means ± standard deviation for monolayers assessed in triplicate. The data shown here are representative of three independent experiments. (C) Representative epifluorescence microscopy images of equine macrophages infected with R. equi strains 103+, 103−, 103+ ΔvapG, and 103+ ΔvapG/vapG taken at 1 h, 24 h, and 48 h postinfection. Scale bar, 10 μm; magnification, ×400.

FIG. 8.

vapG is dispensable for survival in vivo. SCID mice were infected intravenously with 5 × 10⁵ CFU of R. equi 103+, 103−, 103+ ΔvapG, and 103+ ΔvapG/vapG. At 0, 2, 7, and 10 days postinfection, mice were humanely sacrificed and organs removed and homogenized. The total numbers of bacteria in the spleens (A), lungs (B), and livers (C) were determined by dilution plating of the organ homogenates. Each point on the graph represents the mean ± standard deviation of bacterial CFU of four mice.