IcgA is a virulence factor of Rhodococcus equi that modulates intracellular growth

Abstract

Virulence of the intracellular pathogen Rhodococcus equi depends on a 21.3-kb pathogenicity island located on a conjugative plasmid. To date, the only nonregulatory pathogenicity island-encoded virulence factor identified is the cell envelope-associated VapA protein. Although the pathogenicity islands from porcine and equine R. equi isolates have undergone major rearrangements, the virR operon (virR-icgA-vapH-orf7-virS) is highly conserved in both, suggesting these genes play an important role in pathogenicity. VirR and VirS are transcriptional regulators controlling expression of pathogenicity island genes, including vapA. Here, we show that while vapH and orf7 are dispensable for intracellular growth of R. equi, deletion of icgA, formerly known as orf5, encoding a major facilitator superfamily transport protein, elicited an enhanced growth phenotype in macrophages and a significant reduction in macrophage viability, while extracellular growth in broth remained unaffected. Transcription of virS, located downstream of icgA, and vapA was not affected by the icgA deletion during growth in broth or in macrophages, showing that the enhanced growth phenotype caused by deletion of icgA was not mediated through abnormal transcription of these genes. Transcription of icgA increased 6-fold within 2 h following infection of macrophages and remained significantly higher 48 h postinfection compared to levels at the start of the infection. The major facilitator superfamily transport protein IcgA is the first factor identified in R. equi that negatively affects intracellular replication. Aside from VapA, it is only the second pathogenicity island-encoded structural protein shown to play a direct role in intracellular growth of this pathogenic actinomycete.

FIG 1

Reverse transcriptase PCR of pathogenicity island genes confirming the phenotypes of R. equi ΔvapGΔ(icgA-vapH-orf7) and individual icgA, vapH, and orf7 deletion mutants. Total RNA was extracted and used to generate cDNA from wild-type and mutant strains, followed by PCR using primers (Table 2) that specifically amplify internal regions of icgA, vapH, orf7, vapA, and the chromosomal gene gyrB. Standard DNA markers (M) are indicated on the left of each panel. (A) Lane 1, R. equi 103S; lane 2, R. equi 103S^P−; lane 3, R. equi ΔvapGΔ(icgA-vapH-orf7); lane 4, R. equi ΔvapGΔ(icgA-vapH-orf7)/(icgA-vapH-orf7). (B) Lane 1, R. equi 103S; lane 2, R. equi 103S^P−; lane 3, R. equi ΔicgA; lane 4, R. equi ΔicgA/icgA; lane 5, R. equi ΔvapH; lane 6, R. equi 103S; lane 7, R. equi Δorf7.

FIG 2

Deletion of a DNA fragment containing icgA, vapH, and orf7 results in enhanced intracellular growth. Intracellular growth of R. equi strains was assessed in murine bone marrow-derived macrophage monolayers infected with R. equi 103S, 103S^P−, R. equi ΔvapGΔ(icgA-vapH-orf7), and R. equi ΔvapGΔ(icgA-vapH-orf7)/(icgA-vapH-orf7) (complemented strain) at an MOI of 10. Following incubation for 1 h to allow phagocytosis, monolayers were washed and treated with amikacin to kill any remaining extracellular bacteria (t = 0 h). Macrophage monolayers were lysed in triplicate at 1 h, 24 h, and 48 h postinfection. Lysates were plated onto BHI agar plates to determine the associated CFU. (A) Intracellular growth of R. equi strains following infection of macrophages. (B) Fold change in CFU of intracellular bacteria at 24 h and 48 h postinfection relative to 1 h postinfection (hpi). Error bars represent the standard deviations. Horizontal lines represent the statistical significance of fold changes in CFU per monolayer for R. equi strains represented by bars at the end of each line. N.S., not significant. Data are representative of three independent experiments.

FIG 3

Deletion of icgA results in an enhanced intracellular growth phenotype, whereas vapH and orf7 are dispensable for growth in macrophages. Murine bone marrow-derived macrophage monolayers were infected with R. equi wild-type and mutant strains at an MOI of 10. Following incubation for 1 h to allow phagocytosis, monolayers were washed and treated with amikacin to kill any remaining extracellular bacteria (t = 0 h). Macrophage monolayers were lysed in triplicate at 1 h, 24 h, and 48 h postinfection. Lysates were plated onto BHI agar plates to determine the associated CFU. Shown are the fold changes in CFU of intracellular bacteria at 24 h and 48 h postinfection relative to levels at 1 h postinfection. (A) Infection of macrophage monolayers with R. equi 103S and R. equi ΔvapH. (B) Infection of macrophage monolayers with R. equi 103S and R. equi Δorf7. (C) Infection of macrophage monolayers with R. equi 103S, R. equi 103S^P−, R. equi ΔicgA, and R. equi ΔicgA/icgA. Error bars represent the standard deviations. Horizontal lines represent the statistical significance of fold changes in CFU per monolayer for R. equi strains, represented by bars at the end of each line. Data are representative of three independent experiments. N.S., not significant; hpi, hours postinfection.

FIG 4

Deletion of icgA does not affect extracellular growth of R. equi. R. equi 103S, R. equi ΔicgA, and R. equi ΔicgA/icgA were grown in LB broth under vapA-inducing growth conditions (pH 5.5, 37°C). Data represent averages from three independent experiments. Standard deviations did not exceed 10% and are omitted for clarity.

FIG 5

Enhanced intracellular growth of R. equi ΔicgA is not due to increased transcription of virS or vapA in vitro or in vivo. The transcript levels of virS and vapA were determined following growth of R. equi 103S, R. equi ΔicgA, and R. equi ΔicgA/icgA. The transcript levels of virS and vapA were assessed by quantitative PCR and normalized to 16S rRNA. The data are expressed as a fold change in transcript levels relative to the level for R. equi 103S. (A) R. equi strains were grown in LB broth under vapA-inducing growth conditions (pH 5.5, 37°C). RNA was extracted when the culture reached an OD₆₀₀ of 1.0. The data are representative of three independent experiments in which each sample was measured in triplicate. (B) Murine J774A.1 macrophages were infected with R. equi strains. Following an incubation period (1 h) to allow phagocytosis, monolayers were washed and treated with vancomycin to kill extracellular bacteria (t = 0 h). At 24 h postinfection, bacterial RNA was extracted from lysed macrophages. Data shown are representative of two independent experiments in which each sample was measured in triplicate. Error bars reflect the standard deviations.

FIG 6

Transcriptional profile of the icgA gene during infection of macrophages with R. equi 103S. Following a 1-h incubation period to allow phagocytosis, murine macrophage-like cell line J774A.1 monolayers were washed and treated with vancomycin to kill extracellular bacteria (t = 0 h). The icgA transcript levels were determined by reverse transcriptase quantitative PCR using the transcript levels of the 16S rRNA gene as a reference for normalization. Fold changes in gene expression are relative to t = 0. Error bars denote the standard errors of the means. Results are from two independent experiments in which each sample was analyzed in triplicate (*, P < 0.05; **, P < 0.01).

FIG 7

Enhanced intracellular growth phenotype of R. equi ΔicgA decreases macrophage viability. Murine J774A.1 macrophages were infected with R. equi 103S, R. equi ΔicgA, and R. equi ΔicgA/icgA. Following an incubation period (1 h) to allow phagocytosis, monolayers were washed and treated with vancomycin to kill extracellular bacteria (t = 0 h). Macrophage viability was assessed at 0, 24, and 48 h postinfection using the trypan blue exclusion assay. Results are from three independent experiments (*, P < 0.05). Error bars reflect the standard deviations.