H(2)O(2), which causes macrophage-related stress, triggers induction of expression of virulence-associated plasmid determinants in Rhodococcus equi

Abstract

The response of the intracellular pathogen Rhodococcus equi to H(2)O(2) treatment, a situation potentially encountered after the oxidative burst of alveolar macrophages, was analyzed. Compared to other bacteria, including Deinococcus radiodurans, R. equi showed exceptionally high resistance to this stress. A proteomic approach showed that four polypeptides present in the wild-type strain (85F) are missing in the plasmid-cured strain 85F(P-), and by using a DNA macroarray, we identified two plasmid-encoded vap genes, vapA and vapG, whose expression was highly induced by H(2)O(2) treatment. Whereas the transcript size of vapA was compatible with a monocistronic mRNA, the transcript of vapG was considerably longer. Rapid amplification of cDNA ends PCRs showed that the transcriptional start sites of the two operons were 69 and 269 nucleotides (nt) upstream of the start codon, respectively. Analysis of these leader sequences revealed the presence of a small open reading frame named podG, which encodes a sequence of 55 amino acids preceded by a putative ribosome binding site sequence in the vapG transcript. Taking this result into account, the untranslated leader of the podG/vapG operon is 87 nt. Alignment of this sequence with the leader sequences of vapA and vapD, genes previously shown to be induced by acid, revealed significant homologies. Since our results showed that vapA, vapD, and vapG are genes highly induced by macrophage-related stresses, their gene products may, within the Vap protein family, play a dominant role inside these phagocytic cells and may be the most promising candidates for vaccination strategies.

FIG. 1.

Effect of H₂O₂ treatments on R. equi 85F survival. Cultures were grown to mid-exponential growth phase and either unstressed (•) or treated with H₂O₂ at concentrations of 10 mM (○), 50 mM (▴), 100 mM (▵), and 150 mM (▪) for 60 min. The survival of two reference bacteria, E. coli (□) and E. faecalis (◊) in the presence of the highest H₂O₂ dose (150 mM) after 5, 10, and 20 min, determined under the same conditions, are also shown. The values are the averages of results from three independent experiments. Comparable results were obtained with the plasmid-cured strain.

FIG. 2.

Silver-stained two-dimensional gels of cytoplasmic proteins from the R. equi strains 85F (A and B) and 85F(P-) (C and D). The protein patterns of untreated (A and C) and H₂O₂-treated (10 mM for 45 min) (B and D) cultures are shown. Molecular mass (MW) is shown on the y axis, and pI is shown the x axis. Proteins overexpressed in strains 85F and 85F(P-) (black arrows) and polypeptides whose expression is specifically induced in strain 85F (white arrows) are indicated. Only reproducible changes observed from at least three individual experiments are indicated.

FIG. 3.

Structural organization of the genetic environment of the vapG gene. Large arrows represent the ORFs, and their orientation shows the transcriptional direction. The distances between vapG and ORF3 and the putative Rho-independent terminator (T_G) located downstream of the vapG stop codon are shown.

FIG. 4.

Expression analysis of the R. equi 85F vap genes. Northern blot analysis of vapA (A) and vapG (B) under oxidative stress conditions. Total RNA was isolated from strain 85F after 10-min incubation without H₂O₂ (lane 1) and with H₂O₂ at concentrations of 10, 25, and 50 mM (lanes 2 to 4, respectively). Hybridization was done using vapA- and vapG-specific probes, respectively. Only the relevant parts of autoradiograms are shown.

FIG. 5.

Western blot analysis of VapA. Total proteins were extracted after 15 min without H₂O₂ (lane 1) or 15, 30, and 45 min in the presence of 25 mM H₂O₂ (lanes 2 to 4, respectively). In all cases, only the relevant parts of the autoradiograms are shown.

FIG. 6.

Sequences of the vapA (A), vapG (B), and vapD (C) promoter regions. The transcriptional initiation nucleotides (+1), the putative −10 and −35 boxes, and the RBS sequences are indicated. Electrophoretograms obtained from 5′ RACE PCR experiments are shown. The sequences were obtained using Rho1c, G8, and Dtr2 primers for vapA, vapG, and vapD, respectively, and cDNA from 5′ dA-tailed RNA using RACE kit (Roche Molecular Biochemicals). The TSS (G, A, and G for vapA, vapG, and vapD, respectively) are indicated above. (D) Alignment of the spacers between the transcriptional initiation nucleotide and the RBS sequence of the vap genes. Gaps introduced to maximize alignment are indicated by the dashes.

FIG. 7.

(A) Spacer region of vapG with the amino acid sequence of the putative ORF (podG). The TSS of the vapG mRNA, determined by 5′ RACE PCR, is indicated by the arrow. Putative RBS sequences for vapG and podG are shown in boldface type. A putative −10 box is indicated. (B) Hydrophilicity plot of PodG obtained using MacVector software. Positive and negative values on the Kyte-Doolittle scale correspond to hydrophilic and hydrophobic domains, respectively.