Rhodococcus equi's extreme resistance to hydrogen peroxide is mainly conferred by one of its four catalase genes

Abstract

Rhodococcus equi is one of the most widespread causes of disease in foals aged from 1 to 6 months. R. equi possesses antioxidant defense mechanisms to protect it from reactive oxygen metabolites such as hydrogen peroxide (H(2)O(2)) generated during the respiratory burst of phagocytic cells. These defense mechanisms include enzymes such as catalase, which detoxify hydrogen peroxide. Recently, an analysis of the R. equi 103 genome sequence revealed the presence of four potential catalase genes. We first constructed ΔkatA-, ΔkatB-, ΔkatC-and ΔkatD-deficient mutants to study the ability of R. equi to survive exposure to H(2)O(2)in vitro and within mouse peritoneal macrophages. Results showed that ΔkatA and, to a lesser extent ΔkatC, were affected by 80 mM H(2)O(2). Moreover, katA deletion seems to significantly affect the ability of R. equi to survive within murine macrophages. We finally investigated the expression of the four catalases in response to H(2)O(2) assays with a real time PCR technique. Results showed that katA is overexpressed 367.9 times (± 122.6) in response to exposure to 50 mM of H(2)O(2) added in the stationary phase, and 3.11 times (± 0.59) when treatment was administered in the exponential phase. In untreated bacteria, katB, katC and katD were overexpressed from 4.3 to 17.5 times in the stationary compared to the exponential phase. Taken together, our results show that KatA is the major catalase involved in the extreme H(2)O(2) resistance capability of R. equi.

Figure 1. Phylogenetic relationships between the catalases of Rhodococcus equi 103.

Catalases were aligned using Phylogeny.fr (http://www.phylogeny.fr/) .

Figure 2. Decrease in ΔkatA and ΔkatC survival after exposure to H₂O₂ (80 mM, 30 min).

R. equi WT, ΔkatA, ΔkatB, ΔkatC or ΔkatD were grown to exponential (OD₆₀₀ = 0.2) and stationary growth phases (16 h of growth). The number of bacterial cells was standardized by dilution of stationary phase cells before treatment. Cells were challenged with 80 mM of H₂O₂ for 30 min under agitation. Viability was assayed both before (black bars) and after (grey and white bars) H₂O₂ treatment by plating the bacterial cells on BHI agar. The mean values of three independent experiments are represented, and the standard deviations are indicated.

Figure 3. ΔkatA is the most susceptible to elimination by macrophages.

Mice were infected with 10⁷ to 10⁸ cells of R. equi WT, ΔkatA, ΔkatB, ΔkatC or ΔkatD by intraperitoneal injection. Intramacrophages bacteria viability was evaluated 24-, 48-and 72 h post infection. The data are the mean numbers of viable intracellular bacteria per 10⁵ macrophages ± standard deviations (error bars) for three independent experiments with three wells in each experiment.

Figure 4. katA is overexpressed after H₂O₂ treatment (50 mM).

R. equi WT was treated with 50 mM H₂O₂ in (A) the exponential phase (OD₆₀₀ = 0.4) or (B) the stationary phase. cDNAs derived from total RNA were used for real time PCR. The overexpression factor in treated bacteria vs untreated bacteria, was calculated using the 2^−ΔΔCt method . The data are the mean of overexpression factor ± standard deviation (error bars) of triplicate measurements from four reverse transcriptions of two independent experiments. To evaluate overexpressions in the exponential phase (A), we only considered time points 5, 10 and 20 min post exposure to avoid any effects of bacterial transition in the stationary growth phase.

Figure 5. In untreated bacteria, katB, katC and katD are overexpressed in the stationary growth phase.

R. equi WT was grown in BHI broth without any H₂O₂ treatment and cells were collected at the exponential (OD of 0.4) and stationary (OD of 1) growth phases for total RNA extraction. cDNAs derived from total RNA, were used for real time PCR. The overexpression factor was calculated using the 2^−ΔΔCt method . The data are the mean of overexpression factor in stationary phase collected bacteria compared to exponential phase collected bacteria ± standard deviation of triplicate measurements from four reverse transcriptions of two independent experiments.