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

Bidaud P, Hébert L, Barbey C, Appourchaux AC, Torelli R, Sanguinetti M, Laugier C, Petry S - PLoS ONE (2012)

Bottom Line: Moreover, katA deletion seems to significantly affect the ability of R. equi to survive within murine macrophages.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.

View Article: PubMed Central - PubMed

Affiliation: Dozulé Laboratory for Equine Diseases, Unit Bacteriology and Parasitology, ANSES, Goustranville, France.

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.

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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 [36]. 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.
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pone-0042396-g004: 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 [36]. 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.

Mentions: We evaluated the effect of a sublethal H2O2 concentration (50 mM) defined previously (data not shown) on the expression of katA, katB, katC and katD transcripts during exponential (Figure 4A) and stationary phases (Figure 4B). The expression of catalases was measured at several timepoints: 5, 10 and 20 minutes after treatment in the exponential phase and 5, 10, 20, 30 and 60 minutes after treatment in the stationary phase. Results showed that only the katA gene was overexpressed in response to H2O2 treatment. For example, when H2O2 was added in the exponential phase, katA was overexpressed 3.11 (±0.59) times in bacteria treated for 5 min compared to untreated bacteria (Figure 4A). However, when H2O2 was added in the stationary phase, katA was overexpressed 367.90 (±122.63) times in bacteria treated for 10 min compared to untreated bacteria (Figure 4B). We did not observe any modifications in the rate of transcripts for katB, katC or katD (Figures 4A and 4B).


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

Bidaud P, Hébert L, Barbey C, Appourchaux AC, Torelli R, Sanguinetti M, Laugier C, Petry S - PLoS ONE (2012)

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 [36]. 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.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3412833&req=5

pone-0042396-g004: 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 [36]. 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.
Mentions: We evaluated the effect of a sublethal H2O2 concentration (50 mM) defined previously (data not shown) on the expression of katA, katB, katC and katD transcripts during exponential (Figure 4A) and stationary phases (Figure 4B). The expression of catalases was measured at several timepoints: 5, 10 and 20 minutes after treatment in the exponential phase and 5, 10, 20, 30 and 60 minutes after treatment in the stationary phase. Results showed that only the katA gene was overexpressed in response to H2O2 treatment. For example, when H2O2 was added in the exponential phase, katA was overexpressed 3.11 (±0.59) times in bacteria treated for 5 min compared to untreated bacteria (Figure 4A). However, when H2O2 was added in the stationary phase, katA was overexpressed 367.90 (±122.63) times in bacteria treated for 10 min compared to untreated bacteria (Figure 4B). We did not observe any modifications in the rate of transcripts for katB, katC or katD (Figures 4A and 4B).

Bottom Line: Moreover, katA deletion seems to significantly affect the ability of R. equi to survive within murine macrophages.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.

View Article: PubMed Central - PubMed

Affiliation: Dozulé Laboratory for Equine Diseases, Unit Bacteriology and Parasitology, ANSES, Goustranville, France.

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.

Show MeSH
Related in: MedlinePlus