Limits...
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.

Show MeSH

Related in: MedlinePlus

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.
© Copyright Policy
Related In: Results  -  Collection


getmorefigures.php?uid=PMC3412833&req=5

pone-0042396-g002: 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.

Mentions: To evaluate the effect of exogenous H2O2 stress on the survival of each mutant, cells were challenged with 80 mM of H2O2 under agitation, and CFUs determined 30 min after treatment (Figure 2). ΔkatA was the most sensitive mutant to H2O2 since it did not survive when treated in the stationary phase, and its survival decreased by two orders of magnitude compared to the WT strain when treated in the exponential phase. ΔkatC survived as well as the WT strain in the stationary phase but in contrast, when the cells were in the exponential phase, ΔkatC was the most susceptible to H2O2 exposure with a decrease in survival of over three magnitudes compared to the WT strain. Both ΔkatB and ΔkatD had the same resistance capability as the WT strain, whether in exponential or stationary phases. The drop in survival rates for ΔkatA and ΔkatC after exposure to H2O2 demonstrate that, in these experimental conditions, only KatA and KatC primarily contribute to the oxidative defense system of R. equi.


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)

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.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3412833&req=5

pone-0042396-g002: 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.
Mentions: To evaluate the effect of exogenous H2O2 stress on the survival of each mutant, cells were challenged with 80 mM of H2O2 under agitation, and CFUs determined 30 min after treatment (Figure 2). ΔkatA was the most sensitive mutant to H2O2 since it did not survive when treated in the stationary phase, and its survival decreased by two orders of magnitude compared to the WT strain when treated in the exponential phase. ΔkatC survived as well as the WT strain in the stationary phase but in contrast, when the cells were in the exponential phase, ΔkatC was the most susceptible to H2O2 exposure with a decrease in survival of over three magnitudes compared to the WT strain. Both ΔkatB and ΔkatD had the same resistance capability as the WT strain, whether in exponential or stationary phases. The drop in survival rates for ΔkatA and ΔkatC after exposure to H2O2 demonstrate that, in these experimental conditions, only KatA and KatC primarily contribute to the oxidative defense system of R. equi.

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