Salmonella enterica serovar Typhimurium is an intracellular pathogen that can survive and replicate within macrophages. One of the host defense mechanisms that
Salmonella encounters during infection is the production of reactive oxygen species by the phagocyte NADPH oxidase. Among them, hydrogen peroxide (H
2O
2) can diffuse across bacterial membranes and damage biomolecules. Genome analysis allowed us to identify five genes encoding H
2O
2 degrading enzymes: three catalases (KatE, KatG, and KatN) and two alkyl hydroperoxide reductases (AhpC and TsaA). Inactivation of the five cognate structural genes yielded the HpxF
− mutant, which exhibited a high sensitivity to exogenous H
2O
2 and a severe survival defect within macrophages. When the phagocyte NADPH oxidase was inhibited, its proliferation index increased 3.7-fold. Moreover, the overexpression of
katG or
tsaA in the HpxF
− background was sufficient to confer a proliferation index similar to that of the wild type in macrophages and a resistance to millimolar H
2O
2 in rich medium. The HpxF
− mutant also showed an attenuated virulence in a mouse model. These data indicate that
Salmonella catalases and alkyl hydroperoxide reductases are required to degrade H
2O
2 and contribute to the virulence. This enzymatic redundancy highlights the evolutionary strategies developed by bacterial pathogens to survive within hostile environments.
Salmonella is a facultative intracellular pathogen that is associated with gastroenteritis, septicemia, and typhoid fever. This gram-negative bacterium survives and replicates in macrophages during the course of infection and can be exposed to a number of stressful environments during its life cycle (
16). One of the host defense mechanisms that
Salmonella encounters upon infection is the production of superoxide anion O
2− by the phagocyte NADPH oxidase (
1,
25). This radical can pass the outer membrane of the bacteria and represents one of the major weapons used by the macrophage to kill engulfed pathogens (
18). Evidence that phagocyte-produced superoxide is a key mechanism for avoiding
Salmonella infection is clear: mice and humans who are genetically defective in superoxide production are significantly more susceptible to infection (
36,
38). Superoxide dismutases, located in the bacterial periplasm and in the cytoplasm, dismutate superoxide O
2− to hydrogen peroxide H
2O
2 and molecular oxygen. Unlike superoxide, hydrogen peroxide can diffuse readily across bacterial membranes and form HO hydroxyl radicals in the presence of Fe(II) (
18). These reactive oxygen species (ROS) can oxidize and damage proteins, nucleic acids, and cell membranes.To scavenge and degrade H
2O
2 molecules generated either as a by-product of aerobic metabolism or by the phagocyte NADPH oxidase,
Salmonella has evolved numerous defense mechanisms. The KatE and KatG catalases are involved in H
2O
2 degradation, with
katE being described as a member of the RpoS regulon (
17,
22) and
katG being OxyR dependent (
26,
39). Both enzymes share the ability to reduce hydrogen peroxide to water and molecular oxygen, and their role was shown to be predominant at millimolar concentrations of H
2O
2 since they do not require any reductant (
32). This observation is of particular importance, since these enzymes are not limited by the availability of a reductant, such as NADH, which cannot be generated fast enough to face a burst of H
2O
2. However, the
katG and
katE simple mutants, as well as the
katE katG double mutant, did not show any increased susceptibility in macrophage or virulence attenuation in mice (
5,
27). A possible reason could be the presence of a third nonheme and manganese-dependent catalase called KatN (
30). This enzyme may contribute to hydrogen peroxide resistance under certain environmental conditions, but its involvement in virulence remains unknown. Moreover,
katE,
katG, and
katN single mutants did not show any susceptibility to exogenous millimolar H
2O
2, essentially due to the compensatory function of the remaining catalases (
5,
30).Another family of enzymes was shown to play an alternative role in H
2O
2 scavenging: the alkyl hydroperoxide reductases. These proteins directly convert organic hydroperoxides to alcohols, e.g., hydrogen peroxide to water. The alkyl hydroperoxide reductase AhpC belongs to the two-cysteine peroxiredoxin family, and the gene encoding this enzyme was identified as a member of the OxyR regulon (
26,
39). The redox system consists of two proteins, AhpC and AhpF, with the latter being a thioredoxin reductase-like protein that contains two disulfide centers and transfers electrons from NADH to AhpC (
13). AhpC was shown to be a predominant scavenger at low concentrations of H
2O
2, mainly because its catalytic efficiency was better than those of catalases (
32). Recently the alkyl hydroperoxide reductase from
Helicobacter hepaticus, TsaA (
Thiol-
Specific
Antioxidant), was characterized (
24). The
tsaA mutant was found to be more sensitive to oxidizing agents like superoxide anion or
t-butyl hydroperoxide. Surprisingly, this mutant was more resistant than the wild-type to H
2O
2, essentially because the level of catalase was increased in this background (
24). In gastric pathogens, TsaA plays a critical role in the defense against oxygen toxicity that is essential for survival and growth (
2). Interestingly,
Salmonella contains two genes encoding alkyl hydroperoxide reductases,
ahpC and
tsaA, whereas a single copy was found in
Escherichia coli (
ahpC) or in
Helicobacter pylori (
tsaA).The redundancy of these antioxidant proteins could explain the extremely high resistance of
Salmonella to hydrogen peroxide. It has been shown by Imlay and coworkers that in
E. coli, three genes were involved in H
2O
2 scavenging: two catalase genes (
katE and
katG) and an alkyl hydroperoxide reductase gene (
ahpC) (
32). Simultaneous inactivation of the
katE,
katG, and
ahpCF genes negated H
2O
2 degradation. As a consequence, this triple mutant, called the Hpx
− mutant, accumulates intracellular H
2O
2 (
32). Moreover, H
2O
2 generated by aerobic metabolism was found to be sufficient to create toxic levels of DNA damage in such a background (
28). In the present study, we deleted the
Salmonella katE,
katG, and
ahpCF genes and two more genes absent in
E. coli,
katN and
tsaA, to obtain the HpxF
− mutant, which lacks three catalases and two alkyl hydroperoxide reductases. HpxF
− cells exhibited the incapacity to degrade micromolar concentrations of H
2O
2, whereas this phenotype was not observed for the Kat
− (
katE katG katN) and Ahp
− (
ahpCF tsaA) mutants. Therefore, the HpxF
− mutant exhibited a high sensitivity to this compound. Moreover, this mutant did not show any proliferation within macrophages and presented reduced virulence in mice, suggesting that
Salmonella catalases and alkyl hydroperoxide reductases form a redundant antioxidant arsenal essential for survival and replication within host cells.
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