Redundant hydrogen peroxide scavengers contribute to Salmonella virulence and oxidative stress resistance

Abstract

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(2)O(2)) can diffuse across bacterial membranes and damage biomolecules. Genome analysis allowed us to identify five genes encoding H(2)O(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(2)O(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(2)O(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(2)O(2) and contribute to the virulence. This enzymatic redundancy highlights the evolutionary strategies developed by bacterial pathogens to survive within hostile environments.

FIG. 1.

E. coli Hpx⁻ and Salmonella HpxF⁻ strains share the same growth defect linked to filamentation. S. Typhimurium 12023 (wild type; filled squares), E. coli MG1655 (wild type; filled circles), S. Typhimurium HpxF⁻ (katE katG katN aphCF tsaA; open triangles), S. Typhimurium HpxT⁻ (katE katG aphCF; open squares), and E. coli Hpx⁻ (katE katG aphCF; open circles) cells were grown in LB medium anaerobically until early log phase. The cells were then diluted in aerobic LB medium to an OD₆₀₀ of 0.05, and aerobic growth was monitored at 600 nm (A and B). At OD₆₀₀s of 0.1, 0.5, and 1, cells from the five strains were removed for microscopic observations (C) (magnification, ×100).

FIG. 2.

Endogenous H₂O₂ does not affect HpxF⁻ mutant viability. (A) Cultures of Salmonella Typhimurium 12023 (wild type; filled squares), the Ahp⁻ mutant (aphCF tsaA; filled circles), Kat⁻ (katE katG katN, open squares), and the HpxF⁻ mutant (katE katG katN aphCF tsaA; open circles) were grown overnight aerobically in minimal medium supplemented with Casamino Acids, diluted to an OD of 0.05, and grown for four generations. The cells were then washed in fresh medium and incubated, and aliquots were removed every 5 min to measure H₂O₂ concentrations as described in Materials and Methods. (B) Wild-type, Kat⁻, Ahp⁻, and HpxF⁻ strains were grown in LB medium. At OD₆₀₀s of 0.5 (white bars) and 2 (gray bars), aliquots were removed from each culture and plated on LB agar supplemented with catalase to determine cell viability. Results are the means ± standard deviations for three independent experiments, each in triplicate. (C) Wild-type, Kat⁻, Ahp⁻, and HpxF⁻ cells were grown in minimal medium overnight. The cells were then diluted to an OD₆₀₀ of 0.1, and aerobic growth was monitored at 600 nm.

FIG. 3.

The HpxF⁻ mutant is highly sensitive to exogenous hydrogen peroxide. (A) Wild-type (filled squares), Ahp⁻ (filled circles), Kat⁻ (open squares), HpxF⁻ (open circles), and heat-killed wild-type (filled triangles) cells were grown in minimal medium to an OD₆₀₀ of 0.3 and then diluted at an OD₆₀₀ of 0.1. H₂O₂ was added at a final concentration of 10 μM. Aliquots were removed every 90 s to assay H₂O₂ levels as described in Materials and Methods. (B) Bacterial cultures (wild type, Ahp⁻, Kat⁻, and HpxF⁻) grown to an OD₆₀₀ of 0.3 in LB medium were treated with 1 mM hydrogen peroxide (arrow). The growth was then monitored at 600 nm. (C) Viability was assayed, respectively, before (white bars) and 1 h (gray bars) and 2 h (black bars) after H₂O₂ treatment by plating the bacterial cells on LB agar supplemented with catalase. Results are the means ± standard deviations for three independent experiments, each in triplicate.

FIG. 4.

HpxF⁻ mutations led to a drastic attenuation of intracellular proliferation within bone marrow-derived and RAW 264.7 macrophages. Opsonized bacteria (Wild type or Kat⁻, Ahp⁻, or HpxF⁻ mutant) were phagocytosed by freshly prepared bone marrow-derived macrophages (A) or RAW 264.7 cells (B), both activated with IFN-γ and PMA. Two and sixteen hours postinfection, mouse macrophages were lysed for enumeration of intracellular bacteria (gentamicin protected), determined by CFU counts. The values shown represent the proliferation index, calculated as a ratio of the intracellular bacteria between 16 and 2 h postinfection. (C) The HpxF⁻ mutant and the wild-type strain were tested for infection of activated RAW 267.4 cells without or with 1 μM DPI, which inhibits the phagocyte NADPH oxidase. The proliferation index was calculated as described above. The inset represents the proliferation index ratio (wild type/HpxF⁻) without or with 1 μM DPI. Results presented in the three panels are the means ± standard deviations for at least three independent experiments, each in triplicate.

FIG. 5.

Expression pattern of catalase- and alkyl hydroperoxide reductase-encoding genes in LB and RAW 264.7 macrophages. RNA was extracted from the Salmonella wild-type strain grown either in LB medium to an OD₆₀₀ of 2 or within activated RAW 264.7 cells during 6 h. cDNA were synthesized from 1 μg RNA, and real-time PCR was performed to amplify the katE, katG, katN, ahpC, tsaA genes. A noncoding (n.c.) DNA domain located between STM0413 and STM0414 was used as a control to monitor the basal level of expression due to residual genomic DNA. The results are the means ± standard deviations for three independent experiments.

FIG. 6.

pkatG and ptsaA complement the HpxF⁻ mutant with different efficiencies. Bacterial cultures (wild type, filled symbols; HpxF⁻, open symbols) were transformed with empty vector (squares), the pkatG plasmid (circles), or the ptsaA plasmid (triangles). The cells grown to an OD₆₀₀ of 0.3 and were treated with 1 mM H₂O₂ (arrow). Growth was monitored at an OD of 600 nm (A), and the viability was assayed before (white bars) and 1 h (gray bars) and 2 h (black bars) after H₂O₂ treatment (B). Wild-type and HpxF⁻ strains were transformed with empty vector, pkatG, and ptsaA and were tested for infection within activated RAW 264.7 cells. Bacterial cells were plated and counted on LB agar supplemented with chloramphenicol and catalase. The values shown represent the proliferation index, calculated as a ratio of the intracellular bacteria between 16 and 2 h postinfection (C). Results presented in panels B and C are the means ± standard deviations for at least three independent experiments, each in triplicate.