Listeria monocytogenes Has Both Cytochrome bd-Type and Cytochrome aa 3-Type Terminal Oxidases, Which Allow Growth at Different Oxygen Levels, and Both Are Important in Infection

Abstract

Listeria monocytogenes is a foodborne pathogen responsible for a number of life-threatening infections of humans. During an infection, it invades epithelial cells before spreading from the intestine to the cells of the liver and spleen. This requires an ability to adapt to varying oxygen levels. Here, we demonstrate that L. monocytogenes has two terminal oxidases, a cytochrome bd-type (CydAB) and a cytochrome aa ₃-type menaquinol (QoxAB) oxidase, and that both are used for respiration under different oxygen tensions. Furthermore, we show that possession of both terminal oxidases is important in infection. In air, the CydAB bd-type oxidase is essential for aerobic respiration and intracellular replication, and cydAB mutants are highly attenuated in mice. In contrast, the QoxAB aa ₃-type oxidase is required neither for aerobic respiration in air nor for intracellular growth. However, the qoxAB mutants are attenuated in mice, with a delay in the onset of disease signs and with increased survival time, indicating a role for the QoxAB aa ₃-type oxidase in the initial stages of infection. Growth of bacteria under defined oxygen conditions revealed that at 1% (vol/vol), both oxidases are functional, and the presence of either is sufficient for aerobic respiration and intracellular replication. However, at 0.2% (vol/vol), both oxidases are necessary for maximum growth. These findings are consistent with the ability of L. monocytogenes to switch between terminal oxidases under different oxygen conditions, providing exquisite adaptation to different conditions encountered within the infected host.

Keywords: Listeria monocytogenes; cytochromes; foodborne pathogens; host-pathogen interactions; oxygen.

FIG 1

Difference spectra of the strains used in this study. (A) Reduced (dithionite) minus oxidized (persulfate) difference spectra of intact cell suspensions of the indicated strains (WT, wild type). The spectra were recorded at room temperature. (B) Reduced (dithionite) plus CO minus reduced difference spectra of intact cell suspensions of the same strains. The spectra were recorded at room temperature. Distinctive features of the spectra are indicated (nanometers). Other conditions are given in Materials and Methods. Protein concentrations: wild type, 12.97 mg ml⁻¹; ΔcydAB, 7.57 mg ml⁻¹; Δqox, 22.86 mg ml⁻¹; ΔcydABΔqox, 18.54 mg ml⁻¹.

FIG 2

Growth (optical density at 600 nm [OD₆₀₀]) in vitro of the wild type (diamonds) and ΔcydAB (squares) and ΔqoxAB (triangles) mutants in TSB at 37°C in 1% (vol/vol) oxygen (A) or 0.2% (vol/vol) oxygen (B). The data are the means of the results of at least 3 independent experiments ± SE.

FIG 3

Growth (OD₆₀₀) in TSB of the wild type with (filled diamonds) or without (open diamonds) 10 mM ASN, the ΔcydAB mutant with (filled squares) or without (open squares) 10 mM ASN, and the ΔqoxAB mutant with (filled circles) or without (unfilled circles) 10 mM ASN. The data are the means of the results of at least 3 independent experiments ± SE.

FIG 4

Comparison of the intracellular growth of ΔcydAB and ΔqoxAB mutants in epithelial cells. HeLa cells were infected at an MOI of 10 with either the wild type (diamonds) or the ΔcydAB (squares) or ΔqoxAB (triangles) mutant and grown in air (A) or 1% (vol/vol) oxygen (B). Bacterial growth was assessed by lysing the HeLa cells at intervals, followed by serial dilution and counting of viable bacteria. The data are the means of the results of at least six independent experiments ± SE.

FIG 5

Comparison of the intracellular growth of ΔcydAB and ΔqoxAB mutants in macrophages. J774 cells were infected at an MOI of 0.5 with either the wild type (diamonds) or the ΔcydAB (squares) or ΔqoxAB (triangles) mutant and grown in either 20% (vol/vol) oxygen (A) or 1% (vol/vol) oxygen (B). Bacterial growth was assessed by lysing the J774 cells at intervals, followed by serial dilution and counting of viable bacteria. The data are the means of the results of at least five independent experiments ± SE.

FIG 6

Percent survival of mice following intragastric infection. Mice were infected as described in Materials and Methods with 5.0 × 10⁹ CFU of either the wild type (triangles) or the ΔcydAB (inverted triangles), ΔqoxAB (squares), or ΔcydAB ΔqoxAB (circles) mutant. For each bacterial strain, 10 mice were infected. The experiment was ended after 168 h. Significant differences in survival rates were observed between the wild type and the ΔqoxAB mutant (P < 0.001) and compared with either the ΔcydAB mutant (P < 0.001) or the ΔcydAB ΔqoxAB mutant (P < 0.001). There were no significant differences (ns) in survival rates between the ΔcydAB and ΔcydAB ΔqoxAB mutants (P > 0.05). The data were analyzed with a log rank test and a Gehan-Breslow-Wilcoxon test.