Two heme-dependent terminal oxidases power Staphylococcus aureus organ-specific colonization of the vertebrate host

Abstract

Staphylococcus aureus is a significant cause of infections worldwide and is able to utilize aerobic respiration, anaerobic respiration, or fermentation as the means by which it generates the energy needed for proliferation. Aerobic respiration is supported by heme-dependent terminal oxidases that catalyze the final step of aerobic respiration, the reduction of O2 to H2O. An inability to respire forces bacteria to generate energy via fermentation, resulting in reduced growth. Elucidating the roles of these energy-generating pathways during colonization of the host could uncover attractive therapeutic targets. Consistent with this idea, we report that inhibiting aerobic respiration by inactivating heme biosynthesis significantly impairs the ability of S. aureus to colonize the host. Two heme-dependent terminal oxidases support aerobic respiration of S. aureus, implying that the staphylococcal respiratory chain is branched. Systemic infection with S. aureus mutants limited to a single terminal oxidase results in an organ-specific colonization defect, resulting in reduced bacterial burdens in either the liver or the heart. Finally, inhibition of aerobic respiration can be achieved by exposing S. aureus to noniron heme analogues. These data provide evidence that aerobic respiration plays a major role in S. aureus colonization of the host and that this energy-generating process is a viable therapeutic target.

Importance: Staphylococcus aureus poses a significant threat to public health as antibiotic-resistant isolates of this pathogen continue to emerge. Our understanding of the energy-generating processes that allow S. aureus to proliferate within the host is incomplete. Host-derived heme is the preferred source of nutrient iron during infection; however, S. aureus can synthesize heme de novo and use it to facilitate aerobic respiration. We demonstrate that S. aureus heme biosynthesis powers a branched aerobic respiratory chain composed of two terminal oxidases. The importance of having two terminal oxidases is demonstrated by the finding that each plays an essential role in colonizing distinct organs during systemic infection. Additionally, this process can be targeted by small-molecule heme analogues called noniron protoporphyrins. This study serves to demonstrate that heme biosynthesis supports two terminal oxidases that are required for aerobic respiration and are also essential for S. aureus pathogenesis.

FIG 1

Endogenous heme biosynthesis is required for full S. aureus pathogenesis. (A) The growth of the wild type (WT; circles) or an hemA mutant (open squares) of S. aureus was monitored over time by optical density at 600 nm. The final concentration of aminolevulinic acid (ALA) and heme added to the growth medium was 75 µg ml⁻¹ (gray squares) and 2 µM (gray diamonds), respectively. The average from three independent experiments is shown. Error bars represent one standard deviation from the mean. (B) Female BALB/c mice were systemically infected with 10⁷ CFU of the wild type (WT; circles) or an hemA mutant (squares) of S. aureus, and bacterial burdens were determined 96 h postinfection. The dotted line represents the limit of detection. Error bars represent one standard deviation from the mean. The mean is represented by a horizontal bar. *, P < 0.0001, via an unpaired Student t test. Gray symbols represent organs where no bacteria were recovered. None of the mice infected with the hemA mutant had recoverable CFU in the heart. Five mice had recoverable CFU in the kidneys and the liver when infected with the hemA mutant.

FIG 2

Inactivation of cydB and qoxB reduces the growth of S. aureus. (A) Wild-type (WT) and respective mutant strains of S. aureus were grown overnight, subcultured into fresh medium, and incubated at 37°C. The growth of the strains was monitored over time by measuring optical density at 600 nm. The average from three independent experiments is shown. Error bars represent one standard deviation from the mean. (B) Wild-type (WT) S. aureus and the qoxB cydB mutant harboring a plasmid control (pOS) or a plasmid containing the cydAB operon (pcydAB) were grown overnight, subcultured into fresh medium, and incubated at 37°C. The growth of the strains was monitored over time by measuring optical density at 600 nm. The average from three independent experiments is shown. Error bars represent one standard deviation from the mean.

FIG 3

Inactivation of both cydB and qoxB limits the metabolic flexibility of S. aureus. (A) The membrane potential was measured as the mean ratio of red/green fluorescence of wild-type (WT) and mutant strains of S. aureus grown to mid-exponential phase and incubated with 30 µM of the dye 3′3′-diethyloxacarbocyanine iodide (DiOC₂). The average from three independent experiments is shown. Error bars represent one standard deviation from the mean. (B) l-Lactate and d-lactate production in aerobically respiring and fermenting strains of S. aureus after 15 h of growth. The average from three independent experiments is shown. Error bars represent one standard deviation from the mean. (C) The membrane potential of wild-type (WT) S. aureus and the qoxB cydB mutant harboring a plasmid control (pOS) or a plasmid containing the cydAB operon (pcydAB) was measured as the mean ratio of red/green fluorescence when the strains were grown to mid-exponential phase and incubated with 30 µM of the dye 3′3′-diethyloxacarbocyanine iodide (DiOC₂). The average from three independent experiments is shown. Error bars represent one standard deviation from the mean. (D) l-Lactate and d-lactate produced in the supernatants of wild-type (WT) S. aureus and the qoxB cydB mutant harboring a plasmid control (pOS) or a plasmid containing the cydAB operon (pcydAB) grown after 12 h of growth. The average from three independent experiments is shown. Error bars represent one standard deviation from the mean.

FIG 4

Two terminal oxidases power organ-specific colonization of the host. Female BALB/c mice were systemically infected with 10⁷ CFU of the wild type (WT; circles) or cydB (squares) or qoxB (triangles) mutants of S. aureus, and bacterial burdens were determined 96 h postinfection. The dotted line represents the limit of detection. Error bars represent one standard deviation from the mean. The mean is represented by a horizontal bar. *, P < 0.0001, via an unpaired Student t test. Gray symbols represent organs where no bacteria were recovered. One mouse had recoverable CFU in the heart when infected with the cydB mutant, and one mouse had recoverable CFU in the liver when infected with the qoxB mutant.

FIG 5

Noniron metalloporphyrins inhibit staphylococcal growth. (A) Growth of S. aureus in the presence of increasing concentrations of GaPPIX or ZnPPIX. The growth of the strains was monitored over time by measuring optical density at 600 nm. The average from three independent experiments is shown. Error bars represent one standard deviation from the mean. (B) Aerobic (+O₂) or anaerobic (−O₂) growth of S. aureus in the presence of increasing concentrations of GaPPIX or ZnPPIX. Growth was monitored as the optical density at 12 h and compared to that of cells grown in the absence of the noniron PPIXs. The average from three independent experiments is shown. Error bars represent one standard deviation from the mean. (C) S. aureus was grown in the absence or presence of 1 mM 2,2′-dipyridyl (±DIP) and 0.1 µM GaPPIX or ZnPPIX. Growth was monitored as the optical density at 12 h and compared to that of cells grown in the absence of the noniron PPIXs. The average from three independent experiments is shown. Error bars represent one standard deviation from the mean.

FIG 6

GaPPIX and ZnPPIX inhibit S. aureus aerobic respiration and are trafficked to the plasmid membrane. (A) ICP-MS tracking of GaPPIX in S. aureus. Percentage of total gallium atoms measured in the cell wall (W), membrane (M), and cytoplasmic (C) fractions. Error bars represent the standard deviation from three independent samples. Asterisks represent statistically significant differences between GaPPIX and Ga(NO₃)₃ localization, as determined by a Student t test (*, P < 0.02; **, P < 0.05). (B) Mean ratio of red/green fluorescence of S. aureus grown to mid-exponential phase and incubated with 30 µM of the dye 3′3′-diethyloxacarbocyanine iodide (DiOC₂). The average from three independent experiments is shown. Error bars represent one standard deviation from the mean. (C) Measurement of d- and l-lactate in the supernatants of S. aureus grown in the presence of GaPPIX or ZnPPIX after 15 h of growth. The amount of d- and l-lactate accumulated in the supernatants of anaerobically grown S. aureus is also presented for comparison. The average from three independent experiments is shown. Error bars represent one standard deviation from the mean. (D) ICP-MS tracking of GaPPIX in S. aureus (WT) and the isogenic cydB qoxB mutant. Percentage of total gallium atoms measured in the cell wall (W), membrane (M), and cytoplasmic (C) fractions. Error bars represent the standard deviation from three independent samples. Asterisks represent statistically significant differences between GaPPIX and Ga(NO₃)₃ localization, as determined by a Student t test (*, P < 0.003).