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

Hammer ND, Reniere ML, Cassat JE, Zhang Y, Hirsch AO, Indriati Hood M, Skaar EP - MBio (2013)

Bottom Line: Aerobic respiration is supported by heme-dependent terminal oxidases that catalyze the final step of aerobic respiration, the reduction of O2 to H2O.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.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.

View Article: PubMed Central - PubMed

Affiliation: Vanderbilt University School of Medicine, Nashville, Tennessee, USA.

ABSTRACT

Unlabelled: 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.

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Related in: MedlinePlus

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.
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fig3: 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.

Mentions: The reduced growth phenotype of the cydB qoxB mutant suggests that aerobic respiration is inhibited in these cells. In this case, the capacity of cydB qoxB to generate a PMF should be impaired, and this strain should be limited to fermentation to produce energy and maintain redox balance. The ability of these strains to generate a component of the PMF, the membrane potential, was assessed using the fluorescent dye 3′3′-diethyloxacarbocyanine iodide (DiOC2). When bacteria generate a membrane potential, DiOC2 accumulates intracellularly and self-associates, resulting in a shift from emitting green fluorescence to emitting red fluorescence. Cells unable to generate a membrane potential accumulate less dye and emit green fluorescence (29). A shift to the red spectrum, indicative of the presence of a membrane potential, was observed when the WT and cydB and qoxB mutant strains were grown to mid-log phase and incubated in the presence of DiOC2 (Fig. 3A). No shift was observed when the menB, hemB, or cydB qoxB mutant strains were grown in similar conditions, indicating that these strains are significantly impaired for the ability to generate a membrane potential (Fig. 3A). To test the hypothesis that the terminal oxidase cydB qoxB double mutant is fermenting in order to support growth, the amount of l- and d-lactate that accumulated in the supernatant when S. aureus was grown to stationary phase was measured. A significant increase in the amount of both isomers of lactate was observed in the supernatant of the cydB qoxB double mutant strain compared to that of the WT and cydB or qoxB single-mutant strain (Fig. 3B). The amounts of l- and d-lactate produced by cydB qoxB were comparable to either the menB or hemB respiratory-deficient mutants. Expression of the cydAB operon from a plasmid is sufficient to reverse the loss of a membrane potential and reduce the amount of l- and d-lactate produced by the cydB qoxB double mutant (Fig. 3C and D). These results support the conclusion that cytochromes encoded by the qox and cyd operons are the exclusive terminal oxidases utilized by S. aureus to generate a membrane potential and facilitate aerobic respiration.


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

Hammer ND, Reniere ML, Cassat JE, Zhang Y, Hirsch AO, Indriati Hood M, Skaar EP - MBio (2013)

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.
© Copyright Policy - open-access
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC3735196&req=5

fig3: 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.
Mentions: The reduced growth phenotype of the cydB qoxB mutant suggests that aerobic respiration is inhibited in these cells. In this case, the capacity of cydB qoxB to generate a PMF should be impaired, and this strain should be limited to fermentation to produce energy and maintain redox balance. The ability of these strains to generate a component of the PMF, the membrane potential, was assessed using the fluorescent dye 3′3′-diethyloxacarbocyanine iodide (DiOC2). When bacteria generate a membrane potential, DiOC2 accumulates intracellularly and self-associates, resulting in a shift from emitting green fluorescence to emitting red fluorescence. Cells unable to generate a membrane potential accumulate less dye and emit green fluorescence (29). A shift to the red spectrum, indicative of the presence of a membrane potential, was observed when the WT and cydB and qoxB mutant strains were grown to mid-log phase and incubated in the presence of DiOC2 (Fig. 3A). No shift was observed when the menB, hemB, or cydB qoxB mutant strains were grown in similar conditions, indicating that these strains are significantly impaired for the ability to generate a membrane potential (Fig. 3A). To test the hypothesis that the terminal oxidase cydB qoxB double mutant is fermenting in order to support growth, the amount of l- and d-lactate that accumulated in the supernatant when S. aureus was grown to stationary phase was measured. A significant increase in the amount of both isomers of lactate was observed in the supernatant of the cydB qoxB double mutant strain compared to that of the WT and cydB or qoxB single-mutant strain (Fig. 3B). The amounts of l- and d-lactate produced by cydB qoxB were comparable to either the menB or hemB respiratory-deficient mutants. Expression of the cydAB operon from a plasmid is sufficient to reverse the loss of a membrane potential and reduce the amount of l- and d-lactate produced by the cydB qoxB double mutant (Fig. 3C and D). These results support the conclusion that cytochromes encoded by the qox and cyd operons are the exclusive terminal oxidases utilized by S. aureus to generate a membrane potential and facilitate aerobic respiration.

Bottom Line: Aerobic respiration is supported by heme-dependent terminal oxidases that catalyze the final step of aerobic respiration, the reduction of O2 to H2O.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.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.

View Article: PubMed Central - PubMed

Affiliation: Vanderbilt University School of Medicine, Nashville, Tennessee, USA.

ABSTRACT

Unlabelled: 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.

Show MeSH
Related in: MedlinePlus