Role of respiratory NADH oxidation in the regulation of Staphylococcus aureus virulence.

The success of Staphylococcus aureus as a pathogen is due to its capability of fine-tuning its cellular physiology to meet the challenges presented by diverse environments, which allows it to colonize multiple niches within a single vertebrate host. Elucidating the roles of energy-yielding metabolic pathways could uncover attractive therapeutic strategies and targets. In this work, we seek to determine the effects of disabling NADH-dependent aerobic respiration on the physiology of S. aureus. Differing from many pathogens, S. aureus has two type-2 respiratory NADH dehydrogenases (NDH-2s) but lacks the respiratory ion-pumping NDHs. Here, we show that the NDH-2s, individually or together, are not essential either for respiration or growth. Nevertheless, their absence eliminates biofilm formation, production of α-toxin, and reduces the ability to colonize specific organs in a mouse model of systemic infection. Moreover, we demonstrate that the reason behind these phenotypes is the alteration of the fatty acid metabolism. Importantly, the SaeRS two-component system, which responds to fatty acids regulation, is responsible for the link between NADH-dependent respiration and virulence in S. aureus.

The oligomeric state of the Caldivirga maquilingensis type III sulfide:Quinone Oxidoreductase is required for membrane binding.

Sulfide:quinone oxidoreductase (SQR) is a monotopic membrane flavoprotein present in all domains of life, with multiple roles including sulfide detoxification, homeostasis and energy generation by providing electrons to respiratory or photosynthetic electron transport chains. A type III SQR from the hyperthermophilic archeon Caldivirga maquilingensis has been previously characterized, and its C-terminal amphipathic helices were demonstrated to be responsible for membrane binding. Here, the oligomeric state of this protein was experimentally evaluated by size exclusion chromatography, native gels and crosslinking, and found to be a monomer-dimer-trimer equilibrium. Remarkably, mutant and truncated variants unable to bind to the membrane are able to maintain their oligomeric association. Thus, unlike other related monotopic membrane proteins, the region involved in membrane binding does not influence oligomerization. Furthermore, by studying heterodimers between the WT and mutants, it was concluded that membrane binding requires an oligomer with at least two copies of the protein with intact C-terminal amphipathic helices. A structural homology model of the C. maquilingensis SQR was used to define the flavin- and quinone-binding sites. CmGly12, CmGly16, CmAla77 and CmPro44 were determined to be important for flavin binding. Unexpectedly, CmGly299 is only important for quinone reduction despite its proximity to bound FAD. CmPhe337 and CmPhe362 are also important for quinone binding apparently by direct interaction with the quinone ring, whereas CmLys359, postulated to hydrogen bond to the quinone, seems to have a more structural role. The results presented differentiate the Type III CmSQR from some of its counterparts classified as Type I, II and V.

Characterization and X-ray structure of the NADH-dependent coenzyme A disulfide reductase from Thermus thermophilus.

The crystal structure of the enzyme previously characterized as a type-2 NADH:menaquinone oxidoreductase (NDH-2) from Thermus thermophilus has been solved at a resolution of 2.9 Šand revealed that this protein is, in fact, a coenzyme A-disulfide reductase (CoADR). Coenzyme A (CoASH) replaces glutathione as the major low molecular weight thiol in Thermus thermophilus and is maintained in the reduced state by this enzyme (CoADR). Although the enzyme does exhibit NADH:menadione oxidoreductase activity expected for NDH-2 enzymes, the specific activity with CoAD as an electron acceptor is about 5-fold higher than with menadione. Furthermore, the crystal structure contains coenzyme A covalently linked Cys44, a catalytic intermediate (Cys44-S-S-CoA) reduced by NADH via the FAD cofactor. Soaking the crystals with menadione shows that menadione can bind to a site near the redox active FAD, consistent with the observed NADH:menadione oxidoreductase activity. CoADRs from other species were also examined and shown to have measurable NADH:menadione oxidoreductase activity. Although a common feature of this family of enzymes, no biological relevance is proposed. The CoADR from T. thermophilus is a soluble homodimeric enzyme. Expression of the recombinant TtCoADR at high levels in E. coli results in a small fraction that co-purifies with the membrane fraction, which was used previously to isolate the enzyme wrongly identified as a membrane-bound NDH-2. It is concluded that T. thermophilus does not contain an authentic NDH-2 component in its aerobic respiratory chain.

Type 2 NADH Dehydrogenase Is the Only Point of Entry for Electrons into the Streptococcus agalactiae Respiratory Chain and Is a Potential Drug Target.

The opportunistic pathogen Streptococcus agalactiae is the major cause of meningitis and sepsis in a newborn's first week, as well as a considerable cause of pneumonia, urinary tract infections, and sepsis in immunocompromised adults. This pathogen respires aerobically if heme and quinone are available in the environment, and a functional respiratory chain is required for full virulence. Remarkably, it is shown here that the entire respiratory chain of S. agalactiae consists of only two enzymes, a type 2 NADH dehydrogenase (NDH-2) and a cytochrome bd oxygen reductase. There are no respiratory dehydrogenases other than NDH-2 to feed electrons into the respiratory chain, and there is only one respiratory oxygen reductase to reduce oxygen to water. Although S. agalactiae grows well in vitro by fermentative metabolism, it is shown here that the absence of NDH-2 results in attenuated virulence, as observed by reduced colonization in heart and kidney in a mouse model of systemic infection. The lack of NDH-2 in mammalian mitochondria and its important role for virulence suggest this enzyme may be a potential drug target. For this reason, in this study, S. agalactiae NDH-2 was purified and biochemically characterized, and the isolated enzyme was used to screen for inhibitors from libraries of FDA-approved drugs. Zafirlukast was identified to successfully inhibit both NDH-2 activity and aerobic respiration in intact cells. This compound may be useful as a laboratory tool to inhibit respiration in S. agalactiae and, since it has few side effects, it might be considered a lead compound for therapeutics development.IMPORTANCES. agalactiae is part of the human intestinal microbiota and is present in the vagina of ~30% of healthy women. Although a commensal, it is also the leading cause of septicemia and meningitis in neonates and immunocompromised adults. This organism can aerobically respire, but only using external sources of heme and quinone, required to have a functional electron transport chain. Although bacteria usually have a branched respiratory chain with multiple dehydrogenases and terminal oxygen reductases, here we establish that S. agalactiae utilizes only one type 2 NADH dehydrogenase (NDH-2) and one cytochrome bd oxygen reductase to perform respiration. NADH-dependent respiration plays a critical role in the pathogen in maintaining NADH/NAD+ redox balance in the cell, optimizing ATP production, and tolerating oxygen. In summary, we demonstrate the essential role of NDH-2 in respiration and its contribution to S. agalactiae virulence and propose it as a potential drug target.

Alternate pathways for NADH oxidation in Thermus thermophilus using type 2 NADH dehydrogenases.

Type 2 NADH dehydrogenase (NDH-2) is a single-subunit membrane-associated flavoenzyme that is part of the respiratory chain of many prokaryotes. The enzyme catalyzes the electron transfer from NADH to quinone but is not directly coupled to the generation of a proton motive force. The purpose of the current work is to compare two different NDH-2s that are encoded in strains of Thermus thermophilus. The aerobic T. thermophilus HB27 strain expresses one NDH-2 that has been previously isolated and characterized. In this work it is shown that a gene, which is misannotated as an NADH oxidase, encodes this enzyme. Unlike HB27, strain NAR1 of T. thermophilus is capable of partial denitrification, and in addition its genome contains the nrcN gene that encodes a second putative NDH-2. Of particular interest is the fact that nrcN is part of an operon (nrcDEFN) that is proposed to encode a protein complex specifically required for nitrate reduction. In this work, the nrcN gene has the activity expected of a NDH-2, and functions independently of other components of the putative Nrc complex. The biochemical properties of the two NDH-2 enzymes are compared. Efforts to demonstrate that NrcN is part of a multiprotein complex were not successful. However, the NrcE protein was expressed in Escherichia coli and shown to be a membrane-bound protein containing heme B.

Characterization of the Type III sulfide:quinone oxidoreductase from Caldivirga maquilingensis and its membrane binding.

Sulfide:quinone oxidoreductases (SQRs) are ubiquitous enzymes which have multiple roles: sulfide detoxification, energy generation by providing electrons to respiratory or photosynthetic electron transfer chains, and sulfide homeostasis. A recent structure-based classification defines 6 groups of putative SQRs (I-VI), and representatives of all but group III have been confirmed to have sulfide oxidase activity. In the current work, we report the first characterization of a predicted group III SQR from Caldivirga maquilingensis, and confirm that this protein is a sulfide oxidase. The gene encoding the enzyme was cloned, and the protein was expressed in E. coli and purified. The enzyme oxidizes sulfide using decylubiquinone as an electron acceptor, and is inhibited by aurachin C and iodoacetamide. Analysis of the amino acid sequence indicates that the C. maquilingensis SQR has two amphiphilic helices at the C-terminus but lacks any transmembrane helices. This suggests that C. maquilingensis SQR interacts with the membrane surface and that the interactions are mediated by the C-terminal amphiphilic helices. Mutations within the last C-terminal amphiphilic helix resulted in a water-soluble form of the enzyme which, remarkably, retains full SQR activity using decylubiquinone as the electron acceptor. Mutations at one position, L379, also located in the C-terminal amphiphilic helix, inactivated the enzyme by preventing the interaction with decylubiquinone. It is concluded that the C-terminal amphiphilic helix is important for membrane binding and for forming part of the pathway providing access of the quinone substrate to the protein-bound flavin at the enzyme active site.