Cytochrome Bd Research Articles

Identification of Surf1 as an assembly factor of the cytochrome bc1-aa3 supercomplex of Actinobacteria

Respiration in aerobic Actinobacteria involves a cytochrome bc1-aa3 supercomplex with a diheme cytochrome c1, first isolated from Corynebacterium glutamicum. Synthesis of a functional cytochrome c oxidase requires incorporation of CuA, CuB, heme a, and heme a3. In contrast to eukaryotes and α-proteobacteria, this process is poorly understood in Actinobacteria. Here, we analyzed the role of a Surf1 homolog of C. glutamicum in the formation of a functional bc1-aa3 supercomplex. Deletion of the surf1 gene (cg2460) in C. glutamicum caused a growth defect and cytochrome spectra revealed reduced levels of cytochrome c and a and an increased level of cytochrome d. Membranes of the Δsurf1 strain had lost the ability to oxidize the artificial electron donor N,N,N′,N′-tetramethyl-p-phenylenediamine, suggesting that Surf1 is essential for the formation of a functional cytochrome aa3 oxidase. In contrast to the wild type, a bc1-aa3 supercomplex could not be purified from solubilized membranes of the Δsurf1 mutant. A transcriptome comparison revealed that the genes of the SigC regulon including those for cytochrome bd oxidase were upregulated in the Δsurf1 strain as well as the copper deprivation-inducible gene ctiP. Complementation studies showed that the Surf1 homologs of Corynebacterium diphtheriae, Mycobacterium smegmatis and Mycobacterium tuberculosis could at least partially abolish the growth defect of the C. glutamicum Δsurf1 mutant, suggesting that Surf1 is a conserved assembly factor for actinobacterial cytochrome aa3 oxidase.

Carbon metabolism modulates the efficacy of drugs targeting the cytochrome bc1:aa3 in Mycobacterium tuberculosis

The influence of carbon metabolism on oxidative phosphorylation is poorly understood in mycobacteria. M. tuberculosis expresses two respiratory terminal oxidases, the cytochrome bc1:aa3 and the cytochrome bd oxidase, which are jointly required for oxidative phosphorylation and mycobacterial viability. The essentiality of the cytochrome bc1:aa3 for optimum growth is illustrated by its vulnerability to chemical inhibition by the clinical drug candidate Q203 and several other chemical series. The cytochrome bd oxidase is not strictly essential for growth but is required to maintain bioenergetics when the function of the cytochrome bc1:aa3 is compromised. In this study, we observed that the potency of drugs targeting the cytochrome bc1:aa3 is influenced by carbon metabolism. The efficacy of Q203 and related derivatives was alleviated by glycerol supplementation. The negative effect of glycerol supplementation on Q203 potency correlated with an upregulation of the cytochrome bd oxidase-encoding cydABDC operon. Upon deletion of cydAB, the detrimental effect of glycerol on the potency of Q203 was abrogated. The same phenomenon was also observed in recent clinical isolates, but to a lesser extent compared to the laboratory-adapted strain H37Rv. This study reinforces the importance of optimizing in vitro culture conditions for drug evaluation in mycobacteria, a factor which appeared to be particularly essential for drugs targeting the cytochrome bc1:aa3 terminal oxidase.

YtiB and ythA Genes Reduce the Uranium Removal Capacity of Bacillus atrophaeus.

Two Bacillus atrophaeus strains, the first being a highly stress-resistant ATCC 9372 strain and the Ua strain identified from a chromium mine by our lab, differ in their abilities to tolerate and remove Uranium (VI) from contaminated water. An increase in U(VI) concentration in growth media led to a decrease in the tolerance and bio-remedial capacity of both strains. However, under high concentrations of U(VI) in the growth media, the ATCC 9372 strain demonstrated a higher tolerance and a higher removal capacity than the Ua strain. Two approaches, transcriptome sequencing and transgenic technology, were used to elucidate the relationship between particular genes within these two strains and their U(VI) removal capacity. Sequencing confirmed the expression of two genes unique to the Ua strain, previously designated ytiB and ythA. They encode putative proteins that show the highest levels of identity to carbonic anhydrase and cytochrome bd terminal oxidase I, respectively. Using the pBE-S DNA vector, ytiB and ythA were transformed into the ATCC 9372 strain of Bacillus atrophaeus. Under a U(VI) concentration of 120 mg/L, the removal rates of the transgenic ATCC 9372-ytiB and ATCC 9372-ythA strains decreased by 7.55% and 7.43%, respectively, compared to the removal rate of the control strain transformed with empty plasmid. The results suggest that both ythA and ytiB genes have a negative influence on the uranium removing capacity of Bacillus atrophaeus. This finding will help to elucidate the molecular mechanisms of uranium removal by bacteria.

Pyrazolo[1,5- a]pyridine Inhibitor of the Respiratory Cytochrome bcc Complex for the Treatment of Drug-Resistant Tuberculosis.

Respiration is a promising target for the development of new antimycobacterial agents, with a growing number of compounds in clinical development entering this target space. However, more candidate inhibitors are needed to expand the therapeutic options available for drug-resistant Mycobacterium tuberculosis infection. Here, we characterize a putative respiratory complex III (QcrB) inhibitor, TB47: a pyrazolo[1,5- a]pyridine-3-carboxamide. TB47 is active (MIC between 0.016 and 0.500 μg/mL) against a panel of 56 M. tuberculosis clinical isolates, including 37 multi-drug-resistant and two extensively drug-resistant strains. Pharmacokinetic and toxicity studies showed promising profiles, including negligible CYP450 interactions, cytotoxicity, and hERG channel inhibition. Consistent with other reported QcrB inhibitors, TB47 inhibits oxygen consumption only when the alternative oxidase, cytochrome bd, is deleted. A point mutation in the qcrB cd2-loop (H190Y, M. smegmatis numbering) rescues the inhibitory effects of TB47. Metabolomic profiling of TB47-treated M. tuberculosis H37Rv cultures revealed accumulation of steps in the TCA cycle and pentose phosphate pathway that are linked to reducing equivalents, suggesting that TB47 causes metabolic redox stress. In mouse infection models, a TB47 monotherapy was not bactericidal. However, TB47 was strongly synergistic with pyrazinamide and rifampicin, suggesting a promising role in combination therapies. We propose that TB47 is an effective lead compound for the development of novel tuberculosis chemotherapies.

Arylvinylpiperazine Amides, a New Class of Potent Inhibitors Targeting QcrB of Mycobacterium tuberculosis.

New drugs are needed to control the current tuberculosis (TB) pandemic caused by infection with Mycobacterium tuberculosis We report here on our work with AX-35, an arylvinylpiperazine amide, and four related analogs, which are potent antitubercular agents in vitro All five compounds showed good activity against M. tuberculosisin vitro and in infected THP-1 macrophages, while displaying only mild cytotoxicity. Isolation and characterization of M. tuberculosis-resistant mutants to the arylvinylpiperazine amide derivative AX-35 revealed mutations in the qcrB gene encoding a subunit of cytochrome bc1 oxidase, one of two terminal oxidases of the electron transport chain. Cross-resistance studies, allelic exchange, transcriptomic analyses, and bioenergetic flux assays provided conclusive evidence that the cytochrome bc1-aa3 is the target of AX-35, although the compound appears to interact differently with the quinol binding pocket compared to previous QcrB inhibitors. The transcriptomic and bioenergetic profiles of M. tuberculosis treated with AX-35 were similar to those generated by other cytochrome bc1 oxidase inhibitors, including the compensatory role of the alternate terminal oxidase cytochrome bd in respiratory adaptation. In the absence of cytochrome bd oxidase, AX-35 was bactericidal against M. tuberculosis Finally, AX-35 and its analogs were active in an acute mouse model of TB infection, with two analogs displaying improved activity over the parent compound. Our findings will guide future lead optimization to produce a drug candidate for the treatment of TB and other mycobacterial diseases, including Buruli ulcer and leprosy.IMPORTANCE New drugs against Mycobacterium tuberculosis are urgently needed to deal with the current global TB pandemic. We report here on the discovery of a series of arylvinylpiperazine amides (AX-35 to AX-39) that represent a promising new family of compounds with potent in vitro and in vivo activities against M. tuberculosis AX compounds target the QcrB subunit of the cytochrome bc1 terminal oxidase with a different mode of interaction compared to those of known QcrB inhibitors. This study provides the first multifaceted validation of QcrB inhibition by recombineering-mediated allelic exchange, gene expression profiling, and bioenergetic flux studies. It also provides further evidence for the compensatory role of cytochrome bd oxidase upon QcrB inhibition. In the absence of cytochrome bd oxidase, AX compounds are bactericidal, an encouraging property for future antimycobacterial drug development.

The Cytochrome bd Complex Is Essential for Chromate and Sulfide Resistance and Is Regulated by a GbsR-Type Regulator, CydE, in Alishewanella Sp. WH16-1.

Sulfate-reducing bacteria are a group of microorganisms that use sulfate as an electron acceptor. These bacteria are useful in the bioremediation of heavy metal pollution since they can reduce/precipitate metals. Previously, we identified the Alishewanella strain WH16-1 from soil of a copper and iron mine and determined that it can reduce sulfate and chromate and that it was tolerant to many heavy metals. In this study, we investigated the chromate reduction mechanism of strain WH16-1 through Tn5 transposon mutagenesis. A cytochrome bd (cytbd) Tn5 mutant was generated (Δcytbd), and a detail analysis showed that the following: (1) gene cydE (coding for a GbsR-type regulator) was co-transcribed with the two subunits coding genes of the Cytochrome bd complex (Cytbd), namely, cydA and cydB, based on RT-PCR analysis, and similar gene arrangements were also found in other Alteromonadaceae family strains; (2) the chromate resistance level was dramatically decreased and chromate reduction efficiency also decreased in strain Δcytbd compared to the wild-type and a complemented strain (Δcytbd-C); (3) Cytbd could catalyze the decomposition of H2O2 according to the analyses of H2O2 decomposition ability, cellular H2O2 contents, H2O2 inhibition zone, and H2O2 sensitivity tests; (4) surprisingly, chromate was not an inducer of the expression of Cytbd, but sulfate induced expression of Cytbd, and sulfate/sulfide resistance levels were also decreased in the Δcytbd strain; (5) the addition of sulfate enhanced the chromate resistance level and reduction efficiency; (6) Cytbd expression was repressed by CydE and derepressed by sulfate based on an in vivo bacterial one hybrid system and in vitro EMSA tests; and (7) DNA footprinting and short-fragment EMSA tests revealed two binding sites of CydE in its promoter region. All these results showed that Cytbd is negatively regulated by CydE and derepressed by sulfate. In addition, Cytbd contributes to the resistance of sulfate and sulfide, and sulfide could be used as a reductant to reduce chromate. Moreover, Cytbd is essential to decompose H2O2 to decrease cellular oxidative stress. Thus, the regulation and function of Cytbd may explain why sulfate could enhance chromate reduction.

Enhanced production of poly-γ-glutamic acid by improving ATP supply in metabolically engineered Bacillus licheniformis.

Poly-γ-glutamic acid (γ-PGA) is an important multifunctional biopolymer with various applications, for which adenosine triphosphate (ATP) supply plays a vital role in biosynthesis. In this study, the enhancement of γ-PGA production was attempted through various approaches of improving ATP supply in the engineered strains of Bacillus licheniformis. The first approach is to engineer respiration chain branches of B. licheniformis, elimination of cytochrome bd oxidase branch reduced the maintenance coefficient, leading to a 19.27% increase of γ-PGA yield. The second approach is to introduce Vitreoscilla hemoglobin (VHB) into recombinant B. licheniformis, led to a 13.32% increase of γ-PGA yield. In the third approach, the genes purB and adK in ATP-biosynthetic pathway were respectively overexpressed, with the AdK overexpressed strain increased γ-PGA yield by 14.69%. Our study also confirmed that the respiratory nitrate reductase, NarGHIJ, is responsible for the conversion of nitrate to nitrite, and assimilatory nitrate reductase NasBC is for conversion of nitrite to ammonia. Both NarGHIJ and NasBC were positively regulated by the two-component system ResD-ResE, and overexpression of NarG, NasC, and ResD also improved the ATP supply and the consequent γ-PGA yield. Based on the above individual methods, a method of combining the deletion of cydBC gene and overexpression of genes vgB, adK, and resD were used to enhance ATP content of the cells to 3.53 μmol/g of DCW, the mutant WX-BCVAR with this enhancement produced 43.81 g/L of γ-PGA, a 38.64% improvement compared to wild-type strain WX-02. Collectively, our results demonstrate that improving ATP content in B. licheniformis is an efficient strategy to improve γ-PGA production.

Transcriptomic analysis of longitudinal Burkholderia pseudomallei infecting the cystic fibrosis lung

The melioidosis bacterium, Burkholderia pseudomallei, is increasingly being recognised as a pathogen in patients with cystic fibrosis (CF). We have recently catalogued genome-wide variation of paired, isogenic B. pseudomallei isolates from seven Australasian CF cases, which were collected between 4 and 55 months apart. Here, we extend this investigation by documenting the transcriptomic changes in B. pseudomallei in five cases. Following growth in an artificial CF sputum medium, four of the five paired isolates exhibited significant differential gene expression (DE) that affected between 32 and 792 genes. The greatest number of DE events was observed between the strains from patient CF9, consistent with the hypermutator status of the latter strain, which is deficient in the DNA mismatch repair protein MutS. Two patient isolates harboured duplications that concomitantly increased expression of the β-lactamase-encoding gene penA, and a 35 kb deletion in another abolished expression of 29 genes. Convergent expression profiles in the chronically-adapted isolates identified two significantly downregulated and 17 significantly upregulated loci, including the resistance-nodulation-division (RND) efflux pump BpeEF–OprC, the quorum-sensing hhqABCDE operon, and a cyanide- and pyocyanin-insensitive cytochrome bd quinol oxidase. These convergent pathoadaptations lead to increased expression of pathways that may suppress competing bacterial and fungal pathogens, and that enhance survival in oxygen-restricted environments, the latter of which may render conventional antibiotics less effective in vivo. Treating chronically adapted B. pseudomallei infections with antibiotics designed to target anaerobic infections, such as the nitroimidazole class of antibiotics, may significantly improve pathogen eradication attempts by exploiting this Achilles heel.

Extracellular Electron Transfer by the Gram-Positive Bacterium Enterococcus faecalis.

Extracellular electron transfer (EET) in microbial cells is essential for certain biotechnological applications and contributes to the biogeochemical cycling of elements and syntrophic microbial metabolism in complex natural environments. The Gram-positive lactic acid bacterium Enterococcus faecalis, an opportunistic human pathogen, is shown to be able to transfer electrons generated in fermentation metabolism to electrodes directly and indirectly via mediators. By exploiting E. faecalis wild-type and mutant cells, we demonstrate that reduced demethylmenaquinone in the respiratory chain in the bacterial cytoplasmic membrane is crucial for the EET. Heme proteins are not involved, and cytochrome bd oxidase activity was found to attenuate EET. These results are significant for the mechanistic understanding of EET in bacteria and for the design of microbial electrochemical systems. The basic findings infer that in dense microbial communities, such as in biofilm and in the large intestine, metabolism in E. faecalis and similar Gram-positive lactic acid bacteria might be electrically connected to other microbes. Such atranscellular electron transfer might confer syntrophic metabolism that promotes growth and other activities of bacteria in the microbiota of humans and animals.

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

ABSTRACTThe 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.

Investigation of amino acid specificity in the CydX small protein shows sequence plasticity at the functional level.

Small proteins are a new and expanding area of research. Many characterized small proteins are composed of a single hydrophobic α-helix, and the functional requirements of their limited amino acid sequence are not well understood. One hydrophobic small protein, CydX, has been shown to be a component of the cytochrome bd oxidase complex in Escherichia coli, and is required for enzyme function. To investigate small protein sequence specificity, an alanine scanning mutagenesis on the small protein CydX was conducted using mutant alleles expressed from the E. coli chromosome at the wild-type locus. The resulting mutant strains were assayed for CydX function. No single amino acid was required to maintain wild-type resistance to β-mercaptoethanol. However, substitutions of 10-amino acid blocks indicated that the N-terminus of the protein was required for wild-type CydX activity. A series of double mutants showed that multiple mutations at the N-terminus led to β-mercaptoethanol sensitivity in vivo. Triple mutants showed both in vivo and in vitro phenotypes. Together, these data provide evidence suggesting a high level of functional plasticity in CydX, in which multiple amino acids may work cooperatively to facilitate CydX function.

P-391 - Cytochrome bd and bacterial respiration in sulfide rich environments

Cytochrome bd is a prokaryotic respiratory terminal oxidase phylogenetically unrelated to haem-copper oxidases. In addition to sustaining energy metabolism, this enzyme is implicated in bacterial protection against oxidative and nitrosative stress. Cytochrome bd is only found in prokaryotes and, as it promotes virulence in several pathogenic bacteria, it is currently recognized as a potential drug target. Escherichia coli and other microbes, including pathogens, colonize the human gut, that is known to be rich in hydrogen sulfide (H2S), while being a major site of reactive nitrogen species production. Depending on its concentration, H2S can have beneficial or detrimental effects in both prokaryotes and eukaryotes. Among the most well known toxic effects of H2S is the inhibition of cellular respiration due to binding to mitochondrial heme copper-type cytochrome c oxidase. Interestingly, working on E. coli mutant strains and purified proteins, we found that bd-type oxidases are insensitive to H2S inhibition, thereby promoting bacterial respiration and growth in the presence of high levels of H2S. The potential impact of this discovery on human (patho)physiology and its possible applications in the field of infectious diseases will be discussed.

Functional importance of Glutamate-445 and Glutamate-99 in proton-coupled electron transfer during oxygen reduction by cytochrome bd from Escherichia coli

The recent X-ray structure of the cytochrome bd respiratory oxygen reductase showed that two of the three heme components, heme d and heme b595, have glutamic acid as an axial ligand. No other native heme proteins are known to have glutamic acid axial ligands. In this work, site-directed mutagenesis is used to probe the roles of these glutamic acids, E445 and E99 in the E. coli enzyme. It is concluded that neither glutamate is a strong ligand to the heme Fe and they are not the major determinates of heme binding to the protein. Although very important, neither glutamate is absolutely essential for catalytic function. The close interactions between the three hemes in cyt bd result in highly cooperative properties. For example, mutation of E445, which is near heme d, has its greatest effects on the properties of heme b595 and heme b558. It is concluded that 1) O2 binds to the hydrophilic side of heme d and displaces E445; 2) E445 forms a salt bridge with R448 within the O2 binding pocket, and both residues play a role to stabilize oxygenated states of heme d during catalysis; 3) E445 and E99 are each protonated accompanying electron transfer to heme d and heme b595, respectively; 4) All protons used to generate water within the heme d active site come from the cytoplasm and are delivered through a channel that must include internal water molecules to assist proton transfer: [cytoplasm] → E107 → E99 (heme b595) → E445 (heme d) → oxygenated heme d.

The anti-mycobacterial activity of the cytochrome bcc inhibitor Q203 can be enhanced by small-molecule inhibition of cytochrome bd

Mycobacterial energy metabolism currently attracts strong attention as new target space for development of anti-tuberculosis drugs. The imidazopyridine Q203 targets the cytochrome bcc complex of the respiratory chain, a key component in energy metabolism. Q203 blocks growth of Mycobacterium tuberculosis at nanomolar concentrations, however, it fails to actually kill the bacteria, which may limit the clinical applicability of this candidate drug. In this report we show that inhibition of cytochrome bd, a parallel branch of the mycobacterial respiratory chain, by aurachin D invoked bactericidal activity of Q203. In biochemical assays using inverted membrane vesicles from Mycobacterium tuberculosis and Mycobacterium smegmatis we found that inhibition of respiratory chain activity by Q203 was incomplete, but could be enhanced by inactivation of cytochrome bd, either by genetic knock-out or by inhibition with aurachin D. These results indicate that simultaneously targeting the cytochrome bcc and the cytochrome bd branch of the mycobacterial respiratory chain may turn out as effective strategy for combating M. tuberculosis.

Susceptibility of Mycobacterium tuberculosis Cytochrome bd Oxidase Mutants to Compounds Targeting the Terminal Respiratory Oxidase, Cytochrome c.

We deleted subunits I (cydA) and II (cydB) of the Mycobacterium tuberculosis cytochrome bd menaquinol oxidase. The resulting ΔcydA and ΔcydAB mutants were hypersusceptible to compounds targeting the mycobacterial bc1 menaquinol-cytochrome c oxidoreductase and exhibited bioenergetic profiles indistinguishable from strains deficient in the ABC-type transporter, CydDC, predicted to be essential for cytochrome bd assembly. These results confirm CydAB and CydDC as potential targets for drugs aimed at inhibiting a terminal respiratory oxidase implicated in pathogenesis.

A fluorescence-based reporter for monitoring expression of mycobacterial cytochrome bd in response to antibacterials and during infection

Cytochrome bd is a component of the oxidative phosphorylation pathway in many Gram-positive and Gram-negative bacteria. Next to its role as a terminal oxidase in the respiratory chain this enzyme plays an important role as a survival factor in the bacterial stress response. In Mycobacterium tuberculosis and related mycobacterial strains, cytochrome bd is an important component of the defense system against antibacterial drugs. In this report we describe and evaluate an mCherry-based fluorescent reporter for detection of cytochrome bd expression in Mycobacterium marinum. Cytochrome bd was induced by mycolic acid biosynthesis inhibitors such as isoniazid and most prominently by drugs targeting oxidative phosphorylation. We observed no induction by inhibitors of protein-, DNA- or RNA-synthesis. The constructed expression reporter was suitable for monitoring mycobacterial cytochrome bd expression during mouse macrophage infection and in a zebrafish embryo infection model when using Mycobacterium marinum. Interestingly, in both these infection models cytochrome bd levels were considerably higher than during in vitro culturing of M. marinum. The expression reporter described here can be a valuable tool for elucidating the role of cytochrome bd as a survival factor.

Optimization of the purine operon and energy generation in Bacillus amyloliquefaciens for guanosine production.

To deregulate the purine operon of the purine biosynthetic pathway and optimize energy generation of the respiratory chain to improve the yield of guanosine in Bacillus amyloliquefaciens XH7. The 5'-untranslated region of the purine operon, which contains the guanine-sensing riboswitch, was disrupted. The native promoter Pw in B. amyloliquefaciens XH7 was replaced by different strong promoters. Among the promoter replacement mutants, XH7purE::P41 gave the highest guanosine yield (16.3g/l), with an increase of 23% compared with B. amyloliquefaciens XH7. The relative expression levels of the purine operon genes (purE, purF, and purD) in the XH7purE::P41 mutant were upregulated. The concentration of inosine monophosphate (IMP), the primary intermediate in the purine pathway, was also significantly increased in the XH7purE::P41 mutant. Combined modification of the low-coupling branched respiratory chains (cytochrome bd oxidase) improved guanosine production synergistically. The final guanosine yield in the XH7purE::P41△cyd mutant increased by 41% to 19g/l compared with B. amyloliquefaciens XH7. The combined modification strategy used in this study is a novel approach to improve the production of guanosine in industrial bacterial strains.

Bacterial signals promote mosquito development

Microbiota The ways of the microbiota are many and mysterious. All organisms have prokaryote partners that contribute to health, as well as disease. Coon et al. report that larval mosquitos require bacteria in their gut to complete development into an adult insect. In this case, the bacteria are not making a nutritional contribution, however. To be helpful to the insect, this strain of Escherichia coli has to supply cytochrome bd oxidase expression. The respiratory function of the oxidase causes hypoxia, which then acts as a signal to promote host development. The hypoxia initiates hypoxia-inducible transcription factors and promotes molting. Blocking such cues might be a strategy to interrupt the life cycle of disease-carrying mosquitos. Proc. Natl. Acad. Sci. U.S.A. 10.1073/pnas.1702983114 (2017).

Exploiting the synthetic lethality between terminal respiratory oxidases to kill Mycobacterium tuberculosis and clear host infection

The recent discovery of small molecules targeting the cytochrome bc1 :aa3 in Mycobacterium tuberculosis triggered interest in the terminal respiratory oxidases for antituberculosis drug development. The mycobacterial cytochrome bc1 :aa3 consists of a menaquinone:cytochrome c reductase (bc1 ) and a cytochrome aa3 -type oxidase. The clinical-stage drug candidate Q203 interferes with the function of the subunit b of the menaquinone:cytochrome c reductase. Despite the affinity of Q203 for the bc1 :aa3 complex, the drug is only bacteriostatic and does not kill drug-tolerant persisters. This raises the possibility that the alternate terminal bd-type oxidase (cytochrome bd oxidase) is capable of maintaining a membrane potential and menaquinol oxidation in the presence of Q203. Here, we show that the electron flow through the cytochrome bd oxidase is sufficient to maintain respiration and ATP synthesis at a level high enough to protect M. tuberculosis from Q203-induced bacterial death. Upon genetic deletion of the cytochrome bd oxidase-encoding genes cydAB, Q203 inhibited mycobacterial respiration completely, became bactericidal, killed drug-tolerant mycobacterial persisters, and rapidly cleared M. tuberculosis infection in vivo. These results indicate a synthetic lethal interaction between the two terminal respiratory oxidases that can be exploited for anti-TB drug development. Our findings should be considered in the clinical development of drugs targeting the cytochrome bc1 :aa3 , as well as for the development of a drug combination targeting oxidative phosphorylation in M. tuberculosis.

Bacteria-mediated hypoxia functions as a signal for mosquito development

Mosquitoes host communities of microbes in their digestive tract that consist primarily of bacteria. We previously reported that several mosquito species, including Aedes aegypti, do not develop beyond the first instar when fed a nutritionally complete diet in the absence of a gut microbiota. In contrast, several species of bacteria, including Escherichia coli, rescue development of axenic larvae into adults. The molecular mechanisms underlying bacteria-dependent growth are unknown. Here, we designed a genetic screen around E. coli that identified high-affinity cytochrome bd oxidase as an essential bacterial gene product for mosquito growth. Bioassays showed that bacteria in nonsterile larvae and gnotobiotic larvae inoculated with wild-type E. coli reduced midgut oxygen levels below 5%, whereas larvae inoculated with E. coli mutants defective for cytochrome bd oxidase did not. Experiments further supported that hypoxia leads to growth and ecdysone-induced molting. Altogether, our results identify aerobic respiration by bacteria as a previously unknown but essential process for mosquito development.