Kelly S. Davidge

Carbon Monoxide-releasing Antibacterial Molecules Target Respiration and Global Transcriptional Regulators

Carbon monoxide, a classical respiratory inhibitor, also exerts vasodilatory, anti-inflammatory, and antiapoptotic effects. CO-releasing molecules have therapeutic value, increasing phagocytosis and reducing sepsis-induced lethality. Here we identify for the first time the bacterial targets of Ru(CO)3Cl(glycinate) (CORM-3), a ruthenium-based carbonyl that liberates CO rapidly under physiological conditions. Contrary to the expectation that CO would be preferentially inhibitory at low oxygen tensions or anaerobically, Escherichia coli cultures were also sensitive to CORM-3 at concentrations equimolar with oxygen. CORM-3, assayed as ruthenium, was taken up by bacteria and rapidly delivered CO intracellularly to terminal oxidases. Microarray analysis of CORM-3-treated cells revealed extensively modified gene expression, notably down-regulation of genes encoding key aerobic respiratory complexes. Genes involved in metal metabolism, homeostasis, or transport were also differentially expressed, and free intracellular zinc levels were elevated. Probabilistic modeling of transcriptomic data identified the global transcription regulators ArcA, CRP, Fis, FNR, Fur, BaeR, CpxR, and IHF as targets and potential CO sensors. Our discovery that CORM-3 is an effective inhibitor and global regulator of gene expression, especially under aerobic conditions, has important implications for administration of CO-releasing agents in sepsis and inflammation.

A carbon monoxide-releasing molecule (CORM-3) exerts bactericidal activity against Pseudomonas aeruginosa and improves survival in an animal model of bacteraemia

The search for new molecules to fight Pseudomonas aeruginosa is of paramount importance. Carbon monoxide (CO) is known to act as an effective inhibitor of the respiratory chain in P. aeruginosa, but the practical use of this gas as an antibacterial molecule is hampered by its toxicity and difficulty to manipulate. Here, we show that a water‐soluble CO releaser (CORM‐3) possesses bactericidal properties against laboratory and antibiotic‐resistant P. aeruginosa. CORM‐3 reduced the bacterial count by 4 logs 180 min after in vitro treatment. CORM‐3‐treated bacteria had a lower O2 consumption than vehicle‐treated bacteria, and the decrease in O2 consumption temporally preceded the bactericidal action of CORM‐3. These results support the hypothesis that the antimicrobial effect of CORM‐3 is mediated by an interaction of CO liberated by the carrier with the bacterial respiratory chain. The antibacterial effect occurred at concentrations of CORM‐3 that are 50fold lower than toxic concentrations for eukaryotic cells. CORM‐3 treatment compared to vehicle treatment decreased bacterial counts in the spleen and increased survival in immunocompetent and immunosuppressed mice following P. aeruginosa bacteremia. Our results suggest that CORMs could form the basis for developing a new therapeutic strategy against P. aeruginosa‐induced infection.—Desmard, M., Davidge, K. S., Bouvet, O., Morin, D., Roux, D., Foresti, R., Ricard, J. D., Denamur, E., Poole, R. K., Montravers, P., Motterlini, R., Boczkowski, J. A carbon monoxide‐releasing molecule (CORM‐3) exerts bactericidal activity against Pseudomonas aeruginosa and improves survival in an animal model of bacteraemia. FASEB J. 23, 1023–1031 (2009)

Antibacterial effects of carbon monoxide.

Carbon monoxide (CO) is a colourless and odourless gas that has long been considered as a potent respiratory poison. Recent advances have demonstrated its production by haem oxygenases in both mammals and microbes, and it has roles as a gasotransmitter in higher organisms. This review concentrates on the application of CO, via carbon monoxide-releasing molecules (CO-RMs), as an anti-bacterial agent. Currently, the scope of literature on the effects of CO on bacteria is small, and we have included discussions on the production of CO by bacteria via haem oxygenase enzymes, the use of CO as an energy source, and existing knowledge on CO sensors in bacteria. CO is known to target haem proteins and is an effective inhibitor of respiration, even when provided at concentrations much higher than prevailing oxygen. We review here data suggesting that CO-RMs are more effective inhibitors of respiration than is CO gas, perhaps due to the ability of CO-RMs to deliver CO selectively to intracellular targets. We also consider the recently reported transcriptomic consequences of CO-RM treatment of Escherichia coli, revealing a myriad of unexpected targets for CO and potential CO sensors. Finally, we consider the use of CO and CO-RMs as anti-bacterial agents in vivo, and the future prospects for this gaseous molecule.

Nitric oxide reactivities of the two globins of the foodborne pathogen Campylobacter jejuni: roles in protection from nitrosative stress and analysis of potential reductants.

BACKGROUND During infection and pathogenesis, Campylobacter, the leading cause of gastroenteritis, encounters NO and reactive nitrogen species (RNS) derived from the host. To combat these species, Campylobacter jejuni expresses two haemoglobins: the single domain haemoglobin (Cgb) detoxifies NO but the role of the truncated globin (Ctb) is unclear. Confirmation of Cgb activity and more extensive exploration of Ctb function(s) in vivo are restricted due to difficulties in expressing proteins in Campylobacter and our lack of understanding of how the globin haems are re-reduced after ligand reactions. METHODS The cgb and ctb genes were cloned under the control of arabinose-inducible promoters and the globins expressed in an Escherichia coli mutant lacking the main NO detoxification mechanisms (Hmp and the Nor system comprising the transcription regulator NorR, the flavorubredoxin and its reductase (NorVW)); cellular responses under oxidative and nitrosative stress conditions were assessed. Spectroscopic changes of the Cgb and Ctb haems in soluble fractions after oxidation by NO were evaluated. Construction of E. coli nor mutants and a ubiquinone-defective strain allowed the exploration of the flavorubredoxin reductase and the aerobic respiratory chain as candidates for Cgb electron donors in E. coli mutants. RESULTS Cgb, but not Ctb, complements the NO- and RNS-sensitive phenotype of an E. coli hmp mutant in aerobic conditions; however, Cgb fails to protect an hmp norR mutant in the absence of oxygen. Reduction of Cgb and Ctb in E. coli and C. jejuni soluble extracts and turnover after NO oxidation is demonstrated. Finally, we report a minor role for NorW as a Cgb reductase partner in E. coli but no role for respiratory electron flux in globin redox cycling. CONCLUSIONS The NO detoxification capacity of Cgb is confirmed by heterologous expression in E. coli. The reducibility of Cgb and Ctb in E. coli and C. jejuni extracts and the lack of dependence of reduction upon flavorubredoxin reductase and the respiratory chain in E. coli argue in favor of a non-specific reductase system. GENERAL SIGNIFICANCE We present the most persuasive evidence to date that Cgb, but not Ctb, confers tolerance to NO and RNS by reaction with NO. Since certain hypotheses for the mechanism of haem re-reduction in E. coli following the reaction with NO are not proven, the mechanisms of reduction in C. jejuni now require challenging experimental evaluation.

Do Globins in Microaerophilic Campylobacter jejuni Confer Nitrosative Stress Tolerance Under Oxygen Limitation

The microaerophilic pathogen Campylobacter jejuni possesses inducible systems for resisting NO. Two globins--Cgb (a single-domain globin) and Ctb (a truncated globin)--are up-regulated in response to NO via the positively acting transcription factor NssR. Our aims were to determine whether these oxygen-binding globins also function in severely oxygen-limited environments, as in the host. At growth-limiting oxygen transfer rates, bacteria were more S-nitrosoglutathione (GSNO) sensitive, irrespective of the presence of Cgb, Ctb, or NssR. Pregrowth of cells with GSNO enhanced GSNO resistance, even in nssR and cgb mutants, but transcriptomic profiling of oxygen-limited, NO-exposed cells failed to reveal the NssR regulon. Nevertheless, globin expression in an Escherichia coli mutant lacking the NO-detoxifying flavohemoglobin Hmp showed that Cgb and Ctb consume NO aerobically or anoxically and offer some protection to respiratory inhibition by NO. The constitutively expressed nitrite reductase NrfA does not provide resistance under oxygen-limited conditions. We, therefore, hypothesize that, although Cgb and NrfA can detoxify NO, even anoxically, they are neither up-regulated nor functional under physiologically relevant oxygen-limited conditions and, second, responses to NO do not stem from trancriptional regulation.

Ligand uptake in Mycobacterium tuberculosis truncated hemoglobins is controlled by both internal tunnels and active site water molecules

Mycobacterium tuberculosis, the causative agent of human tuberculosis, has two proteins belonging to the truncated hemoglobin (trHb) family. Mt-trHbN presents well-defined internal hydrophobic tunnels that allow O 2 and (•)NO to migrate easily from the solvent to the active site, whereas Mt-trHbO possesses tunnels that are partially blocked by a few bulky residues, particularly a tryptophan at position G8. Differential ligand migration rates allow Mt-trHbN to detoxify (•)NO, a crucial step for pathogen survival once under attack by the immune system, much more efficiently than Mt-trHbO. In order to investigate the differences between these proteins, we performed experimental kinetic measurements, (•)NO decomposition, as well as molecular dynamics simulations of the wild type Mt-trHbN and two mutants, VG8F and VG8W. These mutations introduce modifications in both tunnel topologies and affect the incoming ligand capacity to displace retained water molecules at the active site. We found that a single mutation allows Mt-trHbN to acquire ligand migration rates comparable to those observed for Mt-trHbO, confirming that ligand migration is regulated by the internal tunnel architecture as well as by water molecules stabilized in the active site.

Haemoglobins of Mycobacteria: structural features and biological functions.

The genus Mycobacterium is comprised of Gram-positive bacteria occupying a wide range of natural habitats and includes species that range from severe intracellular pathogens to economically useful and harmless microbes. The recent upsurge in the availability of microbial genome data has shown that genes encoding haemoglobin-like proteins are ubiquitous among Mycobacteria and that multiple haemoglobins (Hbs) of different classes may be present in pathogenic and non-pathogenic species. The occurrence of truncated haemoglobins (trHbs) and flavohaemoglobins (flavoHbs) showing distinct haem active site structures and ligand-binding properties suggests that these Hbs may be playing diverse functions in the cellular metabolism of Mycobacteria. TrHbs and flavoHbs from some of the severe human pathogens such as Mycobacterium tuberculosis and Mycobacterium leprae have been studied recently and their roles in effective detoxification of reactive nitrogen and oxygen species, electron cycling, modulation of redox state of the cell and facilitation of aerobic respiration have been proposed. This multiplicity in the function of Hbs may aid these pathogens to cope with various environmental stresses and survive during their intracellular regime. This chapter provides recent updates on genomic, structural and functional aspects of Mycobacterial Hbs to address their role in Mycobacteria.

Listeria monocytogenes has both a bd-type and an aa3 -type terminal oxidase which allow growth in 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.