There are already several examples of the effectiveness of CORMs as antimicrobials Table 1 , but similar studies remain to be done with H 2 S. In fact, sulfide and CO-based compounds may represent a novel kind of antimicrobials, with modes of action and targets that are different from those of the currently available antibiotics.
And new antimicrobial drugs are urgently needed due to the growing number of infections caused by antibiotic-resistant pathogenic strains. Writing—original draft preparation, review and editing: S. All authors have read and agreed to the published version of the manuscript. National Center for Biotechnology Information , U. Journal List Antioxidants Basel v. Antioxidants Basel.
Published online May 5. Sofia S. Daniele Mancard, Academic Editor. Author information Article notes Copyright and License information Disclaimer. Received Apr 7; Accepted May 3. Abstract Hydrogen sulfide and carbon monoxide share the ability to be beneficial or harmful molecules depending on the concentrations to which organisms are exposed.
Keywords: bacteria, hydrogen sulfide, carbon monoxide, CORMs. Introduction Hydrogen sulfide H 2 S and carbon monoxide CO are small molecules that are historically related with environmental industrial pollution. Bacterial Responses to H 2 S In general, low concentrations of H 2 S in the micromolar range are cytoprotective, but millimolar concentrations are cytotoxic to microbes, and some prokaryotes contain proteins for protection against H 2 S efflux transporters [ 10 , 11 , 12 ].
Open in a separate window. Figure 1. H 2 S and Microbial Antibiotic Resistance A new mode of antibiotic resistance mediated by sulfide was reported in pathogenic bacteria that involves inhibition of the oxidative stress imposed by ROS-generating antibiotics.
H 2 S in Host-Pathogen Interactions Several works indicate that upregulation of genes encoding bacterial enzymes involved in sulfide biogenesis is an important adaptive response of pathogens during the infection process. CO Utilizing Bacteria Carbon monoxide is utilized by several bacteria as energy source, e. Table 1 CORMs used as antimicrobials.
Figure 2. CO and H 2 S in Host-Pathogen Interactions The finding that HO-1 is highly induced in macrophages in response to bacterial infections, and that animals with blocked HO-1 were highly sensitive to bacteria and presented signs of systemic inflammatory response led to the proposal that CO enhances bacterial clearance [ ].
H 2 S and CO Interplay The chemistry and biology of H 2 S and CO are interconnected due to the shared capacity of these molecules to interact with metal centers and cysteine residues in proteins. Conclusions Along with nitric oxide, H 2 S and CO are double-edged molecules—if on the one hand are essential signaling molecules to human cells, on the other hand they can cause death.
Author Contributions Writing—original draft preparation, review and editing: S. Conflicts of Interest The authors declare no conflict of interest. References 1. Predmore B. Hydrogen Sulfide in Biochemistry and Medicine. Redox Signal. Siracusa R. Trends Pharmacol. Zuhra K. Szabo C. Gullotta F. CO Metabolism, Sensing, and Signaling. Carbon Monoxide: An Unusual Drug. Kourti M. Ryter S. Ritter J. Shatalin K. Chemical Foundations of Hydrogen Sulfide Biology. Nitric Oxide Biol.
Kolluru G. Aroca A. Avalos M. Juarez G. Shimizu T. Walsh B. Forte E. Tanous C. Peng H. ACS Infect. Soutourina O. Shen J. Luebke J. Weikum J. Agents Chemother. Mironov A. Ono K. ACS Chem. Rahman M. Pal V. Xie Z. Hydrogen Sulfide and Cellular Redox Homeostasis. Benedetti F. Sun F. Wallace J. Rivers-Auty J. Whiteman M. Aslami H. Bazhanov N. Cell Mol. Citi V. Yang G. J Physiol Cell Physiol. Garg S. Gemici B. Methods Enzymol. Sen N. Saini V. Rowan F. Toliver-kinsky T.
Hampelska K. Diender M. Liu Z. Erratum for Liu et al. Abrini J. Clostridium autoethanogenum , sp. Robb F. Ragsdale S. Life with Carbon Monoxide. Fukuyama Y. Volume Elsevier Inc. Svetlitchnyi V. Youn H. Kim Y. Antonie Van Leeuwenhoek. King G. Henstra A. Plugge C. Wilson J. Antibacterial Effects of Carbon Monoxide. Zegdi R. Intensive Care Med. This ensures that immune cells become fully activated only when confronted with live bacteria; when presented with bacterial fragments, such as the lipid polysaccharides present on bacteria's outer walls, immune cells become only mildly stimulated.
This distinction conserves energy and helps the immune cells to maintain an efficient defense system. We now see that CO plays a critical role in the execution of this two-step process, enabling the immune cells to successfully carry out their attack. Otterbein has been studying carbon monoxide for more than 12 years and his novel studies have revealed promising therapeutic applications for the gas, including treatment of pulmonary hypertension, prevention of organ rejection following transplantation, reduction of vascular restenosis and, most recently, shrinkage of cancerous tumors.
Clinical trials to test inhaled CO for the treatment of lung disease, intestinal dysfunction and organ transplantation are currently underway -- the direct result of Otterbein's pioneering work. We, therefore, hypothesized that CO, generated by HO-1, was acting as a scout for the innate immune system and was somehow providing the signal to the macrophages that bacteria was present and it was time to attack.
The team also made use of novel bacterial strains that were mutated such that they were unable to respond to CO in order to elucidate potential targets where CO was imparting its effects.
CO appeared to be boosting the immune response to enhance clearance of the bacteria and resolution of the ongoing SIRS.
Subsequent experiments in cell culture and confirmed in animal models revealed the mechanisms underlying the immune cells' two-step process. If no active bacteria is present, the CO dissipates.
But if dangerous microbes are lurking, the CO takes on a new responsibility and attaches itself to specific locations on the bacteria. And this, he explains, serves as the second signal to alert the immune cells to take action.
The discovery unveils a new concept and offers a new avenue for the development of therapies to complement or potentially reduce the need for antibiotics and thus limit the development of antibiotic resistance, say the authors.
Predicted TF activity of Fur, a transcriptional repressor of Fe-regulated genes, and corresponding gene changes. C and D map changes in iron-regulated genes aerobically and anaerobically, respectively.
Yellow indicates genes that remain unchanged in the presence of CO, red indicates genes upregulated, and blue indicates genes downregulated, according to the heat map right. The intensity of the color is indicative of the trust placed on that measured level of regulation after three biological repeats. Feature extraction from the scanned arrays and subsequent data analysis used GeneSpring GX v7. The clustering at the left is a GeneSpring gene tree and shows genes organized by their similarity in response to the imposed conditions.
Cells exposed to CO produce higher levels of siderophores as free intracellular iron pools are depleted. The diameter of the cells plus halo and the diameter of the cells alone were measured and the difference plotted in A. B The ferric iron in rhombic coordination in whole cells as measured by electron paramagnetic resonance EPR spectroscopy shows that the iron levels decreased both aerobically and anaerobically on exposure to CO gas.
In contrast, the aerobic downregulation of cyo expression is explicable by rising FNR activity alone. GadE 28 and CRP 75 upregulate cyo expression.
PdhR functions with other central regulators as an additional master switch 46 , negatively regulating expression of the pdh promoter, with pyruvate serving as an inducing coeffector 51 and repressing the cyo and ndh operons The structural genes of the PDH operon Fig. S2 , as was the cyo operon, implying loss of the repressing activity of PdhR, perhaps due to pyruvate TFInfer analysis Fig.
Electron flow into the respiratory chains when CO is present is facilitated both aerobically and anoxically Fig. FNR represses ndh expression anoxically and expression of an ndh-lacZ fusion in an fnr mutant is enhanced by anaerobiosis Therefore, the inferred increase in FNR activity Fig. Other regulators may be implicated, such as Fis, which enhances ndh expression during rapid growth Expression of Ndh is associated with growth where protonmotive force generation is not favored 10 , consistent with the presence of CO.
In contrast to ndh , the nuo genes encoding a multisubunit, proton-pumping NADH dehydrogenase were downregulated 2- to 5 fold in response to CO in anoxic conditions Supplementary Fig. These results together with downregulation of the cyo operon Fig.
Global responses to CO included gross alterations in amino acid metabolism Fig. First, many genes in the cys and tau operons were upregulated, suggesting a shortage of sulfur in response to CO Supplementary Fig. CysB activates the cys regulon for sulfur utilization and uptake, the tau operon for taurine utilization, the cbl gene encoding a regulator of the sulfate starvation response, and, in coordination with the Cbl protein, the sulfonate utilization genes ssuABCDE Many of these genes are upregulated in response to CO Supplementary Fig.
Second, many genes involved in methionine biosynthesis were perturbed by CO Supplementary Fig. S5B , but with complex patterns of expression. Aerobically, a requirement for methionine appears immediate with many met biosynthetic genes upregulated by CO, especially metA , metB , metF , metK , metN , and metR.
MetJ, which represses the expression of genes involved in biosynthesis and transport of methionine 5 , was elevated in the coherence plot Fig. Most arginine biosynthesis genes were highly upregulated aerobically as well as carA encoding carbamoyl synthetase. However, anaerobically, these genes were predominantly downregulated Supplementary Fig. Thus, CO appears to lead to arginine deprivation under aerobic conditions. Interestingly, peroxynitrite also increases arginine biosynthesis transcript levels in E.
However, CO is relatively inert CO dehydrogenase being the only documented biological redox activity and it is unclear how it could modify or degrade ArgR see the Discussion section.
The location of Fur in the upper left quadrant of the coherence plot Fig. Since the primary biological targets of CO are heme- and [Fe-S] cluster-containing proteins 8 , we investigated the role of Fur, the principal TF involved in detecting iron levels and repressing genes for iron acquisition Aerobically Fig. S6B and the fep and fhu genes involved in ferric enterobactin transport and hydroxamate-dependent iron uptake, respectively.
Anoxically, there was extensive and sustained derepression of iron-regulated genes Figs. S6B and inferred Fur activity fell sharply Fig. Overall, the data point to CO-induced iron deprivation, particularly anoxically. The changes in respiratory genes Fig. S2B are also consistent with modulation by iron. Given the changes in ent gene expression, it was important to determine whether cell physiology reflected transcript levels.
Siderophore production on CAS plates caused a clear orange halo around the bacterial growth not shown. Both aerobically and anaerobically, CO increased halo diameter corrected for the spot size, representing siderophore production; Fig. To test whether free intracellular iron pools were depleted upon exposure to CO, EPR analysis of ferric ions in rhombic coordination was performed Importantly, desferrioxamine does not chelate protein-bound iron. Thus, CO depletes free cellular iron, consistent with patterns of gene expression and elevated siderophore synthesis.
Given the changes in iron-related genes and iron pools, we hypothesized that iron chelators would inhibit growth in CO since intracellular iron limitation would be exacerbated by exogenous chelating activity. Bacteria were therefore grown with 8-hydroxyquinoline or citric acid in CO atmospheres Fig. For each, the aerobic left and anoxic data right show the effects of increasing concentrations as a percentage of the no-chelator control in the same gas atmosphere.
CO alone slightly inhibited aerobic growth. These data are not shown as the chelator concentrations in proprietary Biolog plates are unknown.
We did not use desferrioxamine in these experiments designed to test extracellular retention of iron since this chelator is cell permeable. In conclusion, CO exacerbates inhibition of E. Effects of CO gas on bacterial sensitivity to metal chelators and antibiotics. Aliquots of E. With the advent of antibiotic resistance, combinatorial approaches may reduce dependency on the use of single antibiotics This is shown also in the no-chelator plots in Figure 8A and B , but is currently unexplained.
For example, CT Fig. The results were similar, irrespective of the presence of oxygen. Since CO gas is not a potent antimicrobial agent Supplementary Fig. The toxicity of this compound allowed us to use it in agar plates where its effects on the minimal inhibitory concentrations MICs of three well-established antibiotics TR, DC, and CT were determined.
CO delivery to key sites offers novel pharmacological and therapeutic approaches, but we know too little of how CO mediates its multiple biological effects. Although CO's affinity for heme proteins is undisputed, its actions in vivo are remarkably complex 54 , having further roles in NO release, reactive oxygen species formation, and ion channels It has been tacitly assumed that the biological impacts of CORMs are attributable largely to CO delivery to hemes, but microbial 70 and mammalian 54 studies point to greater complexity.
Indeed, bacteria are often resistant to CO gas 71 , but not to certain CORMs, and demonstrate multiple transcriptomic changes that cannot be understood in terms of known CO biochemistry [ e. Most tellingly, cells lacking all hemes are also inhibited by CORM-3 and reveal multiple transcriptomic changes Finally, other compounds of Ru are taken up and have antimicrobial properties, even though they are not CORMs [ e.
We therefore characterized the effects of CO gas per se , without mediation of a CORM, on bacterial growth, gene expression, and physiology. HO-derived CO induces the dormancy regulon in Mycobacterium tuberculosis The CO-oxidizing Archaeon Archaeoglobus fulgidus responds to CO with only limited transcriptional effects 26 : of 52 genes observed to alter, inorganic ion transport was the most prominent category, along with cooF , encoding a CO dehydrogenase Fe-S enzyme.
Bacterial mechanisms for resisting CO are unclear. Zacharia et al. We demonstrate here for the first time that genes encoding energy transduction are significantly affected by CO via the master regulators, ArcA and FNR. In the case of Arc, CO inhibition of respiration promotes pyruvate accumulation Fig. These changes indirectly result in modulation of Arc activity 6 , 29 , 31 Fig.
In contrast, FNR is primarily a direct sensor of oxygen, although the 4Fe-4S cluster of the transcriptionally active factor is also sensitive to NO, rendering it inactive as a repressor Schematic diagram of the global impacts of CO gas on E.
Inhibition of the electron transfer chain results in stimuli that cause ArcA phosphorylation, namely over-reduction of the quinone pool and formation of the fermentation product, pyruvate, in concentrations that can be assayed extracellularly. Transcriptomic responses are consistent with direct or indirect modulation by CO of Fnr and Fur activities. A striking consequence of CO exposure was enhanced expression of iron acquisition genes and changes in Fur activity.
The reason for the loss of total free iron in cells is unclear, but CO may result in more iron being protein bound, perhaps in inactive states. Bacteria thus respond by producing more iron-containing proteins, further draining iron resources. The change in Fur activity may also be due to a direct effect of CO on the protein since the active form of Fur contains a nonheme ferrous iron site with oxygen and nitrogen donor ligands 1. Binding of CO to Fur-associated heme would be more likely anoxically, consistent with the upregulation of Fe-related genes under anoxic conditions.
The upregulation of genes involved in amino acid metabolism was unexpected Supplementary Figs. S5 and S6 , although a transcriptomic study with CORM-3 showed similar results, with sulfur metabolism being a prime target The introduction of carbonyl groups into proteins by oxidation of amino acid side chains or by oxidative cleavage of proteins is also possible.
Protein carbonylation is metal-catalyzed oxidation, which occurs when reduced metal ions e. This in turn oxidizes amino acid side chains or causes protein backbone cleavage, both resulting in the formation of carbonyl groups. In bacteria, metal-catalyzed oxidations appear to be the predominant source of carbonylated proteins Arginine is one of the four most commonly carbonylated amino acids. Interestingly, the present data point to iron deprivation on CO treatment and could reflect loss of iron due to carbonylation.
Contrary to recent findings with NO 24 and H 2 S 59 , we found no evidence that CO significantly protected bacteria against antibiotics, nor did it potentiate their effect. The mechanisms underlying these effects require urgent investigation to facilitate future applications of combination therapy. For transcriptomic studies, cells were grown in Evans medium 41 with glucose as the carbon source.
Experiments were performed and analyzed as before 41 on samples from chemostat cultures immediately before CO gas addition and at time intervals thereafter. Regulatory proteins for each gene were identified using EcoCyc. Mass flow controllers as above controlled gas mixing and delivery. Anaerobic samples were removed using a sterile syringe and needle through a tube without the need to remove the lid. ArcA protein was purified as before 6. Values were corrected for the OD of the culture at the point of harvest.
ArcA phosphorylation of samples in each of two independent cultures biological repeats was analyzed in duplicate and the results of one experiment are shown in Figure 5. Quinone extraction and measurement methods were adapted from Concentrations for each quinone type were determined using extinction coefficients 27 , The quinone content of two independent cultures was analyzed in duplicate. Concentrations were corrected for the OD of the culture at harvest. Pyruvate content of two independent cultures was analyzed in duplicate.
A vacuum was first used to withdraw air and the vacuum gauge was read; the first gas e. Siderophore production was determined by measuring halo diameters Fig. The method was derived from 34 , Aliquots 0. Aliquots of a cell suspension OD 0. Preliminary testing of the combined effects of CO and antimicrobial compounds was done using Biolog Phenotype Microarrays.
National Center for Biotechnology Information , U. Antioxid Redox Signal. Lauren K. Jesse , 1 Johan W. Hellingwerf , 3 Guido Sanguinetti , 2 and Robert K.
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