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CO-Releasing Molecules Have Nonheme Targets in Bacteria: Transcriptomic, Mathematical Modeling and Biochemical Analyses of CORM-3 [Ru(CO)3Cl(glycinate)] Actions on a Heme-Deficient Mutant of Escherichia coli.

Wilson JL, Wareham LK, McLean S, Begg R, Greaves S, Mann BE, Sanguinetti G, Poole RK - Antioxid. Redox Signal. (2015)

Bottom Line: Carbon monoxide-releasing molecules (CORMs) are being developed with the ultimate goal of safely utilizing the therapeutic potential of CO clinically, including applications in antimicrobial therapy.A full understanding of the actions of CORMs is vital to understand their toxic effects.This is a vital step in exploiting the potential, already demonstrated, for using optimized CORMs in antimicrobial therapy.

View Article: PubMed Central - PubMed

Affiliation: 1 Department of Molecular Biology and Biotechnology, The University of Sheffield , Sheffield, United Kingdom .

ABSTRACT

Aims: Carbon monoxide-releasing molecules (CORMs) are being developed with the ultimate goal of safely utilizing the therapeutic potential of CO clinically, including applications in antimicrobial therapy. Hemes are generally considered the prime targets of CO and CORMs, so we tested this hypothesis using heme-deficient bacteria, applying cellular, transcriptomic, and biochemical tools.

Results: CORM-3 [Ru(CO)3Cl(glycinate)] readily penetrated Escherichia coli hemA bacteria and was inhibitory to these and Lactococcus lactis, even though they lack all detectable hemes. Transcriptomic analyses, coupled with mathematical modeling of transcription factor activities, revealed that the response to CORM-3 in hemA bacteria is multifaceted but characterized by markedly elevated expression of iron acquisition and utilization mechanisms, global stress responses, and zinc management processes. Cell membranes are disturbed by CORM-3.

Innovation: This work has demonstrated for the first time that CORM-3 (and to a lesser extent its inactivated counterpart) has multiple cellular targets other than hemes. A full understanding of the actions of CORMs is vital to understand their toxic effects.

Conclusion: This work has furthered our understanding of the key targets of CORM-3 in bacteria and raises the possibility that the widely reported antimicrobial effects cannot be attributed to classical biochemical targets of CO. This is a vital step in exploiting the potential, already demonstrated, for using optimized CORMs in antimicrobial therapy.

No MeSH data available.


Related in: MedlinePlus

CORM-3 perturbs the outer membrane ofE. coliin both wild-type andhemAcells. Wild-type (A–D) and hemA cells (E–H) were washed, resuspended in PBS, adjusted to an OD600 of ∼0.5, and then exposed to NPN alone (squares), NPN+30 μM CORM-3 (circles), or NPN+100 μM CORM-3 (triangles) (A, C, E, G). Data are shown for measurements in the absence (left) or presence (right) of cyanide (KCN). In control experiments (B, D, F, H), the compounds used were 100 μM iCORM-3 (open circles) or 100 μM CO gas in solution (open squares). All concentrations given are final concentrations in the fluorescence cuvette. Data are representative of ≥2 biological replicates.
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f8: CORM-3 perturbs the outer membrane ofE. coliin both wild-type andhemAcells. Wild-type (A–D) and hemA cells (E–H) were washed, resuspended in PBS, adjusted to an OD600 of ∼0.5, and then exposed to NPN alone (squares), NPN+30 μM CORM-3 (circles), or NPN+100 μM CORM-3 (triangles) (A, C, E, G). Data are shown for measurements in the absence (left) or presence (right) of cyanide (KCN). In control experiments (B, D, F, H), the compounds used were 100 μM iCORM-3 (open circles) or 100 μM CO gas in solution (open squares). All concentrations given are final concentrations in the fluorescence cuvette. Data are representative of ≥2 biological replicates.

Mentions: The effects of CORM-3 on cell outer membranes were assayed using the fluorescent probe N-phenyl-1-napthylamine (NPN) (42), a membrane-impermeable dye that has a weak fluorescence emission in buffer but increased fluorescence on exposure to a hydrophobic environment. Thus, when the bacterial membrane becomes perturbed (for example, by addition of an antibiotic or, here, CORM-3), the dye partitions into the outer membrane, leading to an increase in fluorescence. The potent respiratory inhibitor, potassium cyanide (KCN) was added, where indicated, to cell suspensions to prevent the expulsion of the dye by E. coli cells and give simpler fluorescence kinetics (12). CORM-3 perturbed the membrane of wild-type and hemA E. coli cells in the presence and absence of KCN (Fig. 8); a control with NPN incubated with cells alone showed no increase in basal fluorescence levels over 60 min. Since KCN is an inhibitor of terminal heme-mediated respiration, which is lacking in the hemA mutant, it was not surprising that KCN had less effect on the fluorescence profiles in hemA cells (compare Fig. 8E, G) than in wild-type cells (compare Fig. 8A, C). Interestingly, in mutant cell suspensions, basal levels of NPN fluorescence were greater than in the wild-type, reflected in the fluorescence intensity at zero time. Addition of CORM-3 to hemA cells, with or without KCN, led to higher fluorescence than in wild-type cells, suggesting greater damage to the membrane by CORM-3 perhaps due to an already compromised outer membrane. In wild-type cells, neither equimolar CO nor iCORM-3 significantly increased NPN fluorescence (Fig. 8B, D). However, in hemA cells, particularly in the presence of KCN, iCORM-3 but not CO elicited fluorescence increase, suggesting membrane destabilization (Fig. 8F, H).


CO-Releasing Molecules Have Nonheme Targets in Bacteria: Transcriptomic, Mathematical Modeling and Biochemical Analyses of CORM-3 [Ru(CO)3Cl(glycinate)] Actions on a Heme-Deficient Mutant of Escherichia coli.

Wilson JL, Wareham LK, McLean S, Begg R, Greaves S, Mann BE, Sanguinetti G, Poole RK - Antioxid. Redox Signal. (2015)

CORM-3 perturbs the outer membrane ofE. coliin both wild-type andhemAcells. Wild-type (A–D) and hemA cells (E–H) were washed, resuspended in PBS, adjusted to an OD600 of ∼0.5, and then exposed to NPN alone (squares), NPN+30 μM CORM-3 (circles), or NPN+100 μM CORM-3 (triangles) (A, C, E, G). Data are shown for measurements in the absence (left) or presence (right) of cyanide (KCN). In control experiments (B, D, F, H), the compounds used were 100 μM iCORM-3 (open circles) or 100 μM CO gas in solution (open squares). All concentrations given are final concentrations in the fluorescence cuvette. Data are representative of ≥2 biological replicates.
© Copyright Policy - open-access
Related In: Results  -  Collection

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f8: CORM-3 perturbs the outer membrane ofE. coliin both wild-type andhemAcells. Wild-type (A–D) and hemA cells (E–H) were washed, resuspended in PBS, adjusted to an OD600 of ∼0.5, and then exposed to NPN alone (squares), NPN+30 μM CORM-3 (circles), or NPN+100 μM CORM-3 (triangles) (A, C, E, G). Data are shown for measurements in the absence (left) or presence (right) of cyanide (KCN). In control experiments (B, D, F, H), the compounds used were 100 μM iCORM-3 (open circles) or 100 μM CO gas in solution (open squares). All concentrations given are final concentrations in the fluorescence cuvette. Data are representative of ≥2 biological replicates.
Mentions: The effects of CORM-3 on cell outer membranes were assayed using the fluorescent probe N-phenyl-1-napthylamine (NPN) (42), a membrane-impermeable dye that has a weak fluorescence emission in buffer but increased fluorescence on exposure to a hydrophobic environment. Thus, when the bacterial membrane becomes perturbed (for example, by addition of an antibiotic or, here, CORM-3), the dye partitions into the outer membrane, leading to an increase in fluorescence. The potent respiratory inhibitor, potassium cyanide (KCN) was added, where indicated, to cell suspensions to prevent the expulsion of the dye by E. coli cells and give simpler fluorescence kinetics (12). CORM-3 perturbed the membrane of wild-type and hemA E. coli cells in the presence and absence of KCN (Fig. 8); a control with NPN incubated with cells alone showed no increase in basal fluorescence levels over 60 min. Since KCN is an inhibitor of terminal heme-mediated respiration, which is lacking in the hemA mutant, it was not surprising that KCN had less effect on the fluorescence profiles in hemA cells (compare Fig. 8E, G) than in wild-type cells (compare Fig. 8A, C). Interestingly, in mutant cell suspensions, basal levels of NPN fluorescence were greater than in the wild-type, reflected in the fluorescence intensity at zero time. Addition of CORM-3 to hemA cells, with or without KCN, led to higher fluorescence than in wild-type cells, suggesting greater damage to the membrane by CORM-3 perhaps due to an already compromised outer membrane. In wild-type cells, neither equimolar CO nor iCORM-3 significantly increased NPN fluorescence (Fig. 8B, D). However, in hemA cells, particularly in the presence of KCN, iCORM-3 but not CO elicited fluorescence increase, suggesting membrane destabilization (Fig. 8F, H).

Bottom Line: Carbon monoxide-releasing molecules (CORMs) are being developed with the ultimate goal of safely utilizing the therapeutic potential of CO clinically, including applications in antimicrobial therapy.A full understanding of the actions of CORMs is vital to understand their toxic effects.This is a vital step in exploiting the potential, already demonstrated, for using optimized CORMs in antimicrobial therapy.

View Article: PubMed Central - PubMed

Affiliation: 1 Department of Molecular Biology and Biotechnology, The University of Sheffield , Sheffield, United Kingdom .

ABSTRACT

Aims: Carbon monoxide-releasing molecules (CORMs) are being developed with the ultimate goal of safely utilizing the therapeutic potential of CO clinically, including applications in antimicrobial therapy. Hemes are generally considered the prime targets of CO and CORMs, so we tested this hypothesis using heme-deficient bacteria, applying cellular, transcriptomic, and biochemical tools.

Results: CORM-3 [Ru(CO)3Cl(glycinate)] readily penetrated Escherichia coli hemA bacteria and was inhibitory to these and Lactococcus lactis, even though they lack all detectable hemes. Transcriptomic analyses, coupled with mathematical modeling of transcription factor activities, revealed that the response to CORM-3 in hemA bacteria is multifaceted but characterized by markedly elevated expression of iron acquisition and utilization mechanisms, global stress responses, and zinc management processes. Cell membranes are disturbed by CORM-3.

Innovation: This work has demonstrated for the first time that CORM-3 (and to a lesser extent its inactivated counterpart) has multiple cellular targets other than hemes. A full understanding of the actions of CORMs is vital to understand their toxic effects.

Conclusion: This work has furthered our understanding of the key targets of CORM-3 in bacteria and raises the possibility that the widely reported antimicrobial effects cannot be attributed to classical biochemical targets of CO. This is a vital step in exploiting the potential, already demonstrated, for using optimized CORMs in antimicrobial therapy.

No MeSH data available.


Related in: MedlinePlus