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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

Differential expression of genes involved in iron transport and acquisition. The heat map quantifies the changes elicited by CORM-3 and iCORM3 in selected genes; it should be noted that the “heat scale” at the right is expressed as the natural logarithm of the fold changes in individual genes of the heme-deficient mutant of E. coli (hemA) and the corresponding wild-type grown anaerobically in defined medium after addition of 100 μM CORM-3 or, for the mutant only, 100 μM iCORM-3.
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f4: Differential expression of genes involved in iron transport and acquisition. The heat map quantifies the changes elicited by CORM-3 and iCORM3 in selected genes; it should be noted that the “heat scale” at the right is expressed as the natural logarithm of the fold changes in individual genes of the heme-deficient mutant of E. coli (hemA) and the corresponding wild-type grown anaerobically in defined medium after addition of 100 μM CORM-3 or, for the mutant only, 100 μM iCORM-3.

Mentions: The category of genes most affected by CORM-3 were those encoding iron transport and acquisition functions; even after 10 min of exposure, ∼60% of such genes increased in expression in both the hemA and wild-type strains (Fig. 3A). The heat map in Figure 4 quantifies the changes elicited by CORM-3 and iCORM3 in selected genes involved in iron acquisition; it should be noted that the “heat scale” at the right is expressed as the natural logarithm of the fold change. Genes involved in the biosynthesis of the catecholate siderophore enterobactin (ent) were the most highly altered, with expression levels reaching 80-fold in the mutant and 10-fold in the wild-type strain (Fig. 4). Upregulated genes across all conditions tested also included the following: (i) fepA, which encodes an outer membrane (OM) protein that actively transports ferric enterobactin into the periplasm; (ii) fepBCDG, which encodes an ABC transporter that transports the iron(III)-bound siderophore through the cytoplasmic membrane (9); and (iii) fes, which encodes an enterobactin/ferric enterobactin esterase for intracellular breakdown of the ferrated carrier (8). The fes gene was also more highly upregulated in the mutant (19–35-fold) compared with the wild-type (2–6-fold) in response to CORM-3. Genes encoding the hydroxamate siderophore uptake system (fhu) that enables utilization of ferrichrome, ferric coprogen, and ferrioxamine B as sources of iron under low iron conditions were upregulated, as were genes encoding the ferric citrate (fec) and ferrous iron-uptake (feoA) systems (Fig. 4). Overall, the transcriptomic analysis reveals a marked enhancement of the expression of genes involved in iron scavenging in the hemA mutant when treated with CORM-3.


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)

Differential expression of genes involved in iron transport and acquisition. The heat map quantifies the changes elicited by CORM-3 and iCORM3 in selected genes; it should be noted that the “heat scale” at the right is expressed as the natural logarithm of the fold changes in individual genes of the heme-deficient mutant of E. coli (hemA) and the corresponding wild-type grown anaerobically in defined medium after addition of 100 μM CORM-3 or, for the mutant only, 100 μM iCORM-3.
© Copyright Policy - open-access
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC4492677&req=5

f4: Differential expression of genes involved in iron transport and acquisition. The heat map quantifies the changes elicited by CORM-3 and iCORM3 in selected genes; it should be noted that the “heat scale” at the right is expressed as the natural logarithm of the fold changes in individual genes of the heme-deficient mutant of E. coli (hemA) and the corresponding wild-type grown anaerobically in defined medium after addition of 100 μM CORM-3 or, for the mutant only, 100 μM iCORM-3.
Mentions: The category of genes most affected by CORM-3 were those encoding iron transport and acquisition functions; even after 10 min of exposure, ∼60% of such genes increased in expression in both the hemA and wild-type strains (Fig. 3A). The heat map in Figure 4 quantifies the changes elicited by CORM-3 and iCORM3 in selected genes involved in iron acquisition; it should be noted that the “heat scale” at the right is expressed as the natural logarithm of the fold change. Genes involved in the biosynthesis of the catecholate siderophore enterobactin (ent) were the most highly altered, with expression levels reaching 80-fold in the mutant and 10-fold in the wild-type strain (Fig. 4). Upregulated genes across all conditions tested also included the following: (i) fepA, which encodes an outer membrane (OM) protein that actively transports ferric enterobactin into the periplasm; (ii) fepBCDG, which encodes an ABC transporter that transports the iron(III)-bound siderophore through the cytoplasmic membrane (9); and (iii) fes, which encodes an enterobactin/ferric enterobactin esterase for intracellular breakdown of the ferrated carrier (8). The fes gene was also more highly upregulated in the mutant (19–35-fold) compared with the wild-type (2–6-fold) in response to CORM-3. Genes encoding the hydroxamate siderophore uptake system (fhu) that enables utilization of ferrichrome, ferric coprogen, and ferrioxamine B as sources of iron under low iron conditions were upregulated, as were genes encoding the ferric citrate (fec) and ferrous iron-uptake (feoA) systems (Fig. 4). Overall, the transcriptomic analysis reveals a marked enhancement of the expression of genes involved in iron scavenging in the hemA mutant when treated with CORM-3.

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