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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

CO retention and access of CORM-3 to the cell interior is dependent on heme. (A) Myoglobin (10 μM) and CORM-3 (8 μM) were added to buffer only (circles), wild-type (squares), or hemA mutant (triangles) cells in the presence of Na dithionite. The concentration of Mb-CO accumulated was measured at several time points in CO difference spectra. Data are plotted as means±SEM from ≥5 replicates. (B) CO-reduced minus reduced spectra of myoglobin (10 μM) and CORM-3 (8 μM) added to buffer only (solid line), wild-type (dotted line), or hemA mutant (dashed line) cells in the presence of Na dithionite at t=5 min. (C, D) Intracellular ruthenium levels in hemA (closed circles) and wild-type (open circles) cells were measured by ICP-AES over 120 min after exposure of cultures to 100 μM CORM-3 under anaerobic (C) or aerobic (D) conditions. Data are plotted as means±SEM from ≥3 biological replicates.
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f2: CO retention and access of CORM-3 to the cell interior is dependent on heme. (A) Myoglobin (10 μM) and CORM-3 (8 μM) were added to buffer only (circles), wild-type (squares), or hemA mutant (triangles) cells in the presence of Na dithionite. The concentration of Mb-CO accumulated was measured at several time points in CO difference spectra. Data are plotted as means±SEM from ≥5 replicates. (B) CO-reduced minus reduced spectra of myoglobin (10 μM) and CORM-3 (8 μM) added to buffer only (solid line), wild-type (dotted line), or hemA mutant (dashed line) cells in the presence of Na dithionite at t=5 min. (C, D) Intracellular ruthenium levels in hemA (closed circles) and wild-type (open circles) cells were measured by ICP-AES over 120 min after exposure of cultures to 100 μM CORM-3 under anaerobic (C) or aerobic (D) conditions. Data are plotted as means±SEM from ≥3 biological replicates.

Mentions: To assess CO removal from CORM-3 and intracellular binding of CO in wild-type and heme-deficient E. coli, we followed formation of extracellular carboxymyoglobin (Mb-CO) over time after adding CORM-3 to bacterial suspensions in buffer in the presence of exogenously added myoglobin (Fig. 2A, B). Myoglobin cannot enter cells and so acts as a “sink” for unbound CO that would otherwise freely diffuse through membranes. Wild-type cells retained a significant amount of the CO released from CORM-3 (∼50%), making it unavailable to the extracellular myoglobin (Fig. 2B). However, in the heme-deficient strain, the accumulation of Mb-CO mirrored the pattern observed after addition of CORM-3 to myoglobin in buffer alone. That is, CO is not retained by hemA bacteria that lack a “CO trap.”


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)

CO retention and access of CORM-3 to the cell interior is dependent on heme. (A) Myoglobin (10 μM) and CORM-3 (8 μM) were added to buffer only (circles), wild-type (squares), or hemA mutant (triangles) cells in the presence of Na dithionite. The concentration of Mb-CO accumulated was measured at several time points in CO difference spectra. Data are plotted as means±SEM from ≥5 replicates. (B) CO-reduced minus reduced spectra of myoglobin (10 μM) and CORM-3 (8 μM) added to buffer only (solid line), wild-type (dotted line), or hemA mutant (dashed line) cells in the presence of Na dithionite at t=5 min. (C, D) Intracellular ruthenium levels in hemA (closed circles) and wild-type (open circles) cells were measured by ICP-AES over 120 min after exposure of cultures to 100 μM CORM-3 under anaerobic (C) or aerobic (D) conditions. Data are plotted as means±SEM from ≥3 biological replicates.
© Copyright Policy - open-access
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC4492677&req=5

f2: CO retention and access of CORM-3 to the cell interior is dependent on heme. (A) Myoglobin (10 μM) and CORM-3 (8 μM) were added to buffer only (circles), wild-type (squares), or hemA mutant (triangles) cells in the presence of Na dithionite. The concentration of Mb-CO accumulated was measured at several time points in CO difference spectra. Data are plotted as means±SEM from ≥5 replicates. (B) CO-reduced minus reduced spectra of myoglobin (10 μM) and CORM-3 (8 μM) added to buffer only (solid line), wild-type (dotted line), or hemA mutant (dashed line) cells in the presence of Na dithionite at t=5 min. (C, D) Intracellular ruthenium levels in hemA (closed circles) and wild-type (open circles) cells were measured by ICP-AES over 120 min after exposure of cultures to 100 μM CORM-3 under anaerobic (C) or aerobic (D) conditions. Data are plotted as means±SEM from ≥3 biological replicates.
Mentions: To assess CO removal from CORM-3 and intracellular binding of CO in wild-type and heme-deficient E. coli, we followed formation of extracellular carboxymyoglobin (Mb-CO) over time after adding CORM-3 to bacterial suspensions in buffer in the presence of exogenously added myoglobin (Fig. 2A, B). Myoglobin cannot enter cells and so acts as a “sink” for unbound CO that would otherwise freely diffuse through membranes. Wild-type cells retained a significant amount of the CO released from CORM-3 (∼50%), making it unavailable to the extracellular myoglobin (Fig. 2B). However, in the heme-deficient strain, the accumulation of Mb-CO mirrored the pattern observed after addition of CORM-3 to myoglobin in buffer alone. That is, CO is not retained by hemA bacteria that lack a “CO trap.”

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