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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

Levels of Spy and CpxP protein are altered in response to CORM-3 in wild-type andhemAcells. Western blotting of subcellular fractions was carried out in the absence and presence of 20 μM CORM-3. (A) A typical Western blot is shown in the absence of CORM-3 (lane 1), or with 1, 2, or 4 h of incubation for wild-type or hemA cells with anti-Spy and (B) anti-CpxP. Bands of nonspecific binding of antibody are shown to demonstrate equal loading of protein in each lane.
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f7: Levels of Spy and CpxP protein are altered in response to CORM-3 in wild-type andhemAcells. Western blotting of subcellular fractions was carried out in the absence and presence of 20 μM CORM-3. (A) A typical Western blot is shown in the absence of CORM-3 (lane 1), or with 1, 2, or 4 h of incubation for wild-type or hemA cells with anti-Spy and (B) anti-CpxP. Bands of nonspecific binding of antibody are shown to demonstrate equal loading of protein in each lane.

Mentions: It is clear that upregulation of spy at the transcriptomic level, along with transcripts such as cpxP, is indicative of the upregulation of the Cpx, or “cell envelope stress,” response when cells are exposed to CORM-3. Although these effects at the transcriptomic level are marked, it was important to determine whether the rise in level of transcripts reflects physiological production of protein. Western blot assays were therefore carried out using antisera to two key players in the response: Spy and CpxP. Spy was detected in periplasmic fractions of wild-type cells as expected. However, it was only readily detected in soluble, presumably cytoplasm-derived, fractions of hemA cells; the reason is unclear. For immunoblotting with CpxP, soluble fractions were used for hemA and the isogenic wild-type strain. Loading controls confirmed equal loading of protein in each well (Coomassie-stained gels, not shown), and nonspecific binding in the case of the Spy blots also indicate equal sample loading. As shown in Figure 7A, addition of only 20 μM CORM-3 leads to a large increase in cellular protein after 1 h of incubation of the compound in wild-type cells; further incubation did not significantly increase Spy levels. The control in the absence of CORM shows very little Spy protein. Interestingly, the level of Spy protein is also increased with time after addition of CORM to hemA cells.


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)

Levels of Spy and CpxP protein are altered in response to CORM-3 in wild-type andhemAcells. Western blotting of subcellular fractions was carried out in the absence and presence of 20 μM CORM-3. (A) A typical Western blot is shown in the absence of CORM-3 (lane 1), or with 1, 2, or 4 h of incubation for wild-type or hemA cells with anti-Spy and (B) anti-CpxP. Bands of nonspecific binding of antibody are shown to demonstrate equal loading of protein in each lane.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

f7: Levels of Spy and CpxP protein are altered in response to CORM-3 in wild-type andhemAcells. Western blotting of subcellular fractions was carried out in the absence and presence of 20 μM CORM-3. (A) A typical Western blot is shown in the absence of CORM-3 (lane 1), or with 1, 2, or 4 h of incubation for wild-type or hemA cells with anti-Spy and (B) anti-CpxP. Bands of nonspecific binding of antibody are shown to demonstrate equal loading of protein in each lane.
Mentions: It is clear that upregulation of spy at the transcriptomic level, along with transcripts such as cpxP, is indicative of the upregulation of the Cpx, or “cell envelope stress,” response when cells are exposed to CORM-3. Although these effects at the transcriptomic level are marked, it was important to determine whether the rise in level of transcripts reflects physiological production of protein. Western blot assays were therefore carried out using antisera to two key players in the response: Spy and CpxP. Spy was detected in periplasmic fractions of wild-type cells as expected. However, it was only readily detected in soluble, presumably cytoplasm-derived, fractions of hemA cells; the reason is unclear. For immunoblotting with CpxP, soluble fractions were used for hemA and the isogenic wild-type strain. Loading controls confirmed equal loading of protein in each well (Coomassie-stained gels, not shown), and nonspecific binding in the case of the Spy blots also indicate equal sample loading. As shown in Figure 7A, addition of only 20 μM CORM-3 leads to a large increase in cellular protein after 1 h of incubation of the compound in wild-type cells; further incubation did not significantly increase Spy levels. The control in the absence of CORM shows very little Spy protein. Interestingly, the level of Spy protein is also increased with time after addition of CORM to hemA cells.

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