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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

Functional categories of genes affected by CORM-3 in the heme-deficient mutant and wild-type strains and a comparison of gene changes in thehemAmutant treated with CORM-3versusiCORM-3. Cultures were grown anaerobically in defined medium. The bars show the percentage of genes in each group that exhibit altered expression after treatment with 100 μM CORM-3. (A) Data are shown for the hemA mutant (left of the midline in each panel) and the wild-type (WT, right of the midline in each panel). Data are shown for cells at 10 min (i), 20 min (ii), 40 min (iii), 60 min (iv), and 120 min (v) after addition of CORM-3. (B) The bars show the percentage of genes in each group that exhibit altered expression after treatment with 100 μM CORM-3 (left of the midline in each panel) and iCORM-3 (right of the midline in each panel) in the hemA mutant. Data are shown for cells at 10 min (i), 20 min (ii), 40 min (iii), 60 min (iv), and 120 min (v) after addition of CORM-3. For each group of data (WT vs. hemA or CORM-3 vs. iCORM-3), the darker bars in each category indicate the percentage of upregulated genes and the paler bars indicate the percentage of downregulated genes.
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f3: Functional categories of genes affected by CORM-3 in the heme-deficient mutant and wild-type strains and a comparison of gene changes in thehemAmutant treated with CORM-3versusiCORM-3. Cultures were grown anaerobically in defined medium. The bars show the percentage of genes in each group that exhibit altered expression after treatment with 100 μM CORM-3. (A) Data are shown for the hemA mutant (left of the midline in each panel) and the wild-type (WT, right of the midline in each panel). Data are shown for cells at 10 min (i), 20 min (ii), 40 min (iii), 60 min (iv), and 120 min (v) after addition of CORM-3. (B) The bars show the percentage of genes in each group that exhibit altered expression after treatment with 100 μM CORM-3 (left of the midline in each panel) and iCORM-3 (right of the midline in each panel) in the hemA mutant. Data are shown for cells at 10 min (i), 20 min (ii), 40 min (iii), 60 min (iv), and 120 min (v) after addition of CORM-3. For each group of data (WT vs. hemA or CORM-3 vs. iCORM-3), the darker bars in each category indicate the percentage of upregulated genes and the paler bars indicate the percentage of downregulated genes.

Mentions: To provide an in-depth, time-resolved assessment of the response of the heme-deficient mutant and wild-type strains to CORM-3 and iCORM-3, we performed transcriptomic analyses, sampling cultures after CORM-3 addition to both wild-type and hemA mutant cultures. The CORM-3 added (100 μM) was sufficient to challenge cells without significantly reducing viability within the timeframe of the experiment (Fig. 1D, E). The genome-wide effects of CORM-3 are revealed for each sampling point by the percentages of up- and downregulated genes in a number of functional categories (Fig. 3). Based on the proportions of genes in each class, the wild-type initially (20–60 min after CORM-3 addition) responds to CORM-3 more than the mutant but, by 120 min, the responses are similar (Fig. 3A). The upregulation at 10–60 min in both strains of genes involved in iron transport and acquisition is striking. However, at 20–120 min after CORM-3 addition, genes in most functional classes are down-, not up-, regulated in both strains (Fig. 3A).


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)

Functional categories of genes affected by CORM-3 in the heme-deficient mutant and wild-type strains and a comparison of gene changes in thehemAmutant treated with CORM-3versusiCORM-3. Cultures were grown anaerobically in defined medium. The bars show the percentage of genes in each group that exhibit altered expression after treatment with 100 μM CORM-3. (A) Data are shown for the hemA mutant (left of the midline in each panel) and the wild-type (WT, right of the midline in each panel). Data are shown for cells at 10 min (i), 20 min (ii), 40 min (iii), 60 min (iv), and 120 min (v) after addition of CORM-3. (B) The bars show the percentage of genes in each group that exhibit altered expression after treatment with 100 μM CORM-3 (left of the midline in each panel) and iCORM-3 (right of the midline in each panel) in the hemA mutant. Data are shown for cells at 10 min (i), 20 min (ii), 40 min (iii), 60 min (iv), and 120 min (v) after addition of CORM-3. For each group of data (WT vs. hemA or CORM-3 vs. iCORM-3), the darker bars in each category indicate the percentage of upregulated genes and the paler bars indicate the percentage of downregulated genes.
© Copyright Policy - open-access
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC4492677&req=5

f3: Functional categories of genes affected by CORM-3 in the heme-deficient mutant and wild-type strains and a comparison of gene changes in thehemAmutant treated with CORM-3versusiCORM-3. Cultures were grown anaerobically in defined medium. The bars show the percentage of genes in each group that exhibit altered expression after treatment with 100 μM CORM-3. (A) Data are shown for the hemA mutant (left of the midline in each panel) and the wild-type (WT, right of the midline in each panel). Data are shown for cells at 10 min (i), 20 min (ii), 40 min (iii), 60 min (iv), and 120 min (v) after addition of CORM-3. (B) The bars show the percentage of genes in each group that exhibit altered expression after treatment with 100 μM CORM-3 (left of the midline in each panel) and iCORM-3 (right of the midline in each panel) in the hemA mutant. Data are shown for cells at 10 min (i), 20 min (ii), 40 min (iii), 60 min (iv), and 120 min (v) after addition of CORM-3. For each group of data (WT vs. hemA or CORM-3 vs. iCORM-3), the darker bars in each category indicate the percentage of upregulated genes and the paler bars indicate the percentage of downregulated genes.
Mentions: To provide an in-depth, time-resolved assessment of the response of the heme-deficient mutant and wild-type strains to CORM-3 and iCORM-3, we performed transcriptomic analyses, sampling cultures after CORM-3 addition to both wild-type and hemA mutant cultures. The CORM-3 added (100 μM) was sufficient to challenge cells without significantly reducing viability within the timeframe of the experiment (Fig. 1D, E). The genome-wide effects of CORM-3 are revealed for each sampling point by the percentages of up- and downregulated genes in a number of functional categories (Fig. 3). Based on the proportions of genes in each class, the wild-type initially (20–60 min after CORM-3 addition) responds to CORM-3 more than the mutant but, by 120 min, the responses are similar (Fig. 3A). The upregulation at 10–60 min in both strains of genes involved in iron transport and acquisition is striking. However, at 20–120 min after CORM-3 addition, genes in most functional classes are down-, not up-, regulated in both strains (Fig. 3A).

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