Constitutive Activation of RpoH and the Addition of L-arabinose Influence Antibiotic Sensitivity of PHL628 <i>E. coli</i>
Antibiotics are used to combat the ever-present threat of infectious diseases, but bacteria are continually evolving an assortment of defenses that enable their survival against even the most potent treatments. While the demand for novel antibiotic agents is high, the discovery of a new agent is exc...
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| Format: | Article |
| Language: | English |
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MDPI AG
2024-02-01
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| Series: | Antibiotics |
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| Online Access: | https://www.mdpi.com/2079-6382/13/2/143 |
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| author | Jenna K. Frizzell Ryan L. Taylor Lisa M. Ryno |
| author_facet | Jenna K. Frizzell Ryan L. Taylor Lisa M. Ryno |
| author_sort | Jenna K. Frizzell |
| collection | DOAJ |
| description | Antibiotics are used to combat the ever-present threat of infectious diseases, but bacteria are continually evolving an assortment of defenses that enable their survival against even the most potent treatments. While the demand for novel antibiotic agents is high, the discovery of a new agent is exceedingly rare. We chose to focus on understanding how different signal transduction pathways in the gram-negative bacterium <i>Escherichia coli</i> (<i>E. coli</i>) influence the sensitivity of the organism to antibiotics from three different classes: tetracycline, chloramphenicol, and levofloxacin. Using the PHL628 strain of <i>E. coli</i>, we exogenously overexpressed two transcription factors, FliA and RpoH.I54N (a constitutively active mutant), to determine their influence on the minimum inhibitory concentration (MIC) and minimum duration of killing (MDK) concentration for each of the studied antibiotics. We hypothesized that activating these pathways, which upregulate genes that respond to specific stressors, could mitigate bacterial response to antibiotic treatment. We also compared the exogenous overexpression of the constitutively active RpoH mutant to thermal heat shock that has feedback loops maintained. While FliA overexpression had no impact on MIC or antibiotic tolerance, RpoH.I54N overexpression reduced the MIC for tetracycline and chloramphenicol but had no independent impact on antibiotic tolerance. Thermal heat shock alone also did not affect MIC or antibiotic tolerance. L-arabinose, the small molecule used to induce expression in our system, unexpectedly independently increased the MICs for tetracycline (>2-fold) and levofloxacin (3-fold). Additionally, the combination of thermal heat shock and arabinose provided a synergistic, 5-fold increase in MIC for chloramphenicol. Arabinose increased the tolerance, as assessed by MDK<sub>99</sub>, for chloramphenicol (2-fold) and levofloxacin (4-fold). These experiments highlight the potential of the RpoH pathway to modulate antibiotic sensitivity and the emerging implication of arabinose in enhanced MIC and antibiotic tolerance. |
| first_indexed | 2024-03-07T22:44:43Z |
| format | Article |
| id | doaj.art-8a55d817d6ae4642b1f8bcac5ac3107f |
| institution | Directory Open Access Journal |
| issn | 2079-6382 |
| language | English |
| last_indexed | 2024-03-07T22:44:43Z |
| publishDate | 2024-02-01 |
| publisher | MDPI AG |
| record_format | Article |
| series | Antibiotics |
| spelling | doaj.art-8a55d817d6ae4642b1f8bcac5ac3107f2024-02-23T15:05:03ZengMDPI AGAntibiotics2079-63822024-02-0113214310.3390/antibiotics13020143Constitutive Activation of RpoH and the Addition of L-arabinose Influence Antibiotic Sensitivity of PHL628 <i>E. coli</i>Jenna K. Frizzell0Ryan L. Taylor1Lisa M. Ryno2Department of Chemistry and Biochemistry, Oberlin College, Oberlin, OH 44074, USADepartment of Chemistry and Biochemistry, Oberlin College, Oberlin, OH 44074, USADepartment of Chemistry and Biochemistry, Oberlin College, Oberlin, OH 44074, USAAntibiotics are used to combat the ever-present threat of infectious diseases, but bacteria are continually evolving an assortment of defenses that enable their survival against even the most potent treatments. While the demand for novel antibiotic agents is high, the discovery of a new agent is exceedingly rare. We chose to focus on understanding how different signal transduction pathways in the gram-negative bacterium <i>Escherichia coli</i> (<i>E. coli</i>) influence the sensitivity of the organism to antibiotics from three different classes: tetracycline, chloramphenicol, and levofloxacin. Using the PHL628 strain of <i>E. coli</i>, we exogenously overexpressed two transcription factors, FliA and RpoH.I54N (a constitutively active mutant), to determine their influence on the minimum inhibitory concentration (MIC) and minimum duration of killing (MDK) concentration for each of the studied antibiotics. We hypothesized that activating these pathways, which upregulate genes that respond to specific stressors, could mitigate bacterial response to antibiotic treatment. We also compared the exogenous overexpression of the constitutively active RpoH mutant to thermal heat shock that has feedback loops maintained. While FliA overexpression had no impact on MIC or antibiotic tolerance, RpoH.I54N overexpression reduced the MIC for tetracycline and chloramphenicol but had no independent impact on antibiotic tolerance. Thermal heat shock alone also did not affect MIC or antibiotic tolerance. L-arabinose, the small molecule used to induce expression in our system, unexpectedly independently increased the MICs for tetracycline (>2-fold) and levofloxacin (3-fold). Additionally, the combination of thermal heat shock and arabinose provided a synergistic, 5-fold increase in MIC for chloramphenicol. Arabinose increased the tolerance, as assessed by MDK<sub>99</sub>, for chloramphenicol (2-fold) and levofloxacin (4-fold). These experiments highlight the potential of the RpoH pathway to modulate antibiotic sensitivity and the emerging implication of arabinose in enhanced MIC and antibiotic tolerance.https://www.mdpi.com/2079-6382/13/2/143antibiotic toleranceheat shock responseminimum inhibitory concentration (MIC)PHL638 <i>E. coli</i>L-arabinose |
| spellingShingle | Jenna K. Frizzell Ryan L. Taylor Lisa M. Ryno Constitutive Activation of RpoH and the Addition of L-arabinose Influence Antibiotic Sensitivity of PHL628 <i>E. coli</i> Antibiotics antibiotic tolerance heat shock response minimum inhibitory concentration (MIC) PHL638 <i>E. coli</i> L-arabinose |
| title | Constitutive Activation of RpoH and the Addition of L-arabinose Influence Antibiotic Sensitivity of PHL628 <i>E. coli</i> |
| title_full | Constitutive Activation of RpoH and the Addition of L-arabinose Influence Antibiotic Sensitivity of PHL628 <i>E. coli</i> |
| title_fullStr | Constitutive Activation of RpoH and the Addition of L-arabinose Influence Antibiotic Sensitivity of PHL628 <i>E. coli</i> |
| title_full_unstemmed | Constitutive Activation of RpoH and the Addition of L-arabinose Influence Antibiotic Sensitivity of PHL628 <i>E. coli</i> |
| title_short | Constitutive Activation of RpoH and the Addition of L-arabinose Influence Antibiotic Sensitivity of PHL628 <i>E. coli</i> |
| title_sort | constitutive activation of rpoh and the addition of l arabinose influence antibiotic sensitivity of phl628 i e coli i |
| topic | antibiotic tolerance heat shock response minimum inhibitory concentration (MIC) PHL638 <i>E. coli</i> L-arabinose |
| url | https://www.mdpi.com/2079-6382/13/2/143 |
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