Gain-of-function mutations in acid stress response (evgs) protect escherichia coli from killing by gallium nitrate, an antimicrobial candidate

Jie Zeng; Liwen Wu; Zhou Liu; Yihua Lv; Jinzhi Feng; Weijie Wang; Yunxin Xue; Dai Wang; Jiabin Li; Karl Drlica; Xilin Zhao

doi:10.1128/AAC.01595-20

Gain-of-function mutations in acid stress response (evgs) protect escherichia coli from killing by gallium nitrate, an antimicrobial candidate

Jie Zeng, Liwen Wu, Zhou Liu, Yihua Lv, Jinzhi Feng, Weijie Wang, Yunxin Xue, Dai Wang, Jiabin Li, Karl Drlica, Xilin Zhao

New Jersey Medical School (NJMS), Public Health Research Institute Center

Rutgers, The State University

Research output: Contribution to journal › Article › peer-review

3 Scopus citations

Abstract

Widespread antimicrobial resistance encourages repurposing/refining of nonantimicrobial drugs for antimicrobial indications. Gallium nitrate (GaNt), an FDAapproved medication for cancer-related hypercalcemia, recently showed good activity against several clinically significant bacteria. However, the mechanism of GaNt antibacterial action is still poorly understood. In the present work, resistant and tolerant mutants of Escherichia coli were sought via multiple rounds of killing by GaNt. Multiround-enrichment yielded no resistant mutant; whole-genome sequencing of one representative GaNt-tolerant mutant uncovered mutations in three genes (evgS, arpA, and kdpD) potentially linked to protection from GaNt-mediated killing. Subsequent genetic analysis ruled out a role for arpA and kdpD, but two gain-of-function mutations in evgS conferred tolerance. The evgS mutation-mediated GaNt tolerance depended on EvgS-to-EvgA phosphotransfer; EvgA-mediated upregulation of GadE. YdeO, and SarfA also contributed to tolerance, the latter two likely through their regulation of GadE. GaNt-mediated killing of wild-type cells correlated with increased intracellular reactive oxygen species (ROS) accumulation that was abolished by the evgS-tolerant mutation. Moreover, GaNt-mediated killing was mitigated by dimethyl sulfoxide, and the evgS-tolerant mutation upregulated genes encoding enzymes involved in ROS detoxification and in the glyoxylate shunt of the tricarboxylic acid (TCA) cycle. Collectively, these findings indicate that GaNt kills bacteria through elevation of ROS; gain-of-function mutations in evgS confer tolerance by constitutively activating the EvgA-YdeO/GadE cascade of acid resistance pathways and by preventing GaNt-stimulated ROS accumulation by upregulating ROS detoxification and shifting TCA cycle carbon flux. The striking lethal activity of GaNt suggests that clinical use of the agent may not quickly lead to resistance.

Original language	American English
Article number	e01595-20
Journal	Antimicrobial agents and chemotherapy
Volume	65
Issue number	3
DOIs	https://doi.org/10.1128/AAC.01595-20
State	Published - Mar 2021

ASJC Scopus subject areas

Pharmacology
Pharmacology (medical)
Infectious Diseases

Keywords

EvgS-EvgA acid stress response
Gallium nitrate
Glyoxylate shunt
Mechanisms of action
ROS detoxification
Reactive oxygen species
Tolerance

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@article{ee894dcd92fb49f38a72b4ac489dfbdd,

title = "Gain-of-function mutations in acid stress response (evgs) protect escherichia coli from killing by gallium nitrate, an antimicrobial candidate",

abstract = "Widespread antimicrobial resistance encourages repurposing/refining of nonantimicrobial drugs for antimicrobial indications. Gallium nitrate (GaNt), an FDAapproved medication for cancer-related hypercalcemia, recently showed good activity against several clinically significant bacteria. However, the mechanism of GaNt antibacterial action is still poorly understood. In the present work, resistant and tolerant mutants of Escherichia coli were sought via multiple rounds of killing by GaNt. Multiround-enrichment yielded no resistant mutant; whole-genome sequencing of one representative GaNt-tolerant mutant uncovered mutations in three genes (evgS, arpA, and kdpD) potentially linked to protection from GaNt-mediated killing. Subsequent genetic analysis ruled out a role for arpA and kdpD, but two gain-of-function mutations in evgS conferred tolerance. The evgS mutation-mediated GaNt tolerance depended on EvgS-to-EvgA phosphotransfer; EvgA-mediated upregulation of GadE. YdeO, and SarfA also contributed to tolerance, the latter two likely through their regulation of GadE. GaNt-mediated killing of wild-type cells correlated with increased intracellular reactive oxygen species (ROS) accumulation that was abolished by the evgS-tolerant mutation. Moreover, GaNt-mediated killing was mitigated by dimethyl sulfoxide, and the evgS-tolerant mutation upregulated genes encoding enzymes involved in ROS detoxification and in the glyoxylate shunt of the tricarboxylic acid (TCA) cycle. Collectively, these findings indicate that GaNt kills bacteria through elevation of ROS; gain-of-function mutations in evgS confer tolerance by constitutively activating the EvgA-YdeO/GadE cascade of acid resistance pathways and by preventing GaNt-stimulated ROS accumulation by upregulating ROS detoxification and shifting TCA cycle carbon flux. The striking lethal activity of GaNt suggests that clinical use of the agent may not quickly lead to resistance.",

keywords = "EvgS-EvgA acid stress response, Gallium nitrate, Glyoxylate shunt, Mechanisms of action, ROS detoxification, Reactive oxygen species, Tolerance",

author = "Jie Zeng and Liwen Wu and Zhou Liu and Yihua Lv and Jinzhi Feng and Weijie Wang and Yunxin Xue and Dai Wang and Jiabin Li and Karl Drlica and Xilin Zhao",

note = "Publisher Copyright: {\textcopyright} 2021 American Society for Microbiology.",

year = "2021",

month = mar,

doi = "10.1128/AAC.01595-20",

language = "American English",

volume = "65",

journal = "Antimicrobial agents and chemotherapy",

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T1 - Gain-of-function mutations in acid stress response (evgs) protect escherichia coli from killing by gallium nitrate, an antimicrobial candidate

AU - Zeng, Jie

AU - Wu, Liwen

AU - Liu, Zhou

AU - Lv, Yihua

AU - Feng, Jinzhi

AU - Wang, Weijie

AU - Xue, Yunxin

AU - Wang, Dai

AU - Li, Jiabin

AU - Drlica, Karl

AU - Zhao, Xilin

PY - 2021/3

Y1 - 2021/3

N2 - Widespread antimicrobial resistance encourages repurposing/refining of nonantimicrobial drugs for antimicrobial indications. Gallium nitrate (GaNt), an FDAapproved medication for cancer-related hypercalcemia, recently showed good activity against several clinically significant bacteria. However, the mechanism of GaNt antibacterial action is still poorly understood. In the present work, resistant and tolerant mutants of Escherichia coli were sought via multiple rounds of killing by GaNt. Multiround-enrichment yielded no resistant mutant; whole-genome sequencing of one representative GaNt-tolerant mutant uncovered mutations in three genes (evgS, arpA, and kdpD) potentially linked to protection from GaNt-mediated killing. Subsequent genetic analysis ruled out a role for arpA and kdpD, but two gain-of-function mutations in evgS conferred tolerance. The evgS mutation-mediated GaNt tolerance depended on EvgS-to-EvgA phosphotransfer; EvgA-mediated upregulation of GadE. YdeO, and SarfA also contributed to tolerance, the latter two likely through their regulation of GadE. GaNt-mediated killing of wild-type cells correlated with increased intracellular reactive oxygen species (ROS) accumulation that was abolished by the evgS-tolerant mutation. Moreover, GaNt-mediated killing was mitigated by dimethyl sulfoxide, and the evgS-tolerant mutation upregulated genes encoding enzymes involved in ROS detoxification and in the glyoxylate shunt of the tricarboxylic acid (TCA) cycle. Collectively, these findings indicate that GaNt kills bacteria through elevation of ROS; gain-of-function mutations in evgS confer tolerance by constitutively activating the EvgA-YdeO/GadE cascade of acid resistance pathways and by preventing GaNt-stimulated ROS accumulation by upregulating ROS detoxification and shifting TCA cycle carbon flux. The striking lethal activity of GaNt suggests that clinical use of the agent may not quickly lead to resistance.

AB - Widespread antimicrobial resistance encourages repurposing/refining of nonantimicrobial drugs for antimicrobial indications. Gallium nitrate (GaNt), an FDAapproved medication for cancer-related hypercalcemia, recently showed good activity against several clinically significant bacteria. However, the mechanism of GaNt antibacterial action is still poorly understood. In the present work, resistant and tolerant mutants of Escherichia coli were sought via multiple rounds of killing by GaNt. Multiround-enrichment yielded no resistant mutant; whole-genome sequencing of one representative GaNt-tolerant mutant uncovered mutations in three genes (evgS, arpA, and kdpD) potentially linked to protection from GaNt-mediated killing. Subsequent genetic analysis ruled out a role for arpA and kdpD, but two gain-of-function mutations in evgS conferred tolerance. The evgS mutation-mediated GaNt tolerance depended on EvgS-to-EvgA phosphotransfer; EvgA-mediated upregulation of GadE. YdeO, and SarfA also contributed to tolerance, the latter two likely through their regulation of GadE. GaNt-mediated killing of wild-type cells correlated with increased intracellular reactive oxygen species (ROS) accumulation that was abolished by the evgS-tolerant mutation. Moreover, GaNt-mediated killing was mitigated by dimethyl sulfoxide, and the evgS-tolerant mutation upregulated genes encoding enzymes involved in ROS detoxification and in the glyoxylate shunt of the tricarboxylic acid (TCA) cycle. Collectively, these findings indicate that GaNt kills bacteria through elevation of ROS; gain-of-function mutations in evgS confer tolerance by constitutively activating the EvgA-YdeO/GadE cascade of acid resistance pathways and by preventing GaNt-stimulated ROS accumulation by upregulating ROS detoxification and shifting TCA cycle carbon flux. The striking lethal activity of GaNt suggests that clinical use of the agent may not quickly lead to resistance.

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KW - Gallium nitrate

KW - Glyoxylate shunt

KW - Mechanisms of action

KW - ROS detoxification

KW - Reactive oxygen species

KW - Tolerance

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ER -

Gain-of-function mutations in acid stress response (evgs) protect escherichia coli from killing by gallium nitrate, an antimicrobial candidate

Abstract

ASJC Scopus subject areas

Keywords

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