Mechanisms of chlorate toxicity and resistance in Pseudomonas aeruginosa

Abstract

Pseudomonas aeruginosa is an opportunistic bacterial pathogen that often encounters hypoxic/anoxic environments within the host, which increases its tolerance to many conventional antibiotics. Toward identifying novel treatments, we explored the therapeutic potential of chlorate, a pro‐drug that kills hypoxic/anoxic, antibiotic‐tolerant P. aeruginosa populations. While chlorate itself is relatively nontoxic, it is enzymatically reduced to the toxic oxidizing agent, chlorite, by hypoxically induced nitrate reductase. To better assess chlorate's therapeutic potential, we investigated mechanisms of chlorate toxicity and resistance in P. aeruginosa. We used transposon mutagenesis to identify genes that alter P. aeruginosa fitness during chlorate treatment, finding that methionine sulfoxide reductases (Msr), which repair oxidized methionine residues, support survival during chlorate stress. Chlorate treatment leads to proteome‐wide methionine oxidation, which is exacerbated in a ∆msrA∆msrB strain. In response to chlorate, P. aeruginosa upregulates proteins involved in a wide range of functions, including metabolism, DNA replication/repair, protein repair, transcription, and translation, and these newly synthesized proteins are particularly vulnerable to methionine oxidation. The addition of exogenous methionine partially rescues P. aeruginosa survival during chlorate treatment, suggesting that widespread methionine oxidation contributes to death. Finally, we found that mutations that decrease nitrate reductase activity are a common mechanism of chlorate resistance.

Full text

Molecular Microbiology. 2022;118:321–335. wileyonlinelibrary.com/journal/mmi
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321© 2022 John Wiley & Sons Ltd.
1 | INTRODUCTIO N
It is widely known that many conventional antibiotics are ineffective
at killing slow- or non- growing bacterial cells (Brauner et al., 2016).
One key physiological constraint that dictates the growth rate
of many pathogens is oxygen availability. Many pathogens grow
slower, and thus display increased antibiotic tolerance, under hy-
poxic/anoxic conditions (Gutierrez et al., 2017; Hamad et al., 2011;
Narten et al., 2012). Slow grow th and antibiotic tolerance are also
defining features of biofilms, where bacteria grow as dense multicel-
lular aggregates with oxygen- limited interior populations (Borriello
et al., 2004; Pabst et al., 2016).
The link between hypoxic/anoxic environments and antibiotic
failure has devastating consequences for treating infections. For
example, the mucus that coats the airways of cystic fibrosis (CF) pa-
tients is largely hypoxic/anoxic (Cowley et al., 2015) and suppor ts
biofilm growth of oppor tunistic pathogens, such as Pseudomonas
aeruginosa (Bjarnsholt et al., 2013; DePas et al., 2016). Chronic
Received: 25 April 2022
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Revised: 31 July 2022
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Accepted: 4 Aug ust 2022
DOI : 10.1111/m mi.1497 2
RESEARCH ARTICLE
Mechanisms of chlorate toxicity and resistance in Pseudomonas
aeruginosa
Melanie A. Spero1 | Jeff Jones2 | Brett Lomenick2 | Tsui- Fen Chou2 |
Dianne K. Newman 1,3
1Division of Biology and Biological
Enginee ring, Cal ifornia Ins titute of
Technolog y, Pasadena, C alifornia, USA
2Proteome Exploration Laboratory,
Beckman Institute, Division of B iology and
Biologi cal Engineering, California Ins titute
of Technolog y, Pasadena, California, USA
3Division of Geological and Planetary
Science s, California Instit ute of
Technolog y, Pasadena, C alifornia, USA
Correspondence
Melanie A. Spero and Dianne K. N ewman,
Division of Biology and Biological
Enginee ring, Cal ifornia Ins titute of
Technolog y, Pasadena, C A, USA .
Email: mspero@uoregon.edu and dkn@
caltech.edu
Present address
Melanie A. Spero, Institute of Molecular
Biolog y, Universit y of Oregon, Eugene,
Oregon, USA
Funding information
Beckman Institute, Caltech , Grant/Award
Number : N/A; Betty an d Gordon Moore
Foundation, Grant/Award Number:
GBMF775; Cystic Fibrosis Foundation,
Grant/Award Number: SPERO19F0;
Doren Family Foundation, Grant/Award
Number : N/A; National Institute of Allergy
and Infectious Diseases, Grant/Award
Number : 1R21AI146987- 02; National
Instit utes of Healt h, Grant/Award
Number : OD010788 and OD 020013
Abstract
Pseudomonas aeruginosa is an opportunistic bacterial pathogen that often encounters
hypoxic/anoxic environments within the host, which increases its tolerance to many
conventional antibiotics. Toward identifying novel treatments, we explored the thera-
peutic potential of chlorate, a pro- drug that kills hypoxic/anoxic, antibiotic- tolerant P.
aeruginosa populations. While chlorate itself is relatively nontoxic, it is enzymatically
reduced to the toxic oxidizing agent, chlorite, by hypoxically induced nitrate reduc-
tase. To better assess chlorate's therapeutic potential, we investigated mechanisms
of chlorate toxicity and resistance in P. aeruginosa. We used transposon mutagenesis
to identify genes that alter P. aeruginosa fitness during chlorate treatment, finding
that methionine sulfoxide reductases (Msr), which repair oxidized methionine resi-
dues, support survival during chlorate stress. Chlorate treatment leads to proteome-
wide methionine oxidation, which is exacerbated in a ∆msrA∆msrB strain. In response
to chlorate, P. aeruginosa upregulates proteins involved in a wide range of functions,
including metabolism, DNA replication/repair, protein repair, transcription, and trans-
lation, and these newly synthesized proteins are par ticularly vulnerable to methio-
nine oxidation. The addition of exogenous methionine partially rescues P. aeruginosa
survival during chlorate treatment, suggesting that widespread methionine oxidation
contributes to death. Finally, we found that mutations that decrease nitrate reductase
activity are a common mechanism of chlorate resistance.
KEY WORDS
antibiotic tolerance, chlorate, drug resistance, drug toxicity, Pseudomonas aeruginosa