Abstract
Antimicrobial resistance in Gram-negative bacteria is one of the greatest threats to global health. New antibacterial strategies are urgently needed, and the development of antibiotic adjuvants that either neutralize resistance proteins or compromise the integrity of the cell envelope is of ever-growing interest. Most available adjuvants are only effective against specific resistance proteins. Here, we demonstrate that disruption of cell envelope protein homeostasis simultaneously compromises several classes of resistance determinants. In particular, we find that impairing DsbA-mediated disulfide bond formation incapacitates diverse β-lactamases and destabilizes mobile colistin resistance enzymes. Furthermore, we show that chemical inhibition of DsbA sensitizes multidrug-resistant clinical isolates to existing antibiotics and that the absence of DsbA, in combination with antibiotic treatment, substantially increases the survival of Galleria mellonella larvae infected with multidrug-resistant Pseudomonas aeruginosa. This work lays the foundation for the development of novel antibiotic adjuvants that function as broad-acting resistance breakers.
Highlights
55 Antimicrobial resistance (AMR) is one of the most important public health concerns of our time [1]
120 RESULTS 121 122 The activity of multiple cell envelope resistance proteins is dependent on DsbA 123 124 DsbA has been shown to assist the folding of numerous periplasmic and surface-exposed 125 proteins in Gram-negative bacteria (Figure 1A) [25,26,27]
In addition to β-lactamases, we selected five representative phosphoethanolamine transferases from throughout the mobile colistin resistance (MCR) phylogeny (Figure 1 - figure supplement 1) to gain a comprehensive overview of the contribution of DsbA to the activity of these colistin-resistance determinants. 140 We expressed our panel of 17 discrete resistance enzymes in an Escherichia coli K-12 strain (E. coli MC1000) and its isogenic dsbA mutant (E. coli MC1000 dsbA) and recorded minimum inhibitory concentration (MIC) values for β-lactam or polymyxin antibiotics, as appropriate
Summary
55 Antimicrobial resistance (AMR) is one of the most important public health concerns of our time [1]. Since several cell envelope AMR determinants contain multiple cysteines [18, 28] we hypothesized that interfering with the function of DsbA would compromise bacterial virulence [27], but might offer a broad approach to break resistance across different mechanisms by affecting the stability of resistance proteins We test this hypothesis by investigating the contribution of disulfide bond formation to three of the most important resistance mechanisms in the cell envelope of Enterobacteria: the breakdown of β-lactam antibiotics by β-lactamases, polymyxin resistance arising from the production of MCR enzymes and intrinsic resistance to multiple antibiotic classes due to RND efflux pumps. Our findings prove that targeting protein homeostasis in the cell envelope allows the impairment of diverse AMR proteins and could be a promising avenue for the development of next-generation therapeutic approaches
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