Breaking antimicrobial resistance by disrupting extracytoplasmic protein folding

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.

The biogenesis of β-lactamase enzymes

“…A recent study has demonstrated that disulfide bond formation is essential for many clinically important β-lactamase enzymes that contain two or more cysteine residues and are translocated into the cell envelope through the Sec system [7]. The activity of several ESBL and carbapenemase enzymes from Class A (KPC, GES, FRI, SME families), B3 (L1 family) and D (OXA family) was tested in E.…”

“…freundii and P. aeruginosa expressing these enzymes, either by using a DSB system inhibitor or by deleting dsbA [7]. Generally, the greatest effects were observed for enzymes with broad hydrolytic activities, whilst narrow-spectrum enzymes, such as SHV, were less dependent on their disulfide bonds [7].…”

“…These resistance determinants are found in both Gram-positive (secreted into the extracellular space or embedded into the membrane) and Gram-negative (translocated into the periplasmic space) species and, since they primarily serve as bacterial defence against molecules produced by other microorganisms, they predate the first clinical use of β-lactam antibiotics [4–6]. β-Lactamases are often encoded on plasmids and transposable elements and over six thousand enzymes [7] have now spread through the bacterial phylogeny. In addition to their broad dissemination, their capacity for functional diversification and inhibitor escape constantly complicates the development of agents designed to mitigate their activity [8].…”

“…Class A is the most diverse subgroup of the Ambler classification system [7] and contains some of the most clinically concerning β-lactamases, including TEM and SHV enzymes ( Escherichia coli and Klebsiella pneumoniae , respectively), along with GES ( Pseudomonas spp., Enterobacteriaceae [22–25]), KPC ( K. pneumoniae [26]), and CTX-M enzymes (Enterobacteriaceae [27]) ().…”

“…The most prevalent representatives of this class are the OXA family of mobile β-lactamases found in many Gram-negative pathogens; the inhibitor-resistant carbapenemase OXA-48, initially isolated from K. pneumoniae , is the most clinically challenging enzyme of this group () [7, 56, 57]. With over 750 different enzymes, sub-divided into over 50 phylogenetic sub-families (), OXAs exhibit a diverse range of hydrolytic profiles often originating from single amino acid variations.…”