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

“…In the same study, similar results were obtained in clinical isolates of E. coli, K. pneumoniae, Enterobacter cloacae, C. 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]. This is in agreement with early observations by Schultz et al where removal of the disulfide bond from the narrow-spectrum enzyme TEM-1 did not affect its activity under physiological conditions, but was, nonetheless, detrimental when temperature or pH stresses were applied [241].…”

“…These experiments, mostly performed in the 90s or the early 2000s, form a big part of the knowledge base around the biogenesis of β-lactamases presented in this review. More recent studies, specifically focusing on the translocation or folding of these resistance determinants, have shown that for some β-lactamases, their hydrolytic spectrum depends on their export route [178] or that oxidative folding processes are essential for their activity [7]. These works highlight how an in-depth understanding of the biogenesis process of β-lactamases might prove important as the basis for the development of entirely novel next-generation strategies aiming to abrogate the function of these enzymes, thus potentiating existing invaluable antibiotics.…”

“…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][23][24][25]), KPC (K. pneumoniae [26]), and CTX-M enzymes (Enterobacteriaceae [27]) (Table 1). Several class A β-lactamases are also found in Gram-positive bacteria, for example BCL-1 (Bacillus spp.…”

“…1a), OXAs exhibit a diverse range of hydrolytic profiles often originating from single amino acid variations. The shape of their active site and consequently the hydrolytic spectrum of these enzymes, is also affected by a highly defined Ω-loop harbouring a conserved disulfide bond [7,55,58,59]. Their hydrolytic mechanism largely resembles that of other serine β-lactamases (Fig.…”

“…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][5][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].…”