Highly Contingent Phenotypes of Lon Protease Deficiency in Escherichia coli upon Antibiotic Challenge

Abstract

Evolutionary trajectories and mutational landscapes of drug-resistant bacteria are influenced by cell-intrinsic and extrinsic factors. In this study, I demonstrated that loss of the Lon protease altered susceptibility of Escherichia coli to trimethoprim and that these effects were strongly contingent on the drug concentration and genetic background. Lon, an AAA+ ATPase, is a bacterial master regulator protease involved in cytokinesis, suppression of transposition events, and clearance of misfolded proteins. I show that Lon deficiency enhances intrinsic drug tolerance at sub-MIC levels of trimethoprim. As a result, loss of Lon, though disadvantageous under drug-free conditions, has a selective advantage at low concentrations of trimethoprim. At high drug concentrations, however, Lon deficiency is detrimental for E. coli. I show that the former is explained by suppression of drug efflux by Lon, while the latter can be attributed to SulA-dependent hyperfilamentation. On the other hand, deletion of lon in a trimethoprim-resistant mutant E. coli strain (harboring the Trp30Gly dihydrofolate reductase [DHFR] allele) directly potentiates resistance by enhancing the in vivo stability of mutant DHFR. Using extensive mutational analysis at 3 hot spots of resistance, I show that many resistance-conferring mutations render DHFR prone to proteolysis. This trade-off between gaining resistance and losing in vivo stability limits the number of mutations in DHFR that can confer trimethoprim resistance. Loss of Lon expands the mutational capacity for acquisition of trimethoprim resistance. This paper identifies the multipronged action of Lon in trimethoprim resistance in E. coli and provides mechanistic insight into how genetic backgrounds and drug concentrations may alter the potential for antimicrobial resistance evolution. IMPORTANCE Understanding the evolutionary dynamics of antimicrobial resistance is vital to curb its emergence and spread. Being fundamentally similar to natural selection, the fitness of resistant mutants is a key parameter to consider in the evolutionary dynamics of antimicrobial resistance (AMR). Various intrinsic and extrinsic factors modulate the fitness of resistant bacteria. This study demonstrated that Lon, a bacterial master regulator protease, influences drug tolerance and resistance. Lon is a key regulator of several fundamental processes in bacteria, including cytokinesis. I demonstrated that Lon deficiency produces highly contingent phenotypes in E. coli challenged with trimethoprim and can expand the mutational repertoire available to E. coli to evolve resistance. This multipronged influence of Lon on drug resistance provides an illustrative instance of how master regulators shape the response of bacteria to antibiotics.

Adaptation and Compensation in a Bacterial Gene Regulatory Network Evolving Under Antibiotic Selection

“…Drug concentrations are generally equated with strength of selection for antibiotic resistance [48, 49]. However, selection strength is determined by intrinsic factors as well such as the genetic background [40, 50]. Therefore, we tested whether the link between selection strength and evolutionary trajectory would hold true in another genetic background.…”

“…Therefore, we tested whether the link between selection strength and evolutionary trajectory would hold true in another genetic background. We have shown earlier that Lon protease-deficient E. coli are trimethoprim-tolerant due to a folA -independent mechanism [50]. For E. coli Δlon it is expected, then, that 300 ng/mL of trimethoprim would represent a milder selection strength than wild type.…”

An Intertwined Network of Regulation Controls Membrane Permeability Including Drug Influx and Efflux in Enterobacteriaceae

“…Moreover, we can note that the redundancy and accumulation of these "controllers" of membrane permeability and transporters is a key aspect allowing the bacterial cell to (i) detect/respond to the presence of noxious compounds in the medium and (ii) select an appropriate level of membrane permeation. This large redundancy of controls confers rapid fine-tuned responses, versatility, flexibility, and contributes to a limited cost for efficient adaptation by multiplying the available regulation target for a key function of bacterial metabolism the membrane transport [ 83 , 88 , 97 , 126 , 128 , 129 , 132 ].…”

“…Detection of environmental change is the first adaptation/defense line of bacteria face to detrimental hazards to its basic physiological activities. Gram-negative bacteria have developed several systems that are able to sense the external medium modifications such as pH, osmotic strength, oxidative stress, nutrient starvation, toxic chemicals, and transfer the corresponding signal via transduction to appropriate internal regulators [ 83 , 84 , 85 , 86 , 87 , 88 , 89 , 90 , 91 , 92 , 93 ]. These regulators can directly or indirectly (regulation cascades) manage the expression of various components to adapt the bacterial membrane structure and composition allowing the bacteria to ensure their growth and survival [ 94 , 95 ].…”

“…It must be emphasized how the regulatory network is pleiotropic by (i) the large repertoire of signals triggering regulators, (ii) the substantial number of regulatory partners [ 89 , 118 , 119 ] acting at different levels (post-transcriptional, transcriptional, translational), in which most of them (iii) have multiple targets, and (iv) induce variable responses [ 83 , 110 ]; and (v) the overlap of the pathways leading to cross regulation (CpxAR and EnvZ/OmpR, CpxAR and σE, CpxAR and BaeSR, CpxAR and mar operon, σ E and PhoPQ, Rcs and PhoPQ, Rob and Mar etc.). The complexity of the regulatory network of a bacterium is much greater than what we know.…”