Acidification of Cytoplasm in Escherichia coli Provides a Strategy to Cope with Stress and Facilitates Development of Antibiotic Resistance

Abstract

Awareness of the problem of antimicrobial resistance (AMR) has escalated, and drug-resistant infections are named among the most urgent issues facing clinicians today. Bacteria can acquire resistance to antibiotics by a variety of mechanisms that, at times, involve changes in their metabolic status, thus altering diverse biochemical reactions, many of them pH-dependent. In this work, we found that modulation of the cytoplasmic pH (pH i) of Escherichia coli provides a thus far unexplored strategy to support resistance. We show here that the acidification of the cytoplasmic pH is a previously unrecognized consequence of the activation of the marRAB operon. The acidification itself contributes to the full implementation of the resistance phenotype. We measured the pH i of two resistant strains, developed in our laboratory, that carry mutations in marR that activate the marRAB operon. The pH i of both strains is lower than that of the wild type strain. Inactivation of the marRAB response in both strains weakens resistance, and pH i increases back to wild type levels. Likewise, we showed that exposure of wild type cells to weak acids that caused acidification of the cytoplasm induced a resistant phenotype, independent of the marRAB response. We speculate that the decrease of the cytoplasmic pH brought about by activation of the marRAB response provides a signaling mechanism that modifies metabolic pathways and serves to cope with stress and to lower metabolic costs. Antimicrobial resistance (AMR) is a growing public health threat of broad concern, and drug-resistant infections are named among the most urgent problems facing clinicians today 1-4. Bacteria can acquire resistance to antibiotics by horizontal gene transfer, activation of regulatory loci and genetic mutations that modify the drug target, increase drug efflux, and activate the expression of drug inactivating enzymes 5-8. High-level, clinically relevant resistant strains usually result from the accumulation of several mutations that, at times, involve changes in their metabolic status, thus altering diverse biochemical reactions 9-11. Metabolic rearrangements and changes in rates of reactions necessitate the existence of mechanisms that maintain tight homeostasis of all the physicochemical parameters in the cell within a narrow range of operation, consistent with the survival of the organism. This dynamic state of equilibrium includes many variables such as pH, and solutes and ion concentrations. Because of the accumulating evidence on the metabolic adaptation of antibiotic-resistant cells, we focused our attention on the cytoplasmic pH of these cells. Protons play a crucial role in biochemical networks because changes in intracellular pH affect cell functioning at different levels as it impacts protein folding, enzyme activities, and the protonation of biological macromolecules, lipids, and other metabolites. Moreover, the proton electrochemical gradient across the cell membrane is key to the generation and conversion of cellular energy. It is, therefore, n...

Insights into Emergence of Antibiotic Resistance in Acid-Adapted Enterohaemorrhagic Escherichia coli

“…This system comprises a complex network of two-component regulators with a wide array of interactions to cope with mild to extreme acid stress. The key interacting regulators of this network are GadE, EvgAS, PhoPQ, YdeO, GadW, RcsB, and GadX, which regulate gene expression spatially and temporally ( Figure 2 ) [ 44 , 67 , 87 , 88 , 89 , 90 , 91 , 92 , 93 , 94 , 95 , 96 , 97 ]. This system is activated either by mild acidic pH during the exponential growth phase or by entry into stationary phase.…”

“…When grown at acidic pH, genes involved in metabolism, energy production and class I heat shock proteins are down-regulated to lower metabolic cost [ 128 ]. These strains consume intracellular protons through amino acid decarboxylation during acid stress, which highly acidifies the cytoplasm, resulting in increased acid tolerance [ 44 , 76 ]. During oxidative respiration, electron transport of membrane-bound systems, including the atp operon, is down-regulated to inhibit the import of protons [ 129 ].…”

“…In response to extracellular acid stress, pathogens undergo several cellular and global transcriptional changes that alter their responsiveness to a wide array of antibiotics. These regulatory changes help the organism adapt to extreme environmental stresses and subsequently enable cross-protection consistent with the survival of the organism [ 44 , 60 ].…”

“…While passing through the gastrointestinal tract, EHECs experience anaerobic conditions and nitrosative stress that trigger enhanced expression of the multidrug efflux genes ( mdtEF ) and several two-component signaling systems including EvgAS, PhoPQ, RcsB, PmrAB, ArcAB, BaeSR, KdpA, and CpxAR [ 44 , 130 , 131 , 132 ]. These two-component systems combat cell envelope disruption caused by proton imbalance and antibiotic-induced accumulation of mistranslated peptides that can cause cell damage by disturbing homeostasis [ 133 ].…”

“…EHECs are well known for their adaptation to the acidic environment; their tolerance level is comparable to that of acidophiles. Furthermore, foodborne, acid-adapted strains also confer cross-protection to antibiotics, which plays a vital role in the spread of multidrug-resistant pathotypes [ 16 , 44 , 45 , 46 , 47 , 48 ]. The human gastrointestinal tract is reported to provide the best environment for the emergence, transmission, and spread of antibiotic-resistance genes in bacterial populations.…”