Gain-of-Function Mutations in Acid Stress Response (evgS) Protect Escherichia coli from Killing by Gallium Nitrate, an Antimicrobial Candidate

Abstract

Widespread antimicrobial resistance encourages repurposing/refining of non-antimicrobial drugs for antimicrobial indications. Gallium nitrate (GaNt), an FDA-approved medication for cancer-related hypercalcemia, recently showed good activity against several clinically significant bacteria. However, the mechanism of GaNt antibacterial action is still poorly understood. In the present work, resistant and tolerant mutants of Escherichia coli were sought via multiple rounds of killing by GaNt. Multi-round-enrichment yielded no resistant mutant; whole-genome sequencing of one representative GaNt-tolerant mutant uncovered mutations in three genes (evgS, arpA, kdpD) potentially linked to protection from GaNt-mediated killing. Subsequent genetic analysis ruled out a role for arpA and kdpD, but two gain-of-function mutations in evgS conferred tolerance. The evgS mutation-mediated GaNt tolerance depended on EvgS to EvgA phosphor-transfer; EvgA-mediated up-regulation of GadE. YdeO, and SarfA also contributed to tolerance, the latter two likely through their regulation of GadE. GaNt-mediated killing of wild-type cells correlated with increased intracellular ROS accumulation that was abolished by the evgS-tolerant mutation. Moreover, GaNt-mediated killing was mitigated by dimethyl sulfoxide, and the evgS-tolerant mutation upregulated genes encoding enzymes involved in ROS detoxification and in the glyoxylate shunt of the TCA cycle. Collectively, these findings indicate that GaNt kills bacteria through elevation of ROS; gain-of-function mutations in evgS confer tolerance by constitutively activating the EvgA-YdeO/GadE cascade of acid-resistance pathways and by preventing GaNt-stimulated ROS accumulation by upregulating ROS detoxification and shifting TCA cycle carbon flux. The striking lethal activity of GaNt suggests that clinical use of the agent may not quickly lead to resistance.

It’s Not Easy Being Green: A Narrative Review on the Microbiology, Virulence and Therapeutic Prospects of Multidrug-Resistant Pseudomonas aeruginosa

“…Additionally, it is questionable how long the new agents can manage the worsening resistance situation [146,157,[249][250][251][252][253][254]. With the daunting increase in antimicrobial resistance rates in all types of bacteria, one of the main aims of antimicrobial research is the exploration for new approaches past conventional antibiotics, such as bacteriophages, antimicrobial peptides with diverse structures and mechanisms of action, virulence inhibitors, siderophores, compounds from natural origins (like essential oils), and other adjuvants (e.g., efflux pump inhibitors, monoclonal antibodies) (Table 3) [118,[255][256][257][258][259][260][261][262][263][264][265][266][267][268][269][270]. It is possible that in the next couple of decades, these agents will play a major role in the management of serious bacterial infections caused by P. aeruginosa and other pathogens of critical importance.…”

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

“…Gallium nitrate is an FDA-approved drug widely used for the treatment of carcinogenic hypercalcemia and is effective against several clinically significant MDR bacteria. Advanced genomic techniques confirmed the gain-of-function mutation in the EvgSA two-component system in these tolerant strains [ 91 ]. Deletion mutants of evgS and evgA failed to confer GaNt tolerance.…”

“…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.…”

“…The increased expression of central regulators results in the activation of several acid-resistance genes at different loci. This activation involves more than 20 proteins (CRP, Dps, EvgA/S, GadE, GadX, GadW, H-NS, Lon, PhoP/Q, RNaseE, sigma factor 70, sigma factor RpoS, SspA, TrmE, TopA, TorS/R and YdeO) and several non-coding RNAs (DsrA, GadY, and GcvB) ( Figure 2 ) [ 91 , 99 , 100 , 101 , 102 , 103 , 104 , 105 , 106 , 107 , 108 ].…”

“…Low pH induces the expression of emrAB and emrKY multidrug-resistance efflux genes, which confer a growth advantage and facilitate multidrug resistance against extended-spectrum β-lactamases (ESBLs) ( Table 3 ) [ 150 , 151 , 152 , 153 ]. In addition to the development of resistance to ESBLs, the evgA-ydeO-gadE regulatory cascade also facilitates antimicrobial tolerance to other drugs, including gallium nitrate (GaNt) [ 91 ]. Gallium nitrate is an FDA-approved drug widely used for the treatment of carcinogenic hypercalcemia and is effective against several clinically significant MDR bacteria.…”