Studies on the roles of Lon protease and its substrates during antibiotic resistance in Escherichia coli: modulatory effects of nitroaromatics and nitrofurantoin

Abstract

Bacteria adapt to environmental stresses, including antibiotic stress, by regulating various genes and metabolic pathways. In prokaryotes, the energy-dependent protein degradation is controlled primarily by two ATP-dependent proteases, Lon and Clp. The Lon protease plays a major role in regulating several stress responses by modulating the amounts of substrates, including transcription factors. One such transcription factor is MarA, which is part of the Multiple Antibiotic Resistance operon. Lon and MarA key players in the antimicrobial resistance (AMR) landscape. AMR is one of the major global health concerns according to the World Health Organization (WHO). Also, AMR in Escherichia coli (E.coli) strains contributed to ~829,000 deaths in 2019. The major focus of this thesis study is on the interplay between Lon protease and antibiotic resistance in E.coli linked to nitroaromatics and nitrofurantoin. In the first part of the thesis, we explored the functions of E. coli encoded Lon protease in modulating responses to a toxic nitroaromatic, 2,4-dinitrophenol (2,4-DNP). In the second part, we investigated the roles of Lon protease as well as its substrates in sensitivity to nitrofurantoin, an antibiotic commonly used to treat uncomplicated urinary tract infections (UTI). During the first part of the study, an observation was made that the absence of Lon protease in E. coli resulted in an enhanced conversion of yellow coloured 2,4-DNP to a reddish-brown product. This study aimed to characterise the compound observed in the media with wild-type (WT) and Δlon strains of E. coli, understand the mechanisms of 2,4 DNP conversion and decipher the roles of Lon protease in the conversion of 2,4-DNP. Ultraviolet (UV)-visible and liquid chromatography mass-spectrometry (LC-MS) analyses revealed differences in the conversion products between the WT and Δlon strains. Growth studies with different mutants and trans-complemented strains demonstrated MarA dependent conversion. The bathochromic shift of spectral peaks suggested a reduction process and possible involvement of nitroreductase enzymes. Indeed, the expression of two genes encoding nitroreductases, nfsA and nfsB, increased with 2,4-DNP and was dependent on MarA. Importantly, the production of the reddish-brown product was lower in E. coli strains lacking nfsA or nfsB. Finally, LC-MS analysis identified one of the conversion products of 2,4-DNP to be 4-Amino-2-nitrophenol (4,2-ANP), a dye commonly used for hair colour. Dose studies with purified 4,2-ANP demonstrated that it did not lower the growth of E. coli (unlike 2,4-DNP) but induced phenotypic antibiotic resistance in an acrB dependent (like 2,4-DNP) but marA-independent (unlike 2,4-DNP) manner. This study revealed how bacteria in the environment convert a toxic compound (2,4-DNP) into a less toxic compound (4,2-ANP), also helping the bacteria survive in the presence of antibiotics. This study demonstrates how common pollutants may act as a selective pressure, favouring the survival as well as proliferation of bacteria containing antibiotic resistant genes. In the second part of the study, we explored the molecular mechanisms underlying nitrofurantoin resistance. During an antibiotic screening study, we observed that deletion of lon (Δlon) conferred enhanced susceptibility to nitrofurantoin. This was an interesting observation as the Δlon strain is more resistant to other antibiotic studies (e.g., ciprofloxacin, tetracycline and ampicillin). Investigations into the mechanisms revealed that the lon deletion enhanced the level of MarA and NfsA, subsequently leading to reduced growth. The Δlon strains displayed an elevated amount of Reactive Oxygen species (ROS), membrane thinning and nitrofurantoin-mediated filamentation. The ROS levels and membrane thinning were reversed upon quenching using glutathione, further confirming the role of oxidative stress in mediating the sensitivity to nitrofurantoin. Building on these mechanistic insights, we tested salicylates to synergistically enhance the efficacy of nitrofurantoin by inducing marA. Both sodium salicylate and acetyl salicylate enhanced the potential of nitrofurantoin and reduced the effective dose of nitrofurantoin to lower the growth of the WT strain. Importantly, this synergistic effect extended to nitrofurantoin resistant E. coli clinical isolates, where the combination lowered the effective nitrofurantoin concentration required for growth reduction. Environmental pollutants and antibiotic resistance are two major global burdens. This study highlights the roles of a major ATP-dependent protease, Lon, in the degradation of a toxic environmental pollutant to a less toxic version, as well as the regulation of nitrofurantoin resistance. The first part of the study contributes to our understanding of the biological treatment of nitroaromatics and offers insights into possible environmental pollution mitigation strategies. On the other hand, the second part of the study unravels the uncharacterised roles of Lon protease during nitrofurantoin susceptibility and illustrates the enhanced efficacy of nitrofurantoin–salicylate combinations as a promising therapeutic strategy to overcome emerging resistance in the treatment of pathogens causing UTI infections.