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Cytosolic protein degradation is crucial for cellular homeostasis as it orchestrates protein turnover by destruction of misfolded, unstable and abnormal proteins. This process has two main stages: (i) an ATP-dependent stage mediated by unfoldases and proteases, and (ii) an ATP-independent stage mediated by various peptidases. The ATP dependent proteases recognise target proteins and cleave them into smaller peptides. These enzymes comprise the ATPase-family-associated-with-various-cellular-activities domain that is important for unfolding target proteins. Subsequently, unfolded proteins enter a barrel-shaped proteolytic chamber, an architecture conserved throughout prokaryotes, archea and eukaryotes, where the peptide bond is hydrolysed in an ATP-independent manner. The smaller peptides released are broken down by ATP-independent peptidases into free amino acids recycled into the cellular pool. In prokaryotes, major cellular protein degradation functions are performed by Clp and Lon proteases. Earlier studies in our laboratory have shown the role of an ATP-independent peptidase, AminopeptidaseN, in sodium salicylate (NaSal) induced growth inhibition. NaSal belongs to the family of Non-Steroidal Anti-Inflammatory Drugs and its acetyl ester, Aspirin, is a very widely used analgesic. It is produced by plants as a defence response and is known to cause different effects, including xenobiotic stress i. e. stress mediated by compounds which are not naturally produced or expected to be present in the organism in bacteria. In bacteria, salicylate modulates outer membrane proteins, virulence factors, and reduces motility. In addition, NaSal is able induce “phenotypic antibiotic resistance” by binding to MarR and de-repressing the mar operon. NaSal promotes the dissociation of MarR from the promoter site leading to transcription of MarA, a transcription factor that induces several genes that encode the AcrAB-TolC pump which effluxes multiple antibiotics from the cell. The present study investigates the possible roles of ATP-dependent proteases, Lon and Clp, during growth reduction of E. coli induced with high (2-3 mM) amounts of NaSal. The growth of the Lon mutant (lon), but not clpP, was found to be greatly reduced with high doses of NaSal in the media. Our hypothesis was that the lack of Lon led to the accumulation of high amounts of substrate proteins, which led to its greater sensitivity with high doses of NaSal. To identify the substrate protein/s and to better understand the mechanism of action, single and double mutants (with lon) of E. coli lacking several prominent Lon substrates, i.e. MarA, RcsA, StpA, SulA and UmuD, were generated and screened for rescue of growth with 2-3 mM NaSal. MarA, a transcription factor, was identified to be important during NaSal-induced growth reduction. It modulates outer membrane proteins and induces the AcrAB-TolC pump that increases the efflux of antibiotics from the cell. Also, RT-PCR analysis revealed that the levels of marA and its targets, acrA and acrB, were higher in the lon strain suggesting that the MarA protein levels were stabilised the cell in the absence of Lon. Further studies using approriate strains demonstrated that one of the effectors of MarA, i.e. the AcrAB-TolC efflux pump, was not involved in the NaSal-induced growth inhibition of lon. Therefore, in presence of higher doses of NaSal, MarA is upregulated due to de-repression of the operon. The levels of MarA are regulated by Lon via degradation but in the absence of Lon, MarA levels are stabilised and lead to upregulation of MarA and its target genes like AcrAB-TolC. This study identifies higher amounts of MarA to be responsible for NaSal-induced growth inhibition of lon. Subsequently, experiments were conducted to demonstrate the role of MarA and its targets in antibiotic resistance with low dose (0.5 mM) NaSal that does not affect growth. This low dose of NaSal was able to upregulate marA and its target genes, acrA, acrB and tolC. Quantification of antibiotic resistance further revealed an induction in resistance by 0.5 mM NaSal in a MarA- and AcrB-dependent fashion. Studies using atomic force microscopy demonstrated that ciprofloxacin-induced cell elongation was lower in lon due to higher levels of MarA. Therefore, low dose of NaSal is capable of upregulating MarA and inducing antibiotic resistance but does not affect cell growth. This part of the study addresses the roles of Lon protease, its substrate MarA and MarA-induced targets, e.g. AcrB, during NaSal-mediated growth reduction and antibiotic resistance. The factors contributing to antibiotic resistance in bacteria are an important area of study for the global public health care system. Antibiotic resistance can be acquired by transmittance of genetic material, accumulation of antibiotic resistant mutations in the target molecule or can be induced by certain compounds present in the environment like NaSal. For rapid identification of compounds that may behave in a similar fashion as NaSal, a 96-well plate based screen was developed utilising the growth inhibition feature of the lon strain. The compounds were selected on the basis of their structural (phenolic compounds) and functional (Non-Steroidal Anti Inflammatory Drugs or NSAIDs) similarity to NaSal. Through this screen, four compounds were identified that lowered the growth of lon more than that of wild type strain and may be important in inducing phenotypic antibiotic resistance: Acetaminophen (anti-pyretic), Ibuprofen (NSAID), and two phenolic uncouplers, Carbonyl cyanide m-chlorophenyl hydrazone (CCCP) and 2,4-dinitrophenol (2,4-DNP). Notably, another compound Phenylbutazone (NSAID), which is used to lower inflammation in animals, did not reduce the growth of E. coli. RNA expression analysis revealed that these four compounds, but not phenylbutazone, induced the expression of marA and its target gene involved in antibiotic efflux, acrB. Furthermore, dose dependent and comparative studies with Nasal demonstrated differential effects of these four compounds in inducing antibiotic resistance with respect to ciprofloxacin, tetracycline and nalidixic acid. The two uncouplers were much more effective in inducing antibiotic resistance at lower doses than the NSAIDs. As NSAIDs are clinically important compounds, the study suggests that it would be desirable to screen them for induction of antibiotic resistance. The approach elucidated in this study has the potential to identify additional compounds present in the environment that may contribute to antibiotic resistance in bacteria. Overall, this study delineates the roles of Lon protease and its substrate, MarA, during NaSal-mediated responses, involving antibiotic resistance and/or growth reduction in E. coli. In addition, four other compounds were identified that could induce phenotypic antibiotic resistance in E. coli in a MarA-dependent manner. These observations may have implications in the adaptation of bacteria under different environmental conditions.

The Lon protease is a mitochondrial matrix quality-control protease belonging to the family of AAA+ proteins (ATPases associated with many cellular activities). We had previously found Lon to be upregulated in lung tumours with a non-angiogenic phenotype in a microarray study comparing these to conventional angiogenic tumours. In this project I set out to investigate whether Lon had any role in modulating the hypoxic response of tumour cells. Using a novel monoclonal antibody against Lon, I found that upregulation of Lon was present in breast and lung tumours and that higher levels of Lon are correlated with shorter overall survival in breast cancer patients. Targeting Lon with siRNA and shRNA in tumour cell lines reduced the normoxic and hypoxic stabilisation of HIF-α subunits. This is mediated through a mechanism independent of the activity of HIF-prolyl hydroxylases and independent of any changes in mitochondrial transcription. I found that the pre-imported form of Lon could bind and chaperone VHL in the cytoplasm potentially modulating VHL activity. In cell lines and human tumours, I observed that the proline-hydroxylated form of HIF-1α is induced by hypoxia and the hydroxylated form of HIF-1α is associated with shorter overall survival in breast cancer patients. This observation supports the notion that higher levels of Lon is associated with poor survival by downregulating VHL leading to higher levels of hydroxylated HIF. Finally I show that targeting Lon in cell lines is able to inhibit growth in a cell-line dependent fashion and partially reverses the Warburg effect, increasing oxygen consumption and reducing lactate production. In conclusion, I have demonstrated the broad therapeutic potential of targeting the Lon protease in tumours and highlighted a mechanism of post-hydroxylation HIF-regulation that has not been previously recognised in VHL competent tumours.