Author: Wang, Miaomiao
Title: Mechanisms underlying the active bacterial tolerance response and development of therapies against clinical bacterial persisters
Advisors: Chen, Sheng (ABCT)
Degree: Ph.D.
Year: 2020
Subject: Bacterial genetics
Hong Kong Polytechnic University -- Dissertations
Department: Department of Applied Biology and Chemical Technology
Pages: x, 137 pages : color illustrations
Language: English
Abstract: Bacteria tolerance are largely responsible for the recalcitrance of chronic infections with the underlying mechanisms remains incompletely revealed. Recent studies reveal that physiological dormancy alone is insufficient for maintaining a long-lasting phenotype and nutrient starvation is a known trigger of non-heritable phenotypic antibiotic tolerance in bacteria. In an attempt to investigate if active starvation-induced physiological responses underlie tolerance development, we applied RNA sequencing (RNA-Seq) to investigate whether bacteria actively adjusted gene expression patterns in response to starvation and showed that genes of the phage shock protein (psp) family were consistently over-expressed even under prolonged starvation conditions in an E.coli model. One product of this gene family, PspA, was found to play a pivotal role in maintenance of starvation-induced tolerance by preventing dissipation of the transmembrane proton motive force (PMF). Deletion of the pspA gene resulted in more rapid reduction in tolerance level during starvation process. We found that maintaining the transmembrane PMF is essential for formation of antibiotic tolerance in both Gram-negative and positive bacteria, including E.coli, K.pneumoniae, A.baumannii, P.aeruginosa and S.aureus. Rapid and complete eradication of the entire tolerant sub-population of the test strains could be achieved by complete disruption of PMF by the ionophore CCCP. Most importantly, we found that an FDA-approved antifungal drug, econazole, could also produce such effect in a non-toxic manner. We consider econazole-mediated PMF disruption a feasible strategy for prevention of occurrence of chronic and recurrent bacterial infections, especially among immunocompromised patients. Apart from the investigation of the mechanisms underlying bacterial tolerance response, we aimed to discover nutrient compounds which reverted tolerant cells to become re-susceptible to conventional antibiotics by phenotype microarrays (PM) and found that N-acetyl-D-glucosamine (GlcNAc) or D-glucosamine (GlcN) altered persisters' β-lactam susceptibility. We demonstrated that cell structures completely collapsed after synergistic treatment of β-lactam, GlcNAc and cytoplasmic ß-lactam amount was increased in the presence of GlcNAc. We found that the GlcNAc catabolism pathway was involved in the its resensitization effect as on one hand the amount of fructose-6-phosphate (Fru-6-P), GlcNAc catabolism product and the precursor of glycolysis, increased and another hand resensitization effect diminished after inhibition of NADH oxidation which is the major purpose of glycolysis. Additionally, we detected that GlcNAc triggered the produce of peptidoglycan precursor, UDP- N-acetyl-D-glucosamine (UDP-GlcNAc) and subsequently the reactivation of peptidoglycan biosynthesis which is the target of β-lactam. The mechanism underlying GlcNAc resensitization is complicated and may be the cooperation effect of GlcNAc catabolism and activated peptidoglycan biosynthesis. Our findings imply that GlcNAc or GlcN as adjuvants to β-lactam would be beneficial in the treatment of clinical chronic infections.
Rights: All rights reserved
Access: open access

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