Degree

Doctor of Philosophy (PhD)

Department

Biological Sciences

Document Type

Dissertation

Abstract

Colistin (Polymyxin E) is a “last resort” antibiotic for treatment of infections caused by multidrug resistant Gram-negative bacterial pathogens. Resistance to colistin varies between bacterial species. Some Gram-negative bacteria such as Burkholderia spp. exhibit extremely high intrinsic colistin resistance. The molecular mechanisms behind this are not yet fully understood. The aim of this dissertation is to characterize and propose a role of a Burkholderia thailandensis membrane protein belonging to the DedA superfamily (DbcA; DedA of Burkholderia required for colistin resistance) in maintaining extreme resistance to colistin. Modification of lipopolysaccharide lipid A with the cationic sugar aminoarabinose (Ara4N), encoded by arn operons, is required for colistin resistance. Mass spectrometry of lipid A of ∆dbcA showed a large reduction of Ara4N in lipid A compared to wild type E264. Direct measurement of membrane potential shows that ∆dbcA is partially depolarized, suggesting proton motive force (PMF) dependent processes are compromised in ∆dbcA. There are likely two PMF dependent steps during lipid A modification with Ara4N- the transbilayer movement of undecaprenyl-P-Ara4N to the periplasmic surface of the inner membrane catalyzed by EmrE-like transporters ArnEF and the recycling of undecaprenyl-P back to the cytoplasm possibly by UppP/BacA, the undecaprenyl pyrophosphate phosphatase. Overexpression of arn operons by genomic insertion of inducible promoters partially complemented colistin hypersensitivity of ∆dbcA by increasing Ara4N levels in lipid A. However, overexpression of the UppP homolog of E264 partially complemented colistin sensitivity of ∆dbcA independent of lipid A modification. Artificially increasing the PMF by lowering the pH of the growth media also increased membrane potential, amounts of Ara4N, and colistin resistance of ∆dbcA. ∆dbcA also displayed alkaline pH/bicarbonate sensitivity, suggesting a role of DbcA in cytoplasmic pH homeostasis. While alkaline pH or bicarbonate decreased colistin resistance of E264 and a majority of other Gram-negative and Gram-positive bacterial species tested, hypoxia or glucose improved colistin resistance probably by preventing cytoplasmic alkalinization. We propose that cytoplasmic pH homeostasis is required for colistin resistance. Finally, we propose that DbcA is involved in maintaining optimal PMF, that is required for both lipid A modification dependent colistin resistance as well as lipid A modification independent colistin resistance pathways of B. thailandensis E264.

Date

3-23-2021

Committee Chair

Doerrler, William T.

Available for download on Friday, March 10, 2028

Included in

Bacteriology Commons

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