Stressed Out: Resistance-nodulation-division (RND) and Major Facilitator Superfamily (MFS) Efflux Pump Mediated Survival Strategies of Acinetobacter baumannii

Olvera, Marcela

Stressed Out: Resistance-nodulation-division (RND) and Major Facilitator Superfamily (MFS) Efflux Pump Mediated Survival Strategies of Acinetobacter baumannii

dc.contributor.advisor	Zgurskaya, Helen
dc.contributor.author	Olvera, Marcela
dc.contributor.committeeMember	Rybenkov, Valentin
dc.contributor.committeeMember	Karr, Elizabeth
dc.contributor.committeeMember	Bourne, Christina
dc.date.accessioned	2024-05-10T21:33:15Z
dc.date.available	2024-05-10T21:33:15Z
dc.date.issued	2024-05-11
dc.date.manuscript	2024-05-03
dc.description.abstract	Originally discovered in soil microbial communities, reports of genus Acinetobacter associated infections started to increase around 1977 and continued to climb to frightening heights for the next 20 years resulting in Acinetobacter becoming a high priority target for antibiotic resistance research, leading to the research detailed here. A. baumannii’s exceptional abilities to evade antimicrobials have long been known, but the details of those mechanisms have for the most part remained unclear. The better we can understand the pathogen’s physiology, the more informed our efforts toward alternative treatments can be. This dissertation is focused on the role RND and MFS efflux pumps play in A. baumannii’s survival strategies under various stress conditions. The first goal was to functionally characterize two putative EmrAB-like MFS pumps, identified as being overproduced in RND efflux deficient strains of A. baumannii, and to gain insight into their role in bacterial stress responses. The second goal was to better understand the substrate recognition of two RND pumps in A. baumannii, AdeG of AdeFGH and AdeJ of AdeIJK, and how specific binding pocket residues impact substrate specificity and export efficiency. I used biochemical methods such as cloning, mutagenesis, antibiotic susceptibility assays, inhibition studies, and fluorescence uptake assays to accomplish these goals. I coupled these biochemical results with genetic and structural analyses to compose a more refined picture of how multidrug efflux protects A. baumannii against extracellular stressors. I found that while the two newly characterized MFS family efflux pumps, AmfAB and AmfCD, do not contribute to A. baumannii’s antibiotic non-susceptibility phenotypes, they are critical for A. baumannii’s survival under acidic conditions. These results also suggest that these two MFS transporters contribute to stress survival by modification of A. baumannii’s permeability barrier as a method of keeping stressors out of the cell. I also found several AdeG substrate binding pocket residues that are implicated in the export of nalidixic acid, norfloxacin, and the fluorescent probe NPN. Additionally, I used AdeG to characterize kinetic parameters of the putative efflux pump inhibitor, SLUPP_1377. And lastly, we found that the two AdeJ mutants (R701A and F136A) improved the export efficiency of erythromycin, perhaps by better accommodating the antibiotic into the transporter binding pocket. This work provides new insight into the roles multidrug efflux pumps play in bacterial physiology, potential new drug targets which are vital for survival under acidic stress, and informed EPI development strategies from a more detailed model of the AdeG and AdeJ binding pockets.	en_US
dc.identifier.uri	https://hdl.handle.net/11244/340337
dc.language	en_US	en_US
dc.subject	Biology, Molecular.	en_US
dc.subject	Chemistry, Biochemistry.	en_US
dc.subject	Biology, Microbiology.	en_US
dc.thesis.degree	Ph.D.	en_US
dc.title	Stressed Out: Resistance-nodulation-division (RND) and Major Facilitator Superfamily (MFS) Efflux Pump Mediated Survival Strategies of Acinetobacter baumannii	en_US
ou.group	Dodge Family College of Arts and Sciences::Department of Chemistry and Biochemistry	en_US
shareok.orcid	0009-0003-1808-4572	en_US