Our long-term goal is to elucidate the structures and fundamental mechanisms that give rise to drug transfer across the cell membrane by the AbgT-family of transporters. The primary targets of the proposed work are the Alcanivorax borkumensis YdaH and Neisseria gonorrhoeae MtrF transporters. Approximately 13,000 putative transporters of the family have been identified. However, no structural information has yet been available and even functional data are minimal for this family of membrane proteins. It has been hypothesized that the AbgT-family transporters contribute to the bacterial folate synthesis pathway by importing the catabolite p-aminobenzoyl- glutamate for producing this essential vitamin. To understand the structure and function of the AbgT family of transporters, we have recently cloned, expressed, purified and crystallized the full-length A. borkumensis YdaH and N. gonorrhoeae MtrF membrane proteins. We have also determined the X-ray structures of these two transporters at 2.9 and 3.9 resolutions, respectively. The structures reveal that these two transporters assemble as dimers with architectures distinct from all other families of transporters. Both YdaH and MtrF are bowl- shaped dimers with a solvent-filled basin extending from the cytoplasm halfway across the membrane bilayer. The monomers of YdaH and MtrF contain nine transmembrane helices and two hairpins. These structures directly suggest a plausible pathway for substrate transport. A combination of the crystal structure, genetic analysis and substrate accumulation assay indicates that both YdaH and MtrF behave as exporters, capable of removing the folate metabolite p-aminobenzoic acid from bacterial cells. Further experimental data based on drug susceptibility and radioactive transport assay suggest that both YdaH and MtrF participate as antibiotic efflux pumps, importantly mediating bacterial resistance to sulfonamide antimetabolite drugs. Our hypothesis is that many of these AbgT-family transporters act as exporters, thereby conferring bacterial resistance to sulfonamides.
The specific aim i s to define the structures and transport mechanisms of the AbgT family of transporters. We will use the structural, biochemical and computational approaches to determine the molecular mechanisms of the YdaH and MtrF transporters. Neisseria gonorrhoeae is an obligate human pathogen and the infectious agent for the sexually-transmitted disease gonorrhea. Although gonorrhea is one of the oldest described diseases, it remains a significant global problem with more than 100 million cases reported annually worldwide, with antibiotic resistance increasing at an alarming rate. Sulfonamides were used in the late 1930s and early 1940s to treat gonorrhea, but the rapid emergence of strains resistant to this class of drug resulted in the removal of sulfonamides from lists of recommended therapies. Therefore, elucidating the structures and mechanisms of the AbgT-family transporters will provide important new targets for the rational design of novel antibiotics to combat these bacterial infections.

Public Health Relevance

Drug-resistant pathogens are on the rise. This proposal will determine the molecular mechanism for sulfonamide recognition and extrusion in transmembrane efflux transporters. Thus, the research will establish a body of knowledge about antimetabolite resistance in pathogenic bacteria, which will provide a platform for the design of novel antimetabolite-based antimicrobial therapeutic strategies.

Agency
National Institute of Health (NIH)
Institute
National Institute of Allergy and Infectious Diseases (NIAID)
Type
Research Project (R01)
Project #
1R01AI114629-01A1
Application #
8961200
Study Section
Biochemistry and Biophysics of Membranes Study Section (BBM)
Program Officer
Hiltke, Thomas J
Project Start
2015-09-04
Project End
2018-08-31
Budget Start
2015-09-04
Budget End
2016-08-31
Support Year
1
Fiscal Year
2015
Total Cost
$364,361
Indirect Cost
$114,361
Name
Iowa State University
Department
Physics
Type
Schools of Arts and Sciences
DUNS #
005309844
City
Ames
State
IA
Country
United States
Zip Code
50011
Delmar, Jared A; Yu, Edward W (2018) Crystallographic Analysis of the CusBA Heavy-Metal Efflux Complex of Escherichia coli. Methods Mol Biol 1700:59-70
Kumar, Nitin; Su, Chih-Chia; Chou, Tsung-Han et al. (2017) Crystal structures of the Burkholderia multivorans hopanoid transporter HpnN. Proc Natl Acad Sci U S A 114:6557-6562
Zheng, Fudan; Du, Xiangwei; Chou, Tsung-Han et al. (2017) (S)-5-ethynyl-anabasine, a novel compound, is a more potent agonist than other nicotine alkaloids on the nematode Asu-ACR-16 receptor. Int J Parasitol Drugs Drug Resist 7:12-22
Su, Chih-Chia; Yin, Linxiang; Kumar, Nitin et al. (2017) Structures and transport dynamics of a Campylobacter jejuni multidrug efflux pump. Nat Commun 8:171
Yao, Hong; Shen, Zhangqi; Wang, Yang et al. (2016) Emergence of a Potent Multidrug Efflux Pump Variant That Enhances Campylobacter Resistance to Multiple Antibiotics. MBio 7:
Kumar, Nitin; Radhakrishnan, Abhijith; Su, Chih-Chia et al. (2016) Crystal structure of a conserved domain in the intermembrane space region of the plastid division protein ARC6. Protein Sci 25:523-9
Delmar, Jared A; Yu, Edward W (2016) The AbgT family: A novel class of antimetabolite transporters. Protein Sci 25:322-37
Zheng, Fudan; Robertson, Alan P; Abongwa, Melanie et al. (2016) The Ascaris suum nicotinic receptor, ACR-16, as a drug target: Four novel negative allosteric modulators from virtual screening. Int J Parasitol Drugs Drug Resist 6:60-73