The urinary tract is among the most common sites of bacterial infection and Escherichia coli is by far the most common species infecting this site. Infections are routinely treated with antibiotics including trimethoprim- sulfamethoxazole or quinolones. While vaccine development is in the pipeline, there are no vaccines currently licensed for use in the U.S. In the face of steadily climbing rates of antibiotic resistance and dramatic reduction in development of new antibiotics, isolation of novel small molecule antimicrobials are desperately needed. Lack of such new treatments will result in urinary tract infections (UTI) with multiply antibiotic-resistant strains that are untreatable. Using a series of unbiased screens including transcriptomic, proteomic, immunoproteomic, and qPCR assays, we have identified iron acquisition systems (siderophore- and heme- mediated) as vulnerable targets for inhibition and potential therapy. Our long-term research goal is to prevent and therapeutically treat UTIs in women and men. The objective is to identify and characterize small molecule inhibitors of iron uptake in gram-negative bacterial pathogens. In a pilot high throughput culture-based screen of 6298 compounds, we had an initial hit rate of 2.3% (147 compounds) [overall Z'factor=0.81 (avg Z'per plate=0.9), S/B=6, CV=6%] with a cutoff set at 3 SD below the negative control and inhibitory activity higher than 30%. Five hits were validated by counterscreens, dose response curves, and inhibition with fresh chemical samples. For one compound, data are consistent with inihibition of the TonB system as its target. Our central hypothesis is that specific small molecule inhibitors that do not depend on iron chelation can specifically block iron acquisition in bacteria. This discovery will drive biological investigationof the diverse iron acquisition mechanisms and complement our vaccination efforts using iron receptor protein targets. We will achieve our objective by completing the following specific aims: 1) Develop and implement a high throughput screen to identify small molecule inhibitors of iron acquisition by extraintestinal pathogenic Escherichia coli (ExPEC);2) Conduct counter screens, validate hits using fresh chemical samples, and analyze structure-activity relationships (SAR) for small molecule inhibitors of ExPEC iron acquisition;and 3) Determine common targets used by iron acquisition pathways that are inhibited by each validated small molecule inhibitor. The expected outcomes of these aims will be to identify compounds and develop derivatives that arrest bacterial growth by preventing bacterial iron assimilation. The positive impact of these studies will be substantial. We will identify novel small molecule inhibitors of bacterial iron uptake from among 150,000 chemical compounds and 27,278 natural product extracts, validate these hits with fresh samples, and improve upon these products by assessment of derivatives. This knowledge will provide us with alternate therapeutic agents beyond existing antibiotics and potential vaccines to prevent this public health scourge in women with recurrent UTI and those susceptible to their first UTI.

Public Health Relevance

The urinary tract is among the most common sites of bacterial infection and E. coli is by far the most common species infecting this site. The proposed research is relevant to public health because the discovery of novel small molecule inhibitors of bacterial iron uptake will allow the development of new antimicrobial agents in the face of rising antibiotic resistance in bacterial strains and declining production of new antibiotics. Identifying such novel inhibitory compounds will ultimately allow us to treat and prevent this public health problem in the face of rising antibiotic resistance, extending healthy life and reducing the burdens of illness and disability.

Agency
National Institute of Health (NIH)
Institute
National Institute of Diabetes and Digestive and Kidney Diseases (NIDDK)
Type
Research Project (R01)
Project #
1R01DK097362-01A1
Application #
8577840
Study Section
Special Emphasis Panel (ZRG1-BST-U (55))
Program Officer
Mullins, Christopher V
Project Start
2013-07-15
Project End
2016-06-30
Budget Start
2013-07-15
Budget End
2014-06-30
Support Year
1
Fiscal Year
2013
Total Cost
$383,235
Indirect Cost
$134,515
Name
University of Michigan Ann Arbor
Department
Microbiology/Immun/Virology
Type
Schools of Medicine
DUNS #
073133571
City
Ann Arbor
State
MI
Country
United States
Zip Code
48109