Cell-based HTS for Compounds that Block Virulence Factor Expression in MRSA

Harvard Medical School, Boston MA

Investigators

Abstract

DESCRIPTION (provided by applicant): We aim to discover new classes of antimicrobial compounds that are active against methicillin/multidrug resistant Staphylococcus aureus (MRSA). Currently, MRSA infections cause more deaths each year in the U.S. than HIV/AIDS, and existing therapies are failing. We have uncovered evidence that beta lactam resistance in MRSA, which is mediated in part by the expression of a gene (mecA) encoding a resistant transpeptidase (PBP2A), requires a functional wall teichoic acid (WTA) biosynthetic pathway. Based on this, we have hypothesized that small molecules that prevent the expression of WTAs by inhibiting one of the first two enzymes in the WTA pathway (TarO/TarA) will render MRSA susceptible to beta lactams. Consistent with this hypothesis, preliminary studies have shown that tunicamycin, a natural product that selectively inhibits TarO, indeed sensitizes MRSA to methicillin. Unfortunately, tunicamycin has eukaryotic toxicity and so new compounds are needed in order to evaluate the therapeutic utility of this potential combination therapy. Herein, we propose to develop and carry out a high throughput screen to identify small molecules that inhibit TarO or TarA. This will be done by screening for compounds that antagonize the lethal effects of a WTA-active antibiotic (targocil). This small molecule was discovered in our laboratory using a similar cell-based, pathway-specific high throughput screen in S. aureus. Compounds that rescue growth of S. aureus RN4220 in the presence of lethal concentrations of targocil (4x MIC) will be further examined to assess whether they inhibit WTA biosynthesis. Several techniques will be used to validate hits, including testing for the presence of WTAs through chemical extraction and/or phage infection. We also have established an in vitro assay for TarA inhibition and we have several genetically modified S. aureus strains that will allow us to test for TarO inhibition. Confirmed non-toxic WTA inhibitors would be candidates for evaluating the utility of combination therapy for treating MRSA infections, and could be used as chemical probes to learn more about the roles of WTAs in the physiology of Gram-positive organisms. PUBLIC HEALTH RELEVANCE: Invasive methicillin-resistant Staphylococcus aureus (MRSA) infections pose an ongoing threat to public health in the United States. They caused ~19,000 deaths in this country in 2005, and clinical resistance has already emerged to the two new classes of antibiotics that were introduced to treat them. The proposed research could lead to the discovery of new compounds and strategies to treat invasive MRSA infections.