Bacteria have evolved complex systems to facilitate their survival under conditions of stress such as temperature fluctuations, oxidative challenges and nutrient deprivation. Stress often causes pervasive gene regulatory responses that minimize metabolic activity and result in a dormant state. Dormant bacteria are largely resistant to antimicrobial therapeutics, which complicates treatment of latent bacterial infections. Moreover, it appears that stochastic processes elicit the formation of small numbers of persister cells within growing populations of bacteria. These metabolically dormant cells are resistant to drugs that kill growing cells, which may result in the resurgence of infection after cessation of therapy. Bacterial toxin-antitoxin systems represent one adaptation that down regulates cellular metabolism in response to stress and contributes to the formation of dormant cells and persisters. Type II TA systems feature protein antitoxins that bind to and inactivate protein toxins inside cells. Cells subjected to certain types of stress, such as nutrient deprivation, produce proteases that cleave the antitoxin, thereby releasing the active toxin. Many of the known Type II toxins inhibit protein synthesis, which leads to a dormant physiological state and, if the stress is not relieved, cell death. Since cells continuously synthesize the toxin and antitoxin, and because relief of the stress down regulates expression of the protease, the antitoxin re-accumulates and binds to the toxin thereby reversing growth inhibition. Much of our knowledge of the cellular effects of activating TA systems come from studies where the toxins were produced in non-physiological quantities and under complex physiological conditions, such as amino acid starvation. These stresses also activate pervasive regulatory pathways such as the stringent response, which complicates analysis of the direct effects of TA activity. Moreover, relatively little is known about the details of TA interaction at the molecular level. These deficis could be alleviated by the development and use of specific small molecule inhibitors of TA interaction. Accordingly, we have developed a generalizable, whole cell, high throughput screen for the identification of small molecule inhibitors of TA interactions from any bacterium. The assay features Forster resonance energy transfer quenching (qFRET) as an output signal that measures the binding of antitoxin to toxin in live cells. The signal is stable, highly reproducible and exhibits a relatively high signal to background ratio. Moreover, the assay is applicable, in principle, to any bacterial TA pair.
The Specific Aims of the proposal are to;(1) refine, optimize and generalize the basic qFRET assay, (2) screen small molecule libraries for inhibitors of TA interaction using a TA pair from NTHi as a proof of principle, (3) employ secondary screens to validate the specificity of the inhibitors and (4) genetically map the TA binding surfaces required for inhibitor binding. The compounds identified with this assay will unique tools for the study of TA systems in bacteria.
Bacterial infections pose a major public health challenge, especially in cases where host induced stress causes bacterial dormancy. This application describes the development of a whole cell, high throughput screen for the identification of small molecule inhibitors of systems that contribute to dormancy in pathogenic bacteria. The compounds identified by this method should provide unique tools for the study of these systems in bacteria and may lead to the development of novel antibiotics for clinical use.
|Hamilton, Brooke; Manzella, Alexander; Schmidt, Karyn et al. (2014) Analysis of non-typeable Haemophilous influenzae VapC1 mutations reveals structural features required for toxicity and flexibility in the active site. PLoS One 9:e112921|