Identifying inhibitors of trans‐translation in methicillin resistant Staphylococcus aureus

Open Access
Author:
Rae, Christopher Donald
Area of Honors:
Biochemistry and Molecular Biology
Degree:
Bachelor of Science
Document Type:
Thesis
Thesis Supervisors:
  • Kenneth Charles Keiler, Thesis Supervisor
  • Craig Eugene Cameron, Honors Advisor
  • Scott Brian Selleck, Faculty Reader
Keywords:
  • methicillin resistant Staphylococcus aureus
  • MRSA
  • antibiotic
  • trans-translation
Abstract:
The development of antibiotic resistance among infectious bacteria is a serious public health problem. Methicillin resistant Staphylococcus aureus (MRSA) infections in particular have affected society for generations, demanding the need for new treatment options focused on previously untargeted mechanisms. The trans-translation pathway presents a good target for novel antibiotic development because it is universally conserved among bacteria and is essential for survival. This pathway uses two key components, SmpB and transfer-messenger RNA (tmRNA), which release stalled ribosome complexes that would otherwise accumulate and result in cell death. Identification of small molecule inhibitors against this pathway in MRSA has revealed a set of promising novel antibiotics that could one day treat this deadly infection. Forty-six Kenneth Keiler Laboratory (KKL) compounds that inhibit trans-translation in E. coli were identified in a high-throughput screen. Broth microdilution assays showed that four of these compounds inhibit growth of S. aureus strains, including MRSA, at concentrations as low as 0.7 µM. This is comparable to antibiotics in current use, such as vancomycin or clindamycin. All four compounds are oxadiazoles, characterized by a central five-membered heterocyclic ring and flanking phenyl derivatives. An in vitro trans-translation assay was established using S. aureus tmRNA and SmpB, and was used to evaluate the activity of these compounds. Inhibition of trans-translation was observed at levels nearing 25%. In contrast, no inhibition of translation was observed, indicating that these compounds specifically target trans-translation. Bacterial cytological profiling (BCP) was adapted and optimized for use with suspected trans-translation inhibitors. BCP is a technique that takes advantage of the unique cell morphologies that arise when bacteria are treated with antibiotics. Cell size and shape are visualized using fluorescence microscopy and quantified using image analysis software. Data can then be statistically compared and used to understand the cellular pathways targeted by various antibiotics. This technique was used to eliminate possible drug targets of KKL compounds, further suggesting that trans-translation is the target of inhibition. These results indicate that trans-translation may serve as a target for the development of antibiotics to treat MRSA.