S. aureus bacteria are commonly resistant to methicillin and other cell-wall active antibiotics. The goal of this application is to validate the MazF toxin and specific RNA binding proteins of S. aureus that interfere with specific mRNA degradation as novel targets for antibacterial drug discovery. Toxin antitoxin systems (TA) are common in bacteria. Among the different TA modules that are protein-based, the MazEF system is by far the best characterized. In prior papers that we have published, we have shown that that the MazFsa toxin of S. aureus is a specific endoribonuclease that cuts at the VUUV'site where V or V'can be A, C or G, leading to inhibition of protein synthesis and the ensuing growth arrest. The MazFsa-mediated growth arrest is unique because these cells are viable but cannot replicate. To delineate this unique form of growth arrest, we have shown that MazFsa cleaves most of the cellular mRNAs but spares some housekeeping (e.g. recA, gyrB) and a global regulator (sarA) mRNAs, presumably allowing the bacterium to enter metabolic quiescence without sacrificing viability. We hypothesize that specific RNA-binding protein(s) may protect some of these "important" mRNAs" under MazFsa-mediated stress. A corollary of our hypothesis is that inactivation of these "specific RNA binding proteins", coupled with MazFsa activation, will promote cell death rather than growth arrest since these "essential" mRNA will no longer be protected. Cell fractionation analysis, coupled with Northwestern blots with a labeled sarA mRNA probe, have enabled us to identify CshA, CshB and SA1641 as putative "RNA-binding proteins". CshA and CshB are potential DEAD-box RNA helicases while SA1641 has homology with RNA methyltransferase. Previously, DEAD-box proteins have been known to participate in ribosome biogenesis and mRNA decay. Thus, protection of mRNAs from MazFsa-mediated cleavage by CshA and CshB will be a novel concept for DEAD-box proteins. To validate the above hypotheses, we have developed the following specific aims: I) defining the ability of CshA and CshB to bind and protect selective mRNAs from MazFsa-mediated degradation in vitro;II) assessing the degradation of selective MRNA in single and double cshA and cshB mutants, the viability of these mutants upon MazFsa induction and their sensitivity to selective antibiotics;III) characterization of other factors (e.g. SA1641) that help protect selective mRNA from MazFsa-mediated degradation;IV) evaluating the survival of single and double cshA and cshB mutants with and without MazFsa induction in murine models of infection. The results of these studies will allow us to validate the novel function of DEAD-box and other unique RNA-binding proteins in S. aureus. These studies will also provide us with the validation that activation of MazFsa, coupled with inactivation of CshA, CshB, SA1641 and/or other factors, is a novel approach to kill MSSA and MRSA. Accordingly, MazFsa and "specific RNA binding proteins" represent novel targets for antibiotic development. As TA systems, DEAD-box and other RNA binding proteins are common in prokaryotes, our results may apply to other pathogens.

Public Health Relevance

This project is to study the role of specific RNA-binding proteins in the protection of mRNA when the toxin system called MazF is activated in Staphylococcus aureus. Although the MazF toxin of S. aureus cleaves many cellular mRNAs, it spares mRNAs that are linked to housekeeping activity as well as that of a global regulator called sarA. We have identified some of these RNA-binding proteins that protect selective mRNAs from MazF cleavage in S. aureus. Our goal is to validate these RNA binding proteins as targets for antibiotic development, especially when the MazF toxin is activated under environmental or antibiotic stresses in S. aureus.

Agency
National Institute of Health (NIH)
Institute
National Institute of Allergy and Infectious Diseases (NIAID)
Type
Research Project (R01)
Project #
1R01AI106937-01
Application #
8557227
Study Section
Bacterial Pathogenesis Study Section (BACP)
Program Officer
Huntley, Clayton C
Project Start
2013-05-24
Project End
2017-04-30
Budget Start
2013-05-24
Budget End
2014-04-30
Support Year
1
Fiscal Year
2013
Total Cost
$380,504
Indirect Cost
$145,504
Name
Dartmouth College
Department
Microbiology/Immun/Virology
Type
Schools of Medicine
DUNS #
041027822
City
Hanover
State
NH
Country
United States
Zip Code
03755