The spread of antibiotic resistance among bacterial pathogens is inciting a major public health crisis. The rise in hospital-acquired methicillin- and vancomycin-resistant staphylococci has been directly linked to horizontal gene transfer (HGT), thus warranting the study of pathways that regulate this process. One such pathway, called CRISPR-cas, constitutes a bacterial immune system that protects bacteria from their viruses (phage) and prevents all modes of HGT. CRISPR-Cas immune systems are composed of clustered regularly interspaced short palindromic repeat (CRISPR) loci and CRISPR-associated (cas) genes. CRISPR loci encode small guide RNAs (crRNAs) that, together with Cas proteins, identify and destroy invading nucleic acids in a manner that seems not to discriminate between beneficial and detrimental genetic information. This pathway can therefore have contradicting effects: On the one hand, CRISPR-cas immunity confers a selective advantage for survival by combating phage, but on the other, this pathway also blocks the transfer of useful genetic information such as virulence factors and antibiotic resistance genes. This intriguing paradox supports the hypothesis that CRISPR-cas systems are subject to multiple layers of regulation that likely promote bacterial survival in a given environment. Little is known about how CRISPR-cas systems are regulated in staphylococci. Furthermore, the overall impact of CRISPR-cas immunity on staphylococcal virulence remains obscure. The goal of this study is to characterize the regulation of CRISPR-cas and its influence on the virulence of Staphylococcus epidermidis RP62a, a commensal opportunistic pathogen. S. epidermidis RP62a has been found to harbor a single CRISPR-cas system that targets both staphylococcal phage as well as conjugative plasmids that carry antibiotic resistance genes. Using molecular and genetic approaches, this research aims to 1) Identify the genetic elements required for CRISPR-cas expression in S. epidermidis, 2) Determine how CRISPR-cas expression is regulated under different environmental stimuli, and 3) Evaluate the impact of CRISPR-cas on S. epidermidis fitness and virulence in a simple animal model. CRISPR-cas systems are widespread in the prokaryotic world, residing in nearly all archaea and half of all sequenced bacteria, including many important human pathogens. Advancing our understanding of CRISPR-cas regulation and its resulting influence on bacterial virulence is expected to enable novel strategies to block the spread of antibiotic resistance among bacterial pathogens by manipulating their CRISPR-cas systems.
Staphylococci are among the most common causes of hospital-acquired infections, and the emergence of strains that are resistant to all known antibiotics have become a major threat to public health. CRISPR-cas is a newly discovered bacterial immune system that blocks the uptake of antibiotic resistance genes in staphylococci. The proposed research will explore the potential for CRISPR-cas immunity to be used in a novel strategy to prevent the spread of antibiotic resistance by evaluating CRISPR-cas function and its overall impact on staphylococcal virulence.
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