Luciano A. Marraffini
Prevention of Horizontal Gene Transfer and Antibiotic Resistance in Pathogens
In modern times there has been a relentless battle against bacterial pathogens that constantly evolve to evade our efforts to eradicate them. Pathogens not only have become more virulent but also have acquired resistance to virtually all known antibiotics. The rise of antimicrobial-resistant strains in the hospital setting has become a major health care crisis. To limit the spread of such antibiotic-resistant pathogens, a greater understanding of their means of emergence and survival is required. The major route for the acquisition of antimicrobial resistance is the exchange of genetic material between related or unrelated bacterial species, known as horizontal gene transfer. Our research focuses on the study of mechanisms that prevent horizontal gene transfer among bacterial pathogens.
Recently, clustered regularly interspaced short palindromic repeat (CRISPR) loci have been shown to confer sequence-based immunity against bacteriophage infections and plasmid conjugation in prokaryotes. By interfering with phage infection and plasmid transfer, CRISPR systems block two major routes of horizontal gene transfer (i.e. transduction and conjugation) and thus play a major role in the evolution of prokaryotes and in the acquisition of phage-encoded virulence factors and the transfer of antimicrobial resistance plasmids in bacterial pathogens. Therefore it is anticipated that the study of CRISPR systems will greatly contribute both to the understanding of bacterial evolution and virulence, as well as to the development of new genetic technologies to control the traffic of genetic material.
CRISPR repeats are separated by short "spacers" that match sequences present in phages and plasmids and that specify the targets of CRISPR interference. CRISPR loci are transcribed and processed into small CRISPR RNAs (crRNAs) that retain a full spacer sequence flanked by part of the repeat sequence. By making base pair contacts with the target sequence, crRNAs act as guides for the CRISPR interference machinery. CRISPR function has been proposed to be conceptually analogous to RNAi, but the underlying mechanism remains largely unknown. In our lab we use Staphylococcus epidermidis, a pathogen that constitutes a leading cause of nosocomial infections, as a model system to conduct genetic and biochemical analyses aimed at dissecting the molecular basis of CRISPR function. Our goals are (1) to find the genes required for crRNA biogenesis as well as those responsible for interference against invading nucleic acids, (2) to identify crRNA ribonucleoprotein complexes and characterize their biochemical properties and (3) to manipulate this natural pathway to limit the spread of virulence determinants and antimicrobial resistance genes among bacterial pathogens.