Bacterial infectious diseases are a major cause of mortality worldwide. The rise in antibiotic resistant infections, coupled with the sharp decline in the discovery of new and clinically useful classes of antibiotics, underscores an urgent need for alternative strategies to combat bacterial infections. Small noncoding RNA pathways have recently been recognized as important regulators of bacterial pathogenesis, and the challenge lies in gaining a detailed understanding of these processes. My research uses the tools of biochemistry and molecular genetics to unravel the mechanisms of small RNA-mediated pathways and enable the development of novel anti-microbial therapeutics.
CRISPR-Cas immunity in pathogenic staphylococci
Horizontal gene transfer (HGT), or the direct exchange of genetic material between two species, is a major driving force behind the evolution of bacterial pathogens. CRISPR-Cas is a bacterial immune system that blocks all modes of HGT; CRISPR-Cas systems use small guide RNAs to identify and destroy bacterial viruses (phages) and prevent the stable incorporation of mobile genetic elements. Residing in nearly all archaea and about half of all sequenced bacteria, these systems exhibit remarkable diversity.
CRISPR-Cas systems are composed of clustered regularly interspaced short palindromic repeats and flanking CRISPR-associated (cas) genes (Figure 1). The CRISPR-Cas immunity pathway involves three steps:
Adaptation, CRISPR RNA (crRNA) biogenesis, and Targeting.
1. During adaptation, viral or plasmid challenge stimulates the incorporation of short (24–48 nucleotide) invader-derived sequences in between equally short DNA repeats found in the CRISPR locus. These unique sequences, called spacers, provide a historical account of past invaders and specify future targets of CRISPR immunity.
2. During crRNA biogenesis, transcription of the CRISPR locus generates a long RNA precursor containing repeats and spacers in one contiguous array. This precursor is processed (ie. cleaved) within repeats to separate individual spacers and liberate mature crRNAs that each define a single nucleic acid target.
3. During targeting, crRNAs assemble with Cas proteins to form a surveillance complex, which destroys invading genetic elements antisense to the crRNA it carries.
Pathogenic staphylococci that are resistant to all known antibiotics have recently emerged in both hospital and community settings. Staphylococci disseminate antibiotic resistance primarily through the horizontal transfer of conjugative plasmids. A clinical isolate, Staphylococcus epidermidis RP62a, harbors a CRISPR-Cas system that targets all sequenced staphylococcal conjugative plasmids, thus providing a physiologically-relevant model in which to study CRISPR-Cas biology. Using this organism, our previous work has made three major contributions toward advancing our understanding of its CRISPR-Cas pathway: 1) the first report of a ruler mechanism that measures crRNAs during their processing steps (Hatoum-Aslan, Maniv, and Marraffini 2011), 2) the discovery of a large ribonucleoprotein complex (termed Cas10/Csm) in which crRNA processing takes place (Hatoum-Aslan et al. 2013), and 3) the establishment of genes and motifs required for CRISPR-Cas immunity that play key roles outside of crRNA processing (Hatoum-Aslan et al. 2014). Our current research focuses on unraveling the regulatory mechanisms of CRISPR-Cas pathways and their impact on staphylococcal virulence. This effort will enable the development of novel therapeutic strategies that use CRISPR-Cas immunity to prevent the spread of antibiotic resistance.
Hatoum-Aslan A and Marraffini, LA. (2014). “Impact of CRISPR immunity on the emergence and virulence of bacterial pathogens.” Curr Opin Microbiol, 17, 82-90.
Hatoum-Aslan A, Maniv I, Samai P, and Marraffini, LA. (2014). “Genetic characterization of anti-plasmid immunity by a Type III-A CRISPR-Cas System.” J Bacteriol, 196(2), 310-7.
Hatoum-Aslan A, Samai P, Maniv I, Jiang W, and Marraffini, LA. (2013). “A ruler protein in a complex for antiviral defense determines the length of small interfering CRISPR RNAs.” J Biol Chem, 288(39), 27888-97.
*Highlighted by: Bucci, M. (2013). “A Measure of RNA.”, Nat Chem Biol, 9(12), 754.
Maniv I, Hatoum-Aslan A, and Marraffini, LA. (2013). “CRISPR decoys: competitive inhibitors of CRISPR immunity.” RNA biology, 10(5), 694-9.
Hatoum-Aslan A, Palmer KL, Gilmore MS, and Marraffini LA. (2013). “Type III CRISPR-Cas systems and roles of CRISPR-Cas in Bacterial Virulence.” In R. Barrangou & J. van der Oost (Eds.), CRISPR-Cas Systems. Springer-Verlag Berlin Heidelberg.
Bikard D, Hatoum-Aslan A, Mucida D, and Marraffini LA (2012). “Prevention of horizontal gene transfer during bacterial infection by CRISPR interference”, Cell Host Microbe, 12(2), 177-86.
*Highlighted by: Weinberger AD, Gilmore AS. (2012). “CRISPR-Cas: To take up DNA or not: That is the question.” Cell Host Microbe, 12(2), 125-6.
Hatoum-Aslan A, Maniv I, and Marraffini, LA (2011). “Mature clustered, regularly interspaced, short palindromic repeats RNA (crRNA) length is measured by a ruler mechanism anchored at the precursor processing site”, Proc Natl Acad Sci U S A, 108(52), 21218-21222.
Hatoum A, Roberts JW (2008). “Prevalence of RNA polymerase stalling at E. coli promoters after open complex formation”, Mol Micro, 68(1), 17-28.
*Highlighted by: Artsimovitch I (2008). “Post-initiation control by the initiation factor sigma”, Mol Micro, 68(1), 1-3.
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