Arguably one of the major new findings in microbial genetics in recent years is the discovery of the CRISPRCas system. CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats) loci are novel sequences found in Archaea and Bacteria that are comprised of palindromic repeats separated by unique spacer sequences. Upstream of the CRISPR array are the CRISPR-associated sequences (cas) genes. CRISPRCas loci have been primarily implicated in immunity to phage and plasmid. Overall, CRISPRCas loci have been studied in only a relatively small number of model organisms. However, genomic sequencing indicates that CRISPRCas loci are found in >400 different bacteria, including in a variety of pathogenic organisms and intracellular parasites, and the potential role of CRISPRCas in pathogenicity and intracellular infection has not been directly examined. Legionella pneumophila (Lpn) is the bacterial agent of a severe form of pneumonia called Legionnaires'disease. Humans come in contact with the bacterium by inhaling contaminated water droplets. In its aquatic habitats, Lpn is an intracellular parasite of amoebae, and infection of these protozoa is unquestionably an important step in the transmission and pathogenesis of disease. In the lung, Lpn replicates in macrophages in a process that mimics infection of amoebae. Virulent Lpn strains contain a type of CRISPRCas locus that has not been previously investigated. Given this as well as the pathogenic and intracellular nature of Lpn, we embarked on an examination of the role of CRISPRCas in Lpn biology and virulence. Data presented here show that the CRISPRCas locus of Lpn strain 130b is expressed under extracellular and intracellular growth conditions. Furthermore, loss of cas2 results in a dramatic (approx. 1000-fold) defect in Lpn infection of the amoeba Acanthamoeba castellanii. These observations lead to the unique hypothesis that the Cas protein(s) of Lpn play a heretofore unrecognized role in infection that is outside of a role in immunity to phage and plasmid. Thus, Aim 1 will utilize a full panel of CRISPRCas mutants and complemented mutants in infection assays to determine the importance of the entire CRISPRCas locus in intracellular infection. Given the now established importance of Cas2 in Lpn infection, Aim 2 will use a biochemical approach to elucidate the function of Cas2 by determining if the protein has RNase activity and if this activity is important for infection. Lastly, Aim 3 will identify the stage(s) of infection n which Cas2 plays a role;e.g., the cas2 mutant's infection defect could be indicative of diminished resistance to intracellular killing and/or impaired intracellular replication. Thus, by using Lpn a a new model organism, we will probe for novel functions of the CRISPR/Cas locus that seek to shift current research paradigms.
Legionella pneumophila (Lpn), the bacterial agent of a severe form of pneumonia known as Legionnaires'disease, is an intracellular parasite of amoebae when it is in its natural aquatic habitat and an intracellular parasite of macrophages when it is introduced in the lungs. Data presented here demonstrate that the Lpn CRISPR-Cas locus is expressed during intracellular infection and the Lpn Cas2 protein, encoded within the locus, is required for infection of amoebae. Because Cas2 proteins in other bacteria and archaea have only been linked to immunity to phage and plasmid, the proposed studies aimed at elucidating the mechanism of Lpn Cas2 function will not only further our understanding of Lpn pathogenesis and intracellular parasitism but seek to shift the current research paradigm concerning the CRISPR-Cas system.