Employing CRISPR/Cas for the identification of virulence-critical effectors in an intracellular pathogen
Thursday, September 15, 2016 — Poster Session III
- NA Ellis
- B Kim
- MP Machner
Like other intracellular pathogens, Legionella pneumophila, the causative agent of Legionnaires’ pneumonia, must establish a pathogen-containing vacuole (PCV) within a host cell to replicate. Formation of the PCV depends on the activities of virulence-critical proteins (effectors) that are secreted into the host. It has proven daunting to identify which of the over 300 L. pneumophila effector proteins secreted into alveolar macrophages are critical for infection because the construction of traditional L. pneumophila gene deletion strains is timely and transposon or chemical mutagenesis often disrupts housekeeping genes. To overcome the genetic limitations of interrogating L. pneumophila virulence, we have introduced a CRISPR/Cas platform into L. pneumophila for the direct integration of effector-encoding genes whose products promote PCV formation and intracellular growth. We employ plasmid expressed CRISPR (cr) and a trans-activating (tracr) RNAs to direct a chromosome-expressed copy of dCas9, a nuclease-deficient variant of Cas9 from Streptococcus pyogenes, to effector-encoding genes for down regulation of transcription by preclusion of RNA polymerase. Here we show that our CRISPR/Cas platform is not only tunable, efficient, and specific for the down regulation of effector-encoding genes, but most importantly, is functional in L. pneumophila during intracellular growth. We have targeted both genes encoding components of the Type IV secretion system, necessary for delivery of effector proteins into the host, as well as effector-encoding genes themselves and observed reductions in the intracellular growth of L. pneumophila. The CRISPR/Cas platform made available by this study will be central to elucidating virulence strategies of L. pneumophila and related pathogens.
Category: Microbiology and Infectious Diseases