title: Active and adaptive Legionella CRISPR-Cas reveals a recurrent challenge to the pathogen.
abstract: Clustered regularly interspaced short palindromic repeats with CRISPR-associated gene (CRISPR-Cas) systems are widely recognized as critical genome defense systems that protect microbes from external threats such as bacteriophage infection. Several isolates of the intracellular pathogen Legionella pneumophila possess multiple CRISPR-Cas systems (type I-C, type I-F and type II-B), yet the targets of these systems remain unknown. With the recent observation that at least one of these systems (II-B) plays a non-canonical role in supporting intracellular replication, the possibility remained that these systems are vestigial genome defense systems co-opted for other purposes. Our data indicate that this is not the case. Using an established plasmid transformation assay, we demonstrate that type I-C, I-F and II-B CRISPR-Cas provide protection against spacer targets. We observe efficient laboratory acquisition of new spacers under 'priming' conditions, in which initially incomplete target elimination leads to the generation of new spacers and ultimate loss of the invasive DNA. Critically, we identify the first known target of L. pneumophila CRISPR-Cas: a 30 kb episome of unknown function whose interbacterial transfer is guarded against by CRISPR-Cas. We provide evidence that the element can subvert CRISPR-Cas by mutating its targeted sequences - but that primed spacer acquisition may limit this mechanism of escape. Rather than generally impinging on bacterial fitness, this element drives a host specialization event - with improved fitness in Acanthamoeba but a reduced ability to replicate in other hosts and conditions. These observations add to a growing body of evidence that host range restriction can serve as an existential threat to L. pneumophila in the wild.
authors.affiliation: Department of Molecular Genetics, University of Toronto, Toronto, Ontario, Canada.
authors.forename: Chitong
authors.initials: C
authors.lastname: Rao
authors.affiliation: Public Health Ontario, Toronto, Ontario, Canada.
authors.forename: Cyril
authors.initials: C
authors.lastname: Guyard
authors.affiliation: Centro Nacional de Microbiología, Instituto de Salud Carlos III, Madrid, Spain.
authors.forename: Carmen
authors.initials: C
authors.lastname: Pelaz
authors.affiliation: The McGill University and Génome Québec Innovation Centre, Montreal, Quebec, Canada.
authors.forename: Jessica
authors.initials: J
authors.lastname: Wasserscheid
authors.affiliation: Department of Molecular Genetics, University of Toronto, Toronto, Ontario, Canada.
authors.forename: Joseph
authors.initials: J
authors.lastname: Bondy-Denomy
authors.affiliation: The McGill University and Génome Québec Innovation Centre, Montreal, Quebec, Canada.
authors.forename: Ken
authors.initials: K
authors.lastname: Dewar
authors.affiliation: Department of Molecular Genetics, University of Toronto, Toronto, Ontario, Canada. alex.ensminger@utoronto.ca.|Public Health Ontario, Toronto, Ontario, Canada. alex.ensminger@utoronto.ca.|Department of Biochemistry, University of Toronto, Toronto, Ontario, Canada. alex.ensminger@utoronto.ca.
authors.forename: Alexander W
authors.initials: AW
authors.lastname: Ensminger
mesh_terms: D048248:Acanthamoeba castellanii / Q000382:microbiology; D001483:Base Sequence; D064112:Clustered Regularly Interspaced Short Palindromic Repeats; D017124:Conserved Sequence; D019143:Evolution, Molecular; D005798:Genes, Bacterial; D054884:Host-Pathogen Interactions; D016952:Legionella pneumophila / Q000235:genetics* / Q000254:growth & development; D050296:Microbial Viability; D017422:Sequence Analysis, DNA