Active and adaptive Legionella CRISPR‐Cas reveals a recurrent challenge to the pathogen

Abstract

SummaryClustered 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.

Spacer capture and integration by a type I-F Cas1–Cas2-3 CRISPR adaptation complex

“…Priming occurs in type I-B, I-C, I-E, and I-F systems in multiple genera (28,30,33,34,46), suggesting that this efficient adaptation route is ubiquitous in type I systems. Therefore, the type I-F Cas1-Cas2-3 complex provides a view of adaptation complexes that are recruited to targets by the type I Cascade surveillance machineries.…”

“…Multiple type I systems undergo priming, and for type I-E it requires Cascade, crRNA, Cas1, Cas2, and Cas3 (28,30,33,34). During type I-F priming, new spacers are captured from regions adjacent to the escaped protospacer in a process that involves the 3′-to-5′ translocation of a Cas1-Cas2-3 complex (28,29).…”

CRISPR-Cas and Contact-Dependent Secretion Systems Present on Excisable Pathogenicity Islands with Conserved Recombination Modules

“…In Legionella pneumophila, an intracellular pathogen, multiple CRISPR systems can be present within an isolate, and a noncanonical role in intracellular survival for these systems has been described (40). A recent study has shown that several L. pneumophila isolates contained a type I-C CRISPR-Cas system on the chromosome and a type I-F system on a 114-kb plasmid, and that study also demonstrated a role for these systems in immunity (41). Overall, it appears that many diverse CRISPR-Cas systems are associated with many different types of mobile genetic elements, indicating their ability to be horizontally transferred among isolates.…”

Unusual Legionnaires' outbreak in cool, dry Western Canada: an investigation using genomic epidemiology

“…Briefly, paired-end reads were mapped to the Toronto-2005 outbreak reference genome (Genbank accession no. CP012019) [26] using SMALT v. 0.7.6. High-quality SNVs were identified using two variant callers: FreeBayes v. 0.9.8 and SAMtools v. 1.1 mpileup (mapping and quality scores ⩾30; SNV fraction ⩾0·75; coverage ⩾15).…”