Legionella pneumophila CRISPR-Cas Suggests Recurrent Encounters with One or More Phages in the Family Microviridae

Abstract

Legionella pneumophila is a ubiquitous freshwater pathogen and the causative agent of Legionnaires’ disease. L. pneumophila growth within protists provides a refuge from desiccation, disinfection, and other remediation strategies. One outstanding question has been whether this protection extends to phages. L. pneumophila isolates are remarkably devoid of prophages and to date no Legionella phages have been identified. Nevertheless, many L. pneumophila isolates maintain active CRISPR-Cas defenses. So far, the only known target of these systems is an episomal element that we previously named Legionella Mobile Element-1 (LME-1). The continued expansion of publicly available genomic data promises to further our understanding of the role of these systems. We now describe over 150 CRISPR-Cas systems across 600 isolates to establish the clearest picture yet of L. pneumophila ’s adaptive defenses. By searching for targets of 1,500 unique CRISPR-Cas spacers, LME-1 remains the only identified CRISPR-Cas targeted integrative element. We identified 3 additional LME-1 variants - all targeted by previously and newly identified CRISPR-Cas spacers - but no other similar elements. Notably, we also identified several spacers with significant sequence similarity to microviruses, specifically those within the subfamily Gokushovirinae . These spacers are found across several different CRISPR-Cas arrays isolated from geographically diverse isolates, indicating recurrent encounters with these phages. Our analysis of the extended Legionella CRISPR-Cas spacer catalog leads to two main conclusions: current data argue against CRISPR-Cas targeted integrative elements beyond LME-1, and the heretofore unknown L. pneumophila phages are most likely lytic gokushoviruses. IMPORTANCE Legionnaires’ disease is an often-fatal pneumonia caused by Legionella pneumophila , which normally grows inside amoebae and other freshwater protists. L. pneumophila trades diminished access to nutrients for the protection and isolation provided by the host. One outstanding question is whether L. pneumophila is susceptible to phages, given the protection provided by its intracellular lifestyle. In this work, we use Legionella CRISPR spacer sequences as a record of phage infection to predict that the “missing” L. pneumophila phages belong to the microvirus subfamily Gokushovirinae . Gokushoviruses are known to infect another intracellular pathogen, Chlamydia . How do gokushoviruses access L. pneumophila (and Chlamydia ) inside their “cozy niches”? Does exposure to phages happen during a transient extracellular period (during cell-to-cell spread) or is it indicative of a more complicated environmental lifestyle? One thing is clear, 100 years after their discovery, phages continue to hold important secrets about the bacteria upon which they prey.

Mobile element integration reveals a chromosome dimer resolution system in Legionellales

“…Other invasive elements known to integrate at the dif site of their host (IMEXs) do not encode their own recombinase and instead utilize the host Xer for integration/excision (Das et al, 2013;. Similarly, LME-1 does not appear to encode its own recombinase or integrase (Deecker et al, 2021). Our intermolecular recombination assay indicates that XerL can facilitate integration of circular DNA containing the LME-1 22-bp att site (Fig.…”

“…Several strains of Legionella pneumophila harbor a phage-like integrative mobile genetic element, named Legionella mobile element-1 (LME-1) (Deecker et al, 2021;Lüneberg et al, 2001;Rao et al, 2016). We previously found that integration into the genome requires a 22-bp attachment site (att) in LME-1 that is identical to a sequence on the L. pneumophila genome (Rao et al, 2016).…”

Microviruses: A World Beyond phiX174

“…Based on CRISPR-spacer sequences, it is possible to infer that within the human microbiome, the most commonly infected bacterial genera are Bacteroides, Prevotella, Faecalibacterium, and Phasolarctobacterium (11,90,110). Furthermore, microviral spacers are also present in genera containing pathogens such as Acinetobacter, Burkholderia, Clostridium, Helicobacter, Legionella, and Treponema (11,111). Notably, single-cell viral-tagging has confirmed the CRISPR-based predictions of Gram-positive Actinomycetota (specifically Collinsella) as microviral hosts in the human gut (112).…”

Mobile Element Integration Reveals a Chromosome Dimer Resolution System in Legionellales

“…Several strains of Legionella pneumophila harbor a phage-like integrative mobile genetic element named Legionella mobile element-1 (LME-1) ( 30 – 33 ). We previously found that integration into the genome requires a 22-bp attachment site ( att ) in LME-1 that is identical to a sequence on the L. pneumophila genome ( 33 ).…”