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.
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