# Deciphering how the Cas2 ribonuclease non-canonically controls Legionella pneumophila thermal tolerance and virulence

> **NIH NIH R21** · NORTHWESTERN UNIVERSITY · 2024 · $240,000

## Abstract

PROJECT SUMMARY / ABSTRACT
Legionella pneumophila (Lp) is the agent of Legionnaires’ disease. In water systems, Lp grows in amoebae
and biofilms, and in the lungs, it infects macrophages. CRISPR-Cas systems are a common feature of Lp,
where they expectedly confer immunity to invading genetic elements. Yet, through an analysis of Lp 130b,
which has the most common Lp CRISPR-Cas type (type II-B), we discovered that Cas2 majorly promotes Lp
infection of amoebae. Compatible with this, cas2 mRNA is hyperexpressed during Lp infection of amoebae and
during stationary phase, the growth stage linked to the infectious Lp phenotype. Critically, the infection role of
Cas2 was distinct from adaptive immunity, i.e., i) a cas2 mutant’s infection defect occurred in the absence of
any foreign genetic element, ii) mutants lacking other cas genes or the CRISPR array were not impaired, and
iii) introduction of Cas2 into a Lp strain that naturally lacks a CRISPR-Cas locus caused that strain to be more
infective for amoebae. We purified Lp Cas2 and showed that it has RNase activity, and by testing a mutant
form of Cas2 that lacked the ability to cleave ssRNAs, we showed that RNase activity is critical for the ability of
Cas2 to promote infection of amoebae. This work has endured as the sole example of Cas2 clearly promoting
a non-canonical or infectious phenotype. Thus, we posited that Cas2, by virtue of its ability to cleave/degrade
RNA, influences the amount or configuration of an mRNA that encodes or regulates a key infectivity factor. We
now identify expression of a previously undefined small heat shock protein gene (hspC2) as being increased
by the presence of Cas2. Mutant analysis found that HspC2, along with Cas2, promotes Lp growth in amoebae
and macrophages as well as biofilm formation, thereby revealing a role for a sHsp in pathogenesis and new
insight into how Cas2 impacts Lp infectivity. We also confirmed a role for Cas2 in Lp thermal tolerance and
biofilm formation. Given these data, we propose that the Lp Cas2-HspC2 axis can be a new model or paradigm
for decoding how Cas proteins non-canonically impact gene expression, thermal tolerance, and virulence.
Thus, we now aim to discern whether Cas2 i) acts directly by binding and then cleaving/degrading (only) a part
of hspC2 mRNA such that the transcript is more stable, ii) acts indirectly by binding and degrading wholesale
the RNA for a repressor of hspC2 transcription, or iii) again acts indirectly but by binding and then cleaving a
part of the mRNA for a transcriptional activator of hspC2 such that the resulting RNA is more stable. This
proposal has the potential to i) advance our understanding of Lp physiology and virulence, potentially revealing
new targets for restricting Lp spread, ii) define a new mode of bacterial gene regulation controlled by multi-
purpose Cas2 proteins, and iii) ultimately uncover a form of nuclease activity that might have broad application.

## Key facts

- **NIH application ID:** 10942970
- **Project number:** 1R21AI185007-01
- **Recipient organization:** NORTHWESTERN UNIVERSITY
- **Principal Investigator:** NICHOLAS P CIANCIOTTO
- **Activity code:** R21 (R01, R21, SBIR, etc.)
- **Funding institute:** NIH
- **Fiscal year:** 2024
- **Award amount:** $240,000
- **Award type:** 1
- **Project period:** 2024-07-08 → 2026-05-31

## Primary source

NIH RePORTER: https://reporter.nih.gov/project-details/10942970

## Citation

> US National Institutes of Health, RePORTER application 10942970, Deciphering how the Cas2 ribonuclease non-canonically controls Legionella pneumophila thermal tolerance and virulence (1R21AI185007-01). Retrieved via AI Analytics 2026-05-26 from https://api.ai-analytics.org/grant/nih/10942970. Licensed CC0.

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