# Importance and function of highly conserved substrates of the Legionella pneumophila Type II Secretion System for Infection

> **NIH NIH F31** · NORTHWESTERN UNIVERSITY · 2024 · $48,974

## Abstract

Project Summary
Legionella pneumophila (Lp) is the agent of Legionnaires' disease, an oft-fatal pneumonia that is increasing in
incidence. Lp is ubiquitous in water systems, infecting the lungs after inhalation of contaminated droplets. In
waters, Lp persists as an intracellular parasite of amoebae and constituent of biofilms. In the lungs, it grows in
macrophages, mimicking what it does in amoebae. Learning how Lp survives in water and grows in amoebae
and biofilms is critical to understanding and possibly preventing human disease. In past, the Cianciotto lab
showed that Lp encodes a type II secretion system (T2SS) which mediates secretion of >25 protein substrates.
From mutant analysis, the T2SS was required for infection of at least 4 types of amoebae, macrophages, and
the murine lung. The lab also identified 8 substrates that are required for infection of Acanthamoeba castellanii
and other amoebae. Yet, given the large defect shown by mutants lacking the entire T2SS vs the more modest
defects of mutants lacking individual substrates, I posited there are more important T2SS substrates to be
found. When the lab had used proteomics to define the first 25 substrates, in silico analysis suggested that
there are ~60 more substrates, and recent proteomics on Lp supernatants showed that 47/60 of the predicted
substrates are in fact secreted. Since there had been a positive correlation between a T2SS substrate’s
prevalence across the Legionella genus and the requirement of that substrate for infection of amoebae, I made
mutants lacking each of the 9/47 “new” substrates that occur in > 93% of Legionella species. I then determined
that the new substrate Lpw20501 majorly promotes infection of A. castellanii. After immunoblot confirmation of
the T2SS-dependency of Lpw20501, bioinformatics revealed that the protein represents an uncharacterized
family of polysaccharide deacetylases that is predicted to act on i) N-acetylglucosamine-containing
compounds, which may include chitin, ii) acetyl-containing xylan or cellulose acetate, or iii) other (novel) acetyl-
containing substrates. I further observed that the lpw20501 mutant aggregated more rapidly than WT did,
suggesting that secreted Lpw20501 may uniquely deacetylate the outer surface of Lp and as a result impact
biofilm formation. Thus, I posit that Lpw20501 is a novel secreted protein that enhances the survival of Lp in
multiple intra- and extracellular niches. This proposal will i) purify Lpw20501 and discern its enzyme activity, ii)
further define surface traits tied to Lpw20501, and iii) judge Lpw20501’s impact on Lp growth in various
amoebae, in biofilms, and on acetyl-containing polysaccharides. This work will i) increase our knowledge of a
key pathogen, ii) define a new type of exoenzyme, iii) have implications for other pathogens that use T2SS or
are intracellular parasites, and iv) possibly define a new target for controlling Lp in the built environment.

## Key facts

- **NIH application ID:** 10887423
- **Project number:** 5F31AI172194-02
- **Recipient organization:** NORTHWESTERN UNIVERSITY
- **Principal Investigator:** Carlton Adams
- **Activity code:** F31 (R01, R21, SBIR, etc.)
- **Funding institute:** NIH
- **Fiscal year:** 2024
- **Award amount:** $48,974
- **Award type:** 5
- **Project period:** 2023-07-01 → 2025-06-30

## Primary source

NIH RePORTER: https://reporter.nih.gov/project-details/10887423

## Citation

> US National Institutes of Health, RePORTER application 10887423, Importance and function of highly conserved substrates of the Legionella pneumophila Type II Secretion System for Infection (5F31AI172194-02). Retrieved via AI Analytics 2026-05-24 from https://api.ai-analytics.org/grant/nih/10887423. Licensed CC0.

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