# Defining Mechanisms of NAIP5-independent Flagellin Sensing during Bacterial Infection

> **NIH NIH F32** · UNIVERSITY OF PENNSYLVANIA · 2021 · $65,994

## Abstract

PROJECT SUMMARY/ABSTRACT:
 Antibiotic resistant bacterial infections are an immediate public threat to the United States and global
healthcare systems. We must gain a deeper understanding of the innate immune system’s response to bacterial
pathogens to facilitate development of host-directed therapeutic approaches to combat bacterial infection. Many
clinically relevant bacterial pathogens harbor a flagellum comprised of the structural protein flagellin, which is
recognized by the extracellular Toll-like receptor 5 (TLR5) and the intracellular neuronal apoptosis inhibitory
protein 5 (NAIP5) sensor. NAIP5 sensing of flagellin results in the formation of a multiprotein inflammasome
complex containing the NLR caspase recruitment domain-containing protein 4 (NLRC4) which activates
caspase-1, triggering an inflammatory cell death called pyroptosis. In the course of infection, a number of
pathogens including Salmonella enterica serovar Typhimurium (S. Tm) deliver flagellin into the host cytosol,
leading to activation of the NAIP5-NLRC4 inflammasome. Multiple studies suggest that in mice, NAIP6 also
senses flagellin. Why there are two distinct sensors for cytosolic flagellin, as well as the biological circumstances
under which NAIP6 might sense flagellin is unknown. Intriguingly, bacterial flagellins from many clinically relevant
bacteria that do not activate NAIP5, lack a conserved arginine at the C terminus. Moreover, truncated S. Tm
flagellin with a stop codon inserted in the place of conserved three amino acids in its D0 domain (termed D0STOP)
abrogates recognition of flagellin by the host cell. These observations suggest that the C terminus of flagellin is
essential for NAIP5 sensing and that bacterial pathogens may escape NAIP5 by altering this domain. My
preliminary findings reveal that TLR2 priming of murine bone marrow derived macrophages (BMDMs) leads to
activation of a NLRC4-dependent response to D0STOP flagellin when delivered using the heterologous Type III
secretion system of Yersinia pseudotuberculosis (Yp). Altogether, these findings and my preliminary data
provoke the conceptually novel hypothesis that TLR2 signaling licenses NAIP6-dependent flagellin
sensing to overcome bacterial evasion of NAIP5. In Aim 1, I plan to determine how TLR2 licenses NAIP6-
NLRC4 inflammasome activation in TLR2 primed BMDMs and test if NAIP6 is sufficient to recognize flagellins
that evade NAIP5 sensing using Yp as a delivery system and a retroviral expression vector system to reconstitute
the NAIP5/6-NLRC4 inflammasome in 293T cells. In Aim 2, I will assess the contribution of NAIP6 flagellin
detection in eliciting protective host responses using Yp as a delivery system in Naip5-/- and Naip1-6D/D mice. The
scientific goal of this fellowship is to uncover a new mechanism for innate detection of flagellin and understand
why mice harbor two highly similar cytosolic sensors that detect flagellin. Another goal is to advance my training
as a scientist to propel a ...

## Key facts

- **NIH application ID:** 10313353
- **Project number:** 1F32AI164655-01
- **Recipient organization:** UNIVERSITY OF PENNSYLVANIA
- **Principal Investigator:** James Grayczyk
- **Activity code:** F32 (R01, R21, SBIR, etc.)
- **Funding institute:** NIH
- **Fiscal year:** 2021
- **Award amount:** $65,994
- **Award type:** 1
- **Project period:** 2021-08-01 → 2024-07-31

## Primary source

NIH RePORTER: https://reporter.nih.gov/project-details/10313353

## Citation

> US National Institutes of Health, RePORTER application 10313353, Defining Mechanisms of NAIP5-independent Flagellin Sensing during Bacterial Infection (1F32AI164655-01). Retrieved via AI Analytics 2026-05-27 from https://api.ai-analytics.org/grant/nih/10313353. Licensed CC0.

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