LFn-Needle

Inflammasome activation with Needle

Human NLRC4/NAIP Inflammasome Inducer - LFn-Needle

InvivoGen provides LFn-Needle, an NLRC4/NAIP inflammasome agonist. Needle is a component of the type III secretion systems (T3SS) of intracellular bacteria able to interact with NLRC4 via NAIP [1-4]. 

 More details

LFn-Needle is fused to the amino-terminal domain of B. anthracis lethal factor (LFn). This fusion system, when co-administred with the anthrax toxin’s protective antigen (PA), allows intracellular delivery of the bacterial ligand [4]. The combination of LFn-Needle with the anthrax protective antigen (PA) is named Needle-Tox [5]. Its ability to activate the human NLRC4 inflammasome has been validated using THP1-NLRC4, THP1-KO-NLRC4, and THP1-Null2 cell lines. The subsequent production of IL-1β by these cells was measured using the HEK-Blue™ IL-1β cellular assay.

Key features:

• Needle from B. thailandensis T3SS
• Potent inducer of the human NLRC4 inflammasome in vitro
• Produced in Sf9 insect cells
• Each lot is functionally tested

Download our Practical guide on Inflammasomes.

References:

1. Zhao Y. et al., 2011. The NLRC4 inflammasome receptors for bacterial flagellin and type III secretion apparatus. Nature. 477(7366):596-600.
2. Rauch I. et al., 2016. NAIP proteins are required for cytosolic detection of specific bacterial ligands in vivo. The Journal of Exp. Med. 213(5):657-665.
3. Zhao Y. et al., 2016. Genetic functions of the NAIP family of inflammasome receptors for bacterial ligands in mice. J Exp Med. 213(5):647-656.
4. Worrall L.J. et al., 2011. Structural overview of the bacterial injectisome. Curr Opin Microbiology. 14(1):3-8.
5. Ballard J.D. et al., 1996. Anthrax toxin-mediated delivery of a cytotoxic T-cell epitope in vivo. PNAS. 93(22):12531-12534.

Figures

Secretion of mature IL-1β by THP1-KO-NLRC4 cells upon inflammasome activation.
~3x10⁵ THP1-Null2 (WT) and THP1-KO‑NLRC4 (KO) cells were incubated for 3h at 37°C with LPS-EK (1 μg/ml) (priming) and then incubated (activation) with inflammasome inducers: Nigericin (5 μM), transfected Poly (dA:dT) (1 μg/ml), E. coli outer membrane vesicles (OMVs) (100 μg/ml), or Needle-Tox (4 ng/ml). After 24h, the secretion of mature human (h)IL-1β was assessed in the culture supernatant using HEK-Blue™ IL-1β sensor cells which express an NF-κB SEAP reporter gene. QUANTI-Blue™ Solution was used to measure SEAP activity. Optical density (OD) was read at 630 nm.
 

Note: Needle-Tox is a combination of LFn-Needle (4 ng/ml) with the anthrax protective antigen (PA) (20 ng/ml). PA allows LFn-Needle translocation into the cytosol.

Secretion of mature IL-1β and pyroptosis upon THP1-derived cell activation using LFn-Needle.
~3x10⁵ THP1-Null2 (blue) and THP1-KO-NLRC4 cells (red) were incubated for 3h at 37°C with LPS-EK (1 μg/ml) (priming) and then incubated (activation) with increasing concentration of Needle-Tox. After 24h, mature human (h)IL-1β was assessed in the culture supernatant using HEK-Blue™ IL-1β sensor cells (A), and cell death was assessed using the lactate dehydrogenase (LDH) assay (B).

Note: Needle-Tox is a combination of LFn-Needle (4 ng/ml) with the anthrax protective antigen (PA) (20 ng/ml). PA allows LFn-Needle translocation into the cytosol.

Specifications

Protein construction: T3SS Needle protein [S2-R89] fused to the amino-terminal domain [A43-R296] of anthrax toxin’s lethal factor (LFn) protein (N-terminal).

Accession sequence: WP_009896110 (Needle sequence)

Species: Burkholderia thailandensis

Source: Sf9 insect cells

Tag: N-terminal poly-histidine (6 x His)

Total protein size: 371 a.a. (secreted form)

Molecular weight: ~ 46 kDa (SDS-PAGE gel)

Purification: Ni2+ affinity chromatography

Purity: >90% (SDS-PAGE)

Quality control:

• The biological activity has been validated using cellular assays.
• The absence of bacterial contamination (e.g. lipoproteins and endotoxins) has been confirmed using HEK-Blue™ TLR2 and HEK-Blue™ TLR4 cells.

Contents

Note: B. anthracis protective antigen (PA) is not provided

• 5 μg of lyophilized LFn-Needle protein
• 1.5 ml of endotoxin-free water

 The product is shipped at room temperature.

 Lyophilized protein should be stored at -20 ̊C up to 6 months.

 Resuspended protein is stable up to 6 months when stored at -20°C

Avoid repeated freeze-thaw cycles.

Details

NLRC4 BACKGROUND:

The NLRC4 (aka Ipaf) inflammasome is an intracellular multi-protein complex that plays a central role in innate immunity [1,2]. It is activated by a two-step process; a first signal (‘priming’) is provided by microbial molecules such as TLR ligands, while the second signal is provided by intracellular bacterial molecules such as Flagellin from the motility apparatus, or Inner Rod and Needle proteins from the bacterial type III or IV secretion systems (T3SS or T4SS). NLCR4 is an indirect sensor: it interacts with NAIPs (NLR family apoptosis inhibitory proteins) that directly bind to Flagellin, Needle, and Inner Rod. While a single NAIP operates upstream of NLRC4 in humans and recognizes each of these activators [3], multiple NAIPs have been described in mice with different affinities for each molecule [4-7]. The NLRC4 inflammasome appears to protect mucosal barriers, such as the lung, stomach, and intestine, from invading bacteria [2]. 

References:

1. Platnich J.M. & Muruve D.A., 2019. NOD-like receptors and inflammasomes: A review of their canonical and non-canonical signaling pathways. Arch Biochem Biophys. 670:4-14.
2. Bauer R. & Rauch I., 2020. The NAIP/NLRC4 inflammasome in infection and pathology. Mol Aspects Med. 76:100863.
3. Yang J. et al., 2013. Human NAIP and mouse NAIP1 recognize bacterial type III secretion needle protein for inflammasome activation. PNAS. 110(35):14408-14413.
4. Zhao Y. et al., 2011. The NLRC4 inflammasome receptors for bacterial flagellin and type III secretion apparatus. Nature. 477(7366):596-600.
5. Rauch I. et al., 2016. NAIP proteins are required for cytosolic detection of specific bacterial ligands in vivo. The Journal of Exp. Med. 213(5):657-665.
6. Zhao Y. et al., 2016. Genetic functions of the NAIP family of inflammasome receptors for bacterial ligands in mice. J Exp Med. 213(5):647-656.
7. Kofoed E.M. & Vance R.E., 2011. Innate immune recognition of bacterial ligands by NAIPs determines inflammasome specificity. Nature. 477(7366):592-595.