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LFn-Needle

LFn-Needle Unit size Cat. code Docs Qty Price
T3SS Needle protein fused to Lethal Factor - NLRC4 agonist
5 µg
tlrl-ndl
+-
$306.00
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NLRC4/NAIP inflammasome activation with Needle
NLRC4/NAIP inflammasome activation with Needle

Human NLRC4/NAIP Inflammasome Inducer

InvivoGen provides LFn-Needle, a model of NLRC4/NAIP inflammasome agonist [1-3]. Needle is a component of the type III secretion systems (T3SS) of intracellular bacteria described as an NLRC4/NAIP ligand [1-4]. It 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].

 

InvivoGen's LFn-Needle recombinant protein features B. thailandensis T3SS Needle protein [S2-R89] fused to the amino-terminal domain [A34-R296] of anthrax toxin’s lethal factor (LFn) protein in N-terminal. Its ability to induce the human NLRC4 inflammasome has been validated using THP1-NLRC4, THP1-KO-NLRC4, and THP1-Null2 cell lines. The production of IL-1β by these cells is measured using the HEK-Blue™ IL-1β cellular assay.

LFn-Rod is also available and is a potent inducer of murine NLRC4/NAIP inflammasome.

 

NLRC4 background:

The NLRC4 (aka Ipaf) inflammasome is an intracellular multi-protein complex that plays a central role in innate immunity [6, 7]. 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 [8], multiple NAIPs have been described in mice with different affinities for each molecule [1-3, 9]. The NLRC4 inflammasome appears to protect mucosal barriers, such as the lung, stomach, and intestine, from invading bacteria [7]. 

 

Features of LFn-Needle:

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

 

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.
6Platnich 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.
7. Bauer R. & Rauch I., 2020. The NAIP/NLRC4 inflammasome in infection and pathology. Mol Aspects Med. 76:100863.
8. 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.
9. Kofoed E.M. & Vance R.E., 2011. Innate immune recognition of bacterial ligands by NAIPs determines inflammasome specificity. Nature. 477(7366):592-595.

Figures

Functional validation of LFn-Needle using THP1-derived cells
Functional validation of LFn-Needle using THP1-derived cells

Secretion of mature IL-1β by THP1-KO-NLRC4 cells and their parental THP1-Null2 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.

Human NLRC4 inflammasome response to LFn-Needle
Human NLRC4 inflammasome response to LFn-Needle

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 Needle-Tox (0.006-100 ng/ml). After 24h, mature human (h)IL-1β was assessed in the culture supernatant using HEK-BlueTM IL-1β sensor (A), and cell death was assessed using the lactate dehydrogenase (LDH) assay (B).

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Specifications

LFn-Needle

  • Protein construction: T3SS Needle protein [S2-R89] fused to the amino-terminal domain [A43-R296] of anthrax toxin’s lethal factor (LFn) protein in 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 protein has been validated by functional assays using THP1-NLRC4THP1-KO-NLRC4, and THP1-Null2 cell lines.
    - The absence of bacterial contamination (e.g. lipoproteins and endotoxins) has been confirmed using HEK-Blue™ TLR2 and HEK-Blue™ TLR4 cellular assays.
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Contents

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

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

room temperature The product is shipped at room temperature.

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

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

Avoid repeated freeze-thaw cycles.

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