Part:BBa_K299812
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Creative BioCommons
Invasin + Listeriolysin
Invasin (INV) plays a key role in the initiation of Yersinia enterocolytica and Yersinia pseudotuberculosis infection. Through interaction with a beta1-integrin receptor present on the surface of eucaryotic cell membranes it triggers a signal-transduction pathway leading to internalisation of the whole bacterium in the endocytosis-dependent manner. The strong affinity of invasin to it’s receptor results in highly selective binding to the target molecule. Mammalian cells depleted of beta1-integrin cannot be infected.
Listeriolysin O (LLO) is a member of a widespread cholesterol-dependent pore-forming cytolysins family (CDCs). It's natural role in Listeria monocytogenes is to provide endosomal escape. The first step of the process involves binding of monomeric listeriolysin molecules to lipid bilayer containing cholesterol. The binding induces conformational change that subsequently leads to the formation of a prepores' oligomeric structures (consisting of 33-50 monomers) converting into large (maximum 350A-diameter) pores. This severely disturbs the stability of endosomal membrane and causes it’s rupture.
LLO is a phagosome-specyfic lysin. The acidic pH is necessary for it’s full hemolytic activity. Neutral pH of cytosol causes premature unfolding of TMH domains responsible for aqueous pore formation. This mechanism prevents Listeria spp from killing the host cell and losing the intracellular environment. In case of any tranformed strain it guarantees the lowest possible level of cytotoxicity, incomparable to this involved with the use of any other protein from CDCs family.
Left: Structural model of the LLO monomer from: P. Schnupf , D.A. Portnoy Listeriolysin O: a phagosome-specific lysin, Microbes and infection (2007) 1176-1187
Authors:
Cloned by Joanna Leszczyńska.
Created with the use of parts BBa K299810 and BBa K299811 cloned by Marta Błaszkiewicz.
Construct design.
The part consists of B0032 RBS ligated to inv gene from Yersinia pestis (horizontal gene transfer form Yersinia pseudotuberculosis). Following elements are B0032 RBS ligated to synthetic gene encoding Listeriolysin, codon usage optimized for E. coli. Aminoacid sequence identical with mature form of LLO from Listeria monocytogenes. Original signal sequence (secretion signal) is omitted since it does not work in E. coli. The construct is a fragment of the Invasiveness Operon (BBa_K299813 and BBa_K299815) To find out more about it's background and design click here.Safety
Bacteria transformed with this part are invasive microorganisms. All safety precautions must be taken when manipulating with transformed strain. It involves obligatory use of laboratory gloves. Work under laminar is strongly advised. All waste should be autoclaved to avoid accidental gene transfer to other bacteria and potential rise of pathogenic organism.Function of inv-llo gene on the platform of cyanobacteria
author: Chiu Hsun-I from NYMU.Taipei,2012References revealed when engineered with invasin(inv) from Yersinia pestis and listeriolysin O(llo) from Listeria monocytogenes (both provided by Pamela A. Silver, Harvard Medical school[1]), new S. elongatus was able to invade into cultured mammalian cells and was capable of symbiosis with eukaryotic cells. The symbiosis possibility of induced pluripotent stem cells from mice and J774 mouse macrophage cell line were evaluated.
[1]Towards a Synthetic Chloroplast, Christina M. Agapakis,1 Henrike Niederholtmeyer,1,¤ Ramil R. Noche,1 Tami D. Lieberman,1 Sean G. Megason,1 Jeffrey C. Way,2 and Pamela A. Silver1,2,PLoS One. 2011; 6(4): e18877.
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12INCOMPATIBLE WITH RFC[12]Illegal NheI site found at 3936
- 21INCOMPATIBLE WITH RFC[21]Illegal BglII site found at 770
- 23COMPATIBLE WITH RFC[23]
- 25INCOMPATIBLE WITH RFC[25]Illegal NgoMIV site found at 1387
- 1000COMPATIBLE WITH RFC[1000]
| n/a | Invasin + Listeriolysin |