Welkom bij het Lymeforum

Immediate and effective innate immune response is crucial for the prevention of microbial colonization or dissemination in mammalian host. The defensive actions are enabled by innate immune receptors that sense conserved microbial structures and trigger immediate response by the cells of the immune system. Arguably the most important and so far the most studied group of innate immune receptors are the Toll-like receptors (TLRs). They specifically recognize distinctive microbial molecules, such as double stranded RNA, flagellin or endotoxins

Endotoxins (i.e. lipopolysaccharides, LPS) are the main constituents of the cell envelope of most Gram-negative bacteria (with few exceptions [2]–[4]). They are composed of a highly variable polysaccharide and a lipid A moiety, which is the active principle of endotoxin that is recognized by the TLR4/MD-2 receptor complex. Lipid A is typically composed of a glucosamine disaccharide modified with two phosphates and comprising four primary glucosamine-linked hydroxyacyl chains and two secondary acyl chains [5]. This type of hexaacylated endotoxin can be found in the majority of Gram-negative bacteria, ranging from soil and plant inhabitants to mammalian mucosal pathogens. The hexaacylated endotoxin activates the human TLR4/MD-2 very efficiently, so it is not surprising that some bacteria modify their lipid A structure in order to evade immune recognition [6]. Pseudomonas aeruginosa [7] can produce weakly activating pentaacylated endotoxin and Yersinia pestis and Helicobacter pylori can produce a tetraacylated form of lipid A [8], [9], which cannot activate the human TLR4/MD-2, and can thus avoid activation of the immune response.

Hypoacylated forms of endotoxin are also precursors in the bacterial synthesis of lipid A (i.e. the tetraacylated lipid IVa that has only the primary hydroxyacyl chains) or can be the result of mutations in genes such as msbB, which is a gene in the E. coli endotoxin synthesis pathway that encodes an acyltransferase. Mutation in msbB results in production of a pentaacylated endotoxin that lacks a secondary myristoyl fatty acid [10], [11]. MsbB homologues have been characterized in a variety of pathogenic bacteria (e.g., Salmonella spp., Neisseria spp., Yersinia pestis, Klebsiella pneumoniae) [12]–[15]. Moreover, a second functional paralog of the msbB gene was identified in Shigella and enterohemorrhagic E. coli [16]–[19].

While hypoacylated endotoxins antagonize the activation of human TLR4/MD-2 complex by hexaacylated lipid A, they potently activate the TLR4/MD-2 receptor of several other species (e.g., mouse, rat, horse) [20]–[22]. The molecular difference in TLR4/MD-2 responsible for this discriminating endotoxin recognition is still not clear. Several studies have analyzed the molecular differences in the structure of MD-2 and TLR4 between species and identified regions in the endotoxin receptor complex that are involved in this discrimination [21], [23]–[25].

In the present study we further unravel the structural differences between human, mouse and equine MD-2 that are important for species-specific recognition of endotoxin varieties. We provide evidence that amino acid residues 82 and 122 crucially influence the positioning of endotoxin into MD-2′s hydrophobic pocket and therefore govern the agonistic/antagonistic properties of endotoxin varieties in different species. We were able to confer human MD-2 responsiveness to tetra- and pentaacylated LPS through increasing the hydrophobicity at the edge of the MD-2 binding pocket.

While hypoacylated endotoxins antagonize the activation of human TLR4/MD-2 complex by hexaacylated lipid A, they potently activate the TLR4/MD-2 receptor of several other species (e.g., mouse, rat, horse) [20]–[22]. The molecular difference in TLR4/MD-2 responsible for this discriminating endotoxin recognition is still not clear. Several studies have analyzed the molecular differences in the structure of MD-2 and TLR4 between species and identified regions in the endotoxin receptor complex that are involved in this discrimination [21], [23]–[25].

In the present study we further unravel the structural differences between human, mouse and equine MD-2 that are important for species-specific recognition of endotoxin varieties. We provide evidence that amino acid residues 82 and 122 crucially influence the positioning of endotoxin into MD-2′s hydrophobic pocket and therefore govern the agonistic/antagonistic properties of endotoxin varieties in different species. We were able to confer human MD-2 responsiveness to tetra- and pentaacylated LPS through increasing the hydrophobicity at the edge of the MD-2 binding pocket.


Discussion

We have detailed knowledge of the structure of the activated human and mouse TLR4/MD-2/endotoxin receptor complexes. Since the positioning of endotoxin into MD-2 and its interaction with TLR4 ectodomain is governed by a complex combination of electrostatic as well as hydrophobic interactions, the precise role of the residues mediating the species-specific TLR4/MD-2 activation by hypoacylated endotoxin varieties is still not clear. In the present study we addressed this question by comparing the capacity of MD-2 and TLR4 from three species to activate cells in response to hypoacylated endotoxin stimulation.

Het TLR4 gen bij mensen codeert de Toll-like receptor 4.[1][2] Het detecteert lipopolysacchariden op de buitenmembraan van gram-negatieve bacteriën en is belangrijk voor het activeren van het aangeboren immuunsysteem. TLR4 wordt ook aangeduid met CD284 (cluster of differentiation 284).
Het door dit gen gecodeerde proteïne behoort tot de Toll-like receptor (TLR) familie, dat een fundamentele rol speelt in het herkennen van een ziekteverwekker en het activeren van het aangeboren immuunsysteem.

The MD-2 protein appears to associate with toll-like receptor 4 on the cell surface and confers responsiveness to lipopolysaccaride (LPS), thus providing a link between the receptor and LPS signaling.[3]

Abstract

Lipopolysaccharides (LPS) are a constitutive part of the outer wall of gram negative bacteria. Because many of the symptoms of Lyme disease could be explained by a spirochetal LPS we have subjected Borrelia burgdorferi to standard LPS extraction techniques which yielded a LPS which accounted for 1.5-4% of the dry weight. The LPS was very similar to classical gram negative bacterial LPS both chemically and in its biological activities which included pyrogenicity, mitogenicity for lymphocytes and the induction of Interleukin 1 production by macrophages. In addition, the LPS produced an acute inflammatory reaction when injected intradermally into rabbit skin. It could also prepare a skin site for the production of the local Shwartzman reaction. These results show that the Lyme disease spirochete contains a hitherto unknown LPS that is biologically active in vitro and in vivo. It is likely that this molecule plays an important role in the pathogenesis of Lyme disease.

The LPS-LC extracted from the three genomic groups of B. burgdorferi sensu lato were found to have similar
immunochemical propoperties irrespecive of their genotype origin.
.....
In further experniments IE analysis schowed that the extracted LPS-LC complex of the tested strains has
migration characteristics similar to LPS of other Gram-negative bacteria.
The presence of LPS-LC released into the medium during culture growth was confirmed
by the LAL test.
Table 1 shows the release of endotoxin material into the culture medium
during growth.

Sproetje schreef:Okee, dan komt hier de volgende:
http://www.cssm.info/priloha/fm2004_625 ... ion_detailLipopolysaccharide-like component from B.burgdorferi sensu lato:

Hierin staat:
The LPS-LC extracted from the three genomic groups of B. burgdorferi sensu lato were found to have similar immunochemical propoperties irrespecive of their genotype origin
......
In further experniments IE analysis schowed that the extracted LPS-LC complex of the tested strains has migration characteristics similar to LPS of other Gram-negative bacteria.The presence of LPS-LC released into the medium during culture growth was confirmed by the LAL test.
Table 1 shows the release of endotoxin material into the culture medium during growth.