Dr Tobias Pusterla (10)
Endotoxins are the main component of the outer membrane of the cell wall of Gram-negative bacteria. Typically, the term endotoxin is used synonymously with lipopolysaccharide (LPS), despite the fact that a few endotoxins are not LPS. The main function of endotoxins in bacteria is structural and protective. Gram-negative bacteria are characterised by two membranes: the inner membrane surrounds the cytoplasma whereas the outer membrane separates the bacterial cell wall from the external environment. Thus, the outer membrane serves as the first line of defence against environmental threats. In most cases, the outer membrane is not a common phospholipid bilayer but an asymmetric bilayer comprising LPS in the outer layer and phospholipids in the inner one (fig.1). Lipid A is the toxic component of endotoxins. It is a phosphorylated N-acetylglucosamine disaccharide containing a hydrophobic part (aliphatic chains of fatty acids) that anchors the endotoxin into the bacterial membrane. The rest of the endotoxin projects from the cell surface (fig.1). Although its structure is extremely conserved, it can undergo modifications in response to lớn varying environmental conditions.1 Endotoxins are the main component of the outer membrane of Gram-negative bacteria and of vital importance to lớn their survival. Endotoxins contribute to lớn the structural integrity of bacteria and act as a protective amphipathic barrier, shielding bacteria from chemical attacks. Endotoxins establish a barrier that is permeable only to lớn hydrophilic molecules with low molecular weight, making Gram-negative bacteria resistant to lớn many antimicrobial compounds.3 The reduced permeability to lớn large hydrophilic molecules mainly results from the hydrophobic nature of Lipid A. The hydrophilic nature of the core oligosaccharide and O-antigen additionally make endotoxins impermeable to lớn hydrophobic compounds. In hosts, LPS protects bacteria from killing by phagocytes or serum components. Of notice, variations in the endotoxin structure establish different antigenic strains, increasing their chance of circumventing immunological responses that were previously developed against a specific strain of bacteria, allowing resistance to lớn evolve. As endotoxins are exposed on the surface of bacteria, the innate immune system has evolved to lớn recognise them as a threat and to lớn react accordingly to lớn their presence. Endotoxins are pyrogens, provoking a strong innate immune response. When Gram-negative bacteria are killed by the immune system, fragments of their membrane containing endotoxins are released in the blood stream and may cause fever and diarrhoea. The presence of endotoxins in the blood (endotoxemia) typically leads to lớn hypotension, respiratory failure and reduced oxygen delivery.4 Strong endotoxemia can lead to lớn sepsis and eventually death. Xem thêm: diệu thủ đan tâm Being the most conserved portion of LPS, the immune system has evolved to lớn respond predominantly to lớn Lipid A. The immune reaction is entirely innate and mediated by Toll-Like Receptor 4 (TLR4) in complex with MD2 (fig.4).5 TLR4 stimulates the secretion of pro-inflammatory cytokines and nitric oxide from macrophages and endothelial cells. In addition, endotoxins stimulate B-cell differentiation, proliferation and immunoglobulin secretion (mainly IgG and IgM). In macrophages and monocytes, endotoxins trigger the production of inflammatory cytokines (such as interleukin-1, 6 and 8, TNF and platelet-activating factor) and the consequent release of prostaglandins and leukotrienes.6 Moreover, the complement and coagulation cascades are activated inducing inflammation, vasodilation, chemotaxis of neutrophils, coagulation, bleeding and shock. The O antigen is the immunogenic part of endotoxins, leading to lớn antibody production from the host and contributing to lớn evasion of phagocytosis. The involvement of the O antigen is confirmed by the fact that changes in its polysaccharide sequence significantly affect virulence. However, the mechanism underlying polysaccharide-driven virulence is not fully understood yet. There are several methods to lớn test for the presence of endotoxins. In vitro endotoxin testing methods include LAL assay and ELISA. Both can be run rẩy on microplate readers, significantly increasing throughput and efficiency. The Limulus Amebocyte Lysate (LAL) assay is a common endotoxin testing method. The LAL test is based on an extract of isolated amebocytes from the blood of the horseshoe crab. LAL tests are either chromogenic or turbidimetric. A LAL substitute test based on recombinant proteins and a fluorescent substrate is also available (fig. 5). Find more information on our blog post: “The LAL assay: a living fossil exploited to lớn detect bacterial contamination.” Endotoxins can also be assayed by ELISA which can detect either directly endotoxins or anti-endotoxin antibodies. However, the amphipathic nature of endotoxins negatively affects binding on ELISA plates and results in variable conformations of epitope binding sites. The result is generally low sensitivity and poor reproducibility. Xem thêm: quán cơm tùy hưu BMG LABTECH plate readers that can be used to lớn quantify endotoxins include the SPECTROstar Nano, Omega series, VANTAstar, CLARIOstar Plus and PHERAstar FSX.
The O-antigen is attached to lớn the core polysaccharide and is the outermost part of the molecule. Although not toxic, it is the main immunogenic portion of endotoxins and consequently, it is a recognition target for antibodies and a major antigenic determinant. It is a repetitive glycan polymer made up of 3 to lớn 5 sugars. It is the most diverse component of LPS: composition and length vary among species and even strains of bacteria. Function of endotoxins
Toxicity
Endotoxin testing
All these endotoxin assays can be measured on a microplate reader. These approaches generally require an absorbance microplate reader to detect either a chromogenic reaction (LAL and most typically ELISA), or the changes in turbidity. For assays based on recombinant proteins and a fluorescent substrate, a fluorescence microplate reader is necessary.References
Bạn đang xem: endotoxin la gi
Bình luận