Bacteria are microscopic single celled organisms known to reside in a large proportion of the body as natural flora. They help in normal processes such as digestion and production of nutrients. However if taken out of their natural environment they can become pathogenic and in some cases fatal. These organisms are divided into two main groups namely gram positive and gram negative bacteria (this essay places its emphasis on gram negative bacteria). They can be differentiated into these groups according to their cell wall structure and their ability to retain two stains; safranin in the case of gram negatives and crystal violet in the case of gram positives. This ability lies in the bacterial cell wall (http://micro.digitalproteus.com).
Septic shock is the most common killer in intensive care units and gram negative bacteria are the main known cause of this condition (Engel, C. et al, 2007).
Innate immunity also known as non-specific immunity is the first line defensive response against such an infection and the most basic resistance responsible for defending the body against infections and foreign organisms. In order to do this wholly, it has a number of characteristics which help it summon cells and use different mechanisms. Just as its name suggests, cells and mechanisms of the non-specific immune system act on pathogens in a generic way. Furthermore this branch of the immune system is unable to produce long-lasting immunity against these threats. In the case of gram negative bacterial infection, the innate immune system is activated by certain motifs found on bacteria. Once discovered it calls upon certain molecules which are able to detect and deal with these pathogens appropriately (www.pathmicro.med.sc.edu). As well as fighting foreign pathogens, the innate immune system regulates inflammatory and immune responses tightly in order to prevent overpowering inflammation and or overgrowth of invading pathogens (Norton, J. A. et al, 2008).
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Gram negative bacteria possess specific components in their cell walls that strengthen their capabilities as pathogens. The main components of their cell wall include a variable capsule, a complex lipopolysaccharide layer, a rigid membrane mucopeptide layer and a cytoplasmic membrane. During infection the most important of these constituents is the lipopolysaccharide layer (LPS) also known as the endotoxin layer; so called because its lipid fraction has the ability to act as a toxin (Engel, C. et al, 2007). This conserved bacterial motif consists of a basal portion called lipid A. Lipid A is a glucosamine-based phospholipid, it is linked by keto-deoxyoctonate to the core lipopolysaccharide. It is in fact this component of LPS that is known as the endotoxin because it is this constituent of gram negative bacteria that causes such immense consequences to the immune system. Lipid A remains the most toxic moiety of gram negative toxins produced. This constituent of LPS makes up the outer monolayer of the outer membrane of most gram-negative bacteria. The core of LPS comprises of N-acetyl glucosamine, glucose, galactose and heptose fractions (Raetz, C. R. H., 1990). Finally the terminal segment encompasses repeating oligosaccharide units known to be the cause for O-antigenetic specificity. Unlike lipid A, it was found that the O-antigen does not bring about an inflammatory response; instead it hinders the detection of lipid A (Nishitani C, 2005) which can be very dangerous, as recognition of lipid A is of great importance to the recognition of such an infection. One bacterium is known to contain approximately 2 x 106 lipid A molecules (Fig.1) and about one-quarter of the fatty acyl chains of the bacterial envelope are connected with LPS (Raetz, C. R. H., 1990).
The events leading to the activation of macrophages which in turn leads to the production and release of cytokines is thus very important in the understanding of how things work. As demonstrated by Galanos et al using chemically synthesized material, it can be seen that lipid A brings about most of the effects of endotoxins on these cells (Galanos, C. et al, 1985). The fact that lipid A has such a high potency (Raetz, C. R. H., 1990), coupled with the existence of unresponsive mutants as shown by Sibley et al (Sibley, C. H. et al., 1988), and the detection of an antagonist of endotoxin bioactivity (Takayama, K.. et al, 1989) suggested that a receptor (or receptors) for lipid A exists.
During infection with this type of bacteria, LPS is the main activator of the innate immune response. If this toxin finds its way into the blood stream, a series of events can cause host toxicity which can lead to a condition known as systemic inflammatory response syndrome (sirs) and in some serious cases gram-negative septic shock syndrome a serious condition characterized by a series of clinical conditions caused by the presence of infection which leads to a successive widespread inflammatory response and results in physiologic alterations that occur at the capillary endothelial level. The infected suffers from a sharp rise in temperature, respiration, heart rate and a sudden fall in blood pressure. A combination of these symptoms can be very severe and in some cases fatal (R.L. Paterson and N.R. Webster, 2000).
Sepsis has a similar reaction to infection; however instead of the reaction being contained in one place, its effects are on a systemic level, the consequence being wide spread endothelial dysfunction. Stage one in the development of septic shock is the presence of bacteria in the blood a condition known as bacteremia. The bacterial cells become autolysed, their outer membrane fall apart releasing lipopolysaccharide (LPS) (Baumgarten, G., et al., 2006).
As mentioned before, during gram negative bacterial infection, the provocative cause is the interaction of the host immune cells with the endotoxin LPS. In this process, LPS binds to a serum protein known as LPS binding protein (LBP) forming an LBP-LPS complex. This complex then binds unto receptors on the macrophages and causes regulatory proteins [Nuclear Factor Kappa B (NFkB)] to be activated. This complex is then assembled by the CDreceptors unto the surface of the cell, and finally the signal is translated into the cells by the TLR receptors. This response brings about the production of a number of pro-inflammatory cytokines namely; tumor necrosis factor (TNF), Interleukins 1, 6 and 12 and Interferon gamma (IFN gamma), casing a direct effect on organ function and an indirect one through the use of secondary mediators (Bosshart, H. and M. Heinzelmann, 2007).
Examples of secondary mediators called upon include, complement and platelet-activating factor. Overproduction of these pro-inflammatory cytokines can lead to the production of tissue-factor causing the deposi tion of fibrin which can in turn cause disseminated intravascular coagulation (DIC) (Bosshart, H. and M. Heinzelmann, 2007).
A major advance in our comprehension of the molecular mechanisms of septic shock is the recognition that CD14 is a receptor for LPS. Its accessory molecules and how they can come together to give a tragic result are also important in recognizing how it works. The most important component to take heed of during an infection with gram negative bacteria is CD14. CD14 is the part of the LPS receptor complex which binds ligands, it is made up of two parts namely Toll-like receptor 4 (TLR4) and the extracellular protein myeloid differentiation-2 (MD-2), (Miyake K, 2004). This receptor is a membrane bound glycosyl phosphatidylinositol surface-anchored molecule and a pattern recognition receptor expressed by myeloid cells primarily monocytes and macrophages. It is a critical part of the LPS recognition system which is able to interrelate with a variety of bacterial ligands and is able to recognize major fragments of the gram negative bacterial wall primarily lipopolysaccharide It has a two major roles, firstly it instigates an immune response finally has a fundamental role in systemic inflammation bracause it has the ability to recognize lipopolysaccharide and to a lesser extent other bacterial motifs in the cell wall of gram negative bacteria (SD Wright et al, 1990).
During infection, the first line of defense is the extraction of Lipopolysaccharide (LPS) monomers from the membranes of the bacteria. This is done by the serum protein LPS-binding protein (LBP) an acute-phase protein produced by hepatocytes in the liver as a 50-kDa single polypeptide but released as a larger 60-kDa glycosylated form (Ramadori, G., et al.1990). This protein has a very high affinity for the lipid A moiety in LPS. LPB has two main functions; firstly in the presence of lipopolyscaccharide binding protein, particles containing LPS undergo opsonisation. This process causes leukocytes to be more sensitive to LPS. Secondly, lipopolysaccharide binding protein channels LPS-coated particles to macrophages by binding to the lipid A portion of LPS and then to macrophages (Wright, S. D et al, 1989).
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LBP acts as a ligand for CD14 by transferring the LPS monomer to a lipid-binding site on CD14 in the membrane of phagocytes. Membrane-bound CD14 does not have an intracellular domain, making it incomplete on its own right. Thus it has to interrelate with other cell receptors before signal transduction takes place (Bosshart, H. and M. Heinzelmann, 2007). When LPS is recognised by CD14 the innate immune system is stimulated by TLR4. TLR4 receptors bind the foreign antigen and internalize it resulting in signal transduction and innate immune cell activation the final result being cytokine production. This couple contribute to a valuable host defense mechanism against intact gram-negative bacteria and is so effective that removal of CD14 has been found to aid an over development of a number of gram-negative pathogens in vivo as shown in knockout mice (SD Wright et al, 1990)
CD14 exists in two forms the first being a soluble protein and the second a membrane bound form. Furthermore, two isoforms of the soluble protein have been identified; one form is produced by detaching itself from the cell surface and the other is released before the glycosyl phosphatidylinositol anchor is added to cells (Labeta MO, et al, 1993).
Two further molecules come together to form a complex which is able to recognize a variety of Pathogen-Associated Molecular Patterns (PAMPs), LPS being one of them. PAMPs are relatively invariant molecular structures that the bacteria have but are not found in the host. These structures are recognized by Pattern Recognition Receptors (PRRs. PRRs are transmembrane receptors which are able to distinguish a variety of PAMPs. In the case of gram negative bacterial infection, PRRs are found on cell-surface receptors of cells. They bind the pathogen and set off a signal causing effector molecules to be released. These receptors are Toll-like receptors (TLRs). Toll-like receptors (TLRs) are vital for the regulation of innate immune responses during infection. A number of toll like receptors have been found as well as the PAMPS they are associated with (Takeda K et al, 2003).
The most important TLR in gram negative infection is TLR4 involved in the recognition of the PAMP lipopolyssacharide. With the support of accessory molecules, TLR4 specializes in the recognition of LPS. It requires MD-2 (myeloid differentiation-2) to respond efficiently to LPS. Its amino-terminal region which consists of Glu(24)-Pro(34) is critical for MD-2 binding and LPS signaling(4). This transmembrane protein contains an extracellular region made up of a protein pattern called leucine-rich repeats (LRR). LRR forms a complex with MD-2 an extracellular molecule who has a role in surface expression of TLR4 on cells as well as it’s interaction with LPS. CD14 promotes the binding of LPS to the TLR4–MD-2 complex, which signals to the cell interior. Reseasch has shown that membrane bound TLR4 is the PRR for LPS as it encourages responsiveness of cells to LPS (Nishitani C, 2005).
During gram negative infection, the TLR4–MD-2 complex recognizes gram negative bacteria and activates an effector response causing a signaling cascade which in turn causes NF-κB to be activated. NF-κB is a transcription factor which activates many cytokine genes, examples of which are tumor necrosis factor-alpha (TNF-α) gene, Interleukin-1 (IL-1) and chemokines, (molecules which cause migration of leukocytes to the site of infection), these molecules are all known to cause inflammation at the site of infection. NF-κB is found in the cytosol of cells where it is bound to IκB its inhibitor. Binding of ligands to the receptor causes IκB to be phosphorylation and destroyed. NF-κB can then move into the nucleus where the genes required are activated. Genes encoding IL-1 and other cytokines are turned on by this effector molecule resulting in inflammation and other cell precesses such as processes such as cell adhesion cell proliferation, and angiogenesis (http://users.rcn.com/).
The TLR4-MD-2 complex plays an important role in suppressing Gram-negative bacterial infection by activating innate immune responses (Engel, C. et al, 2007). Even though TLR4-MD-2 recognizes LPS, not much is known about the physical interaction between LPS and TLR4-MD-2. It is known that CD14 significantly enhances the formation of LPS-TLR4-MD-2 complexes by loading LPS onto TLR4-MD-2 complexes. In the absence of CD14, the TLR4–MD-2 complex can still function with some forms of LPS in the presence of high concentrations of LPS (Nishitani C, 2005).
The effect that the presence of endotoxins brings on the immune system is not as important as the effect which overproduction of cytokines has on the host. The latter caused by over reaction of the hosts immune system is what brings about such dire consequences. Prolonged harm to individual organ systems results in multiple organ failure, transitioning into the final stage known as refractory septic shock. Past experiments have shown that protein C levels are low during sepsis. Protein C plays a vital role in the inhibition of coagulation. Low levels thus suggest that during sepsis protein C is inhibited causing coagulation to take place on a systematic level.
The collective consequence of such a cascade is an imbalanced state, where inflammation prevails over anti-inflammation and coagulation prevails over fibrinolysis. The end result being conditions such as ischemia, and high scale tissue destruction; severe sepsis, shock, and multiple organ failure may follow which could eventually lead to death.
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