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121-503: The term Type III secretion system was coined in 1993. This secretion system is distinguished from at least five other secretion systems found in gram-negative bacteria . Many animal and plant associated bacteria possess similar T3SSs. These T3SSs are similar as a result of convergent evolution and phylogenetic analysis supports a model in which gram-negative bacteria can transfer the T3SS gene cassette horizontally to other species. Some of

242-566: A chelator such as EDTA or EGTA ) and by adding the aromatic dye Congo red to the growth medium (for Shigella ), for instance. These methods and other are used in laboratories to artificially induce type III secretion. Induction of secretion by external cues other than contact with host cells also takes place in vivo , in infected organisms. The bacteria sense such cues as temperature , pH , osmolarity and oxygen levels, and use them to "decide" whether to activate their T3SS. For instance, Salmonella can replicate and invade better in

363-477: A brisk host response and multiple types of acute organ failure which can lead to septic shock . In lower levels and over a longer time period, there is evidence LPS may play an important and harmful role in autoimmunity , obesity , depression , and cellular senescence . The toxic activity of LPS was first discovered and termed endotoxin by Richard Friedrich Johannes Pfeiffer . He distinguished between exotoxins , toxins that are released by bacteria into

484-402: A broader physiological role in defense against simple eukaryotic predators and its role in inter-bacteria interactions. The Type VI secretion system gene clusters contain from 15 to more than 20 genes, two of which, Hcp and VgrG, have been shown to be nearly universally secreted substrates of the system. Structural analysis of these and other proteins in this system bear a striking resemblance to

605-419: A challenge for many years. By the end of the 1990s, however, several approaches have been developed for the isolation of T3SS NCs. In 1998 the first NCs were isolated from Salmonella typhimurium . For the isolation, bacteria are grown in a large volume of liquid growth medium until they reach log phase . They are then centrifuged ; the supernatant (the medium) is discarded and the pellet (the bacteria)

726-439: A characteristic which may help in the evasion of bovine host immune defenses. Taken together, these observations suggest that variations in bacterial surface molecules such as LOS can help the pathogen evade both the humoral (antibody and complement-mediated) and the cell-mediated (killing by neutrophils, for example) host immune defenses. Recently, it was shown that in addition to TLR4 mediated pathways, certain members of

847-404: A column coated with particles with high affinity to the tag (in the case of histidine tags: nickel ions ). The tagged protein is retained in the column, and with it the entire needle complex. High degrees of purity can be achieved using such methods. This purity is essential for many delicate assays that have been used for NC characterization. Type III effectors were known since the beginning of

968-492: A common structural architecture. Lipopolysaccharides ( LPS ) are large molecules consisting of three parts: an outer core polysaccharide termed the O- antigen , an inner core oligosaccharide and Lipid A (from which toxicity is largely derived), all covalently linked. In current terminology, the term endotoxin is often used synonymously with LPS, although there are a few endotoxins (in the original sense of toxins that are inside

1089-510: A complete picture of the NC proteome . The T3SS in many bacteria has been manipulated by researchers. Observing the influence of individual manipulations can be used to draw insights into the role of each component of the system. Examples of manipulations are: Manipulation of T3SS components can have influence on several aspects of bacterial function and pathogenicity. Examples of possible influences: A few compounds have been discovered that inhibit

1210-437: A complex microbial flora (including gram-negative bacteria), which outnumber human cells by a factor of 10 to 1. Gram-negative bacterial will shed endotoxins. This host-microbial interaction is a symbiotic relationship which plays a critical role in systemic immunologic homeostasis. When this is disrupted, it can lead to disease such as endotoxemia and endotoxic septic shock. LPS acts as the prototypical endotoxin because it binds

1331-680: A dose up to a thousand times higher. This may relate to differences in the level of circulating natural antibodies between the two species. It may also be linked to multiple immune tactics against pathogens, and part of a multi-faceted anti-microbial strategy that has been informed by human behavioral changes over our species' evolution (e.g., meat eating, agricultural practices, and smoking). Said et al. showed that LPS causes an IL-10 -dependent inhibition of CD4 T-cell expansion and function by up-regulating PD-1 levels on monocytes which leads to IL-10 production by monocytes after binding of PD-1 by PD-L1 . Endotoxins are in large part responsible for

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1452-503: A general response to stress conditions, the process of loading cargo proteins seems to be selective. In some Staphylococcus and Streptococcus species, the accessory secretory system handles the export of highly repetitive adhesion glycoproteins. Lipopolysaccharide Lipopolysaccharide , now more commonly known as endotoxin , is a collective term for components of the outermost membrane of cell envelope of gram-negative bacteria, such as E. coli and Salmonella with

1573-471: A highly evolved process of secretion. Proteins targeted for the outside are synthesized by ribosomes docked to the rough endoplasmic reticulum (ER). As they are synthesized, these proteins translocate into the ER lumen , where they are glycosylated and where molecular chaperones aid protein folding . Misfolded proteins are usually identified here and retrotranslocated by ER-associated degradation to

1694-583: A human patient. The presumed mechanism for the association of endotoxin with obesity is that endotoxin induces an inflammation-mediated pathway accounting for the observed obesity and insulin resistance. Bacterial genera associated with endotoxin-related obesity effects include Escherichia and Enterobacter . There is experimental and observational evidence that LPS might play a role in depression. Administration of LPS in mice can lead to depressive symptoms, and there seem to be elevated levels of LPS in some people with depression. Inflammation may sometimes play

1815-421: A lipopolysaccharide-rich lipid bilayer enclosing periplasmic materials, and are deployed for membrane vesicle trafficking to manipulate environment or invade at host–pathogen interface . Vesicles from a number of bacterial species have been found to contain virulence factors, some have immunomodulatory effects, and some can directly adhere to and intoxicate host cells. release of vesicles has been demonstrated as

1936-411: A molecule called lipid A-Kdo2, which is first created on the surface of the bacterial cell's inner membrane. Then, additional sugars are added to this molecule on the inner membrane before it's moved to the space between the inner and outer membranes ( periplasmic space ) with the help of a protein called MsbA. The O-antigen, another part of LPS, is made by special enzyme complexes on the inner membrane. It

2057-534: A negative cue to the bacteria and inhibit secretion. Cholesterol , a lipid found in most eukaryotic cell membranes, is able to induce secretion in Shigella . The external cues listed above either regulate secretion directly or through a genetic mechanism. Several transcription factors that regulate the expression of T3SS genes are known. Some of the chaperones that bind T3SS effectors also act as transcription factors. A feedback mechanism has been suggested: when

2178-493: A pore or a channel (a translocon ) in the host cell membrane, through which other effectors may enter. Mutated bacteria that lack translocators are able to secrete proteins but are not able to deliver them into host cells. In general each T3SS includes three translocators. Some translocators serve a double role; after they participate in pore formation they enter the cell and act as bona fide effectors. T3SS effectors manipulate host cells in several ways. The most striking effect

2299-482: A potent activator and modulator of the immune system, especially its inducement of inflammation. LPS is directly cytoxic and is highly immunostimulatory – as host immune cells recognize LPS, complement are strongly activated. Complement activation and a rising anti-inflammatory response can lead to immune cell dysfunction, immunosuppression, widespread coagulopathy, serious tissue damage and can progress to multi-system organ failure and death. The presence of endotoxins in

2420-689: A proliferative stress, leading to impair their competitive repopulating ability. Infection in mice using S. typhimurium showed similar results, validating the experimental model also in vivo . O-antigens (the outer carbohydrates) are the most variable portion of the LPS molecule, imparting antigenic specificity. In contrast, lipid A is the most conserved part. However, lipid A composition also may vary (e.g., in number and nature of acyl chains even within or between genera). Some of these variations may impart antagonistic properties to these LPS. For example, diphosphoryl lipid A of Rhodobacter sphaeroides (RsDPLA)

2541-425: A result are able to increase their resistance to complement -mediated killing or even down-regulate complement activation or evade the effects of bactericidal antibodies. Sialylation may also contribute to hindered neutrophil attachment and phagocytosis by immune system cells as well as a reduced oxidative burst. Haemophilus somnus , a pathogen of cattle, has also been shown to display LOS phase variation,

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2662-455: A result of increased populations of endotoxin-producing bacteria in the intestinal tract, is associated with certain obesity-related patient groups. Other studies have shown that purified endotoxin from Escherichia coli can induce obesity and insulin-resistance when injected into germ-free mouse models . A more recent study has uncovered a potentially contributing role for Enterobacter cloacae B29 toward obesity and insulin resistance in

2783-408: A right-handed helical assembly with roughly 11 subunits per two turns, similar to that of the flagellum of Salmonella typhimurium . The model also revealed an extended amino-terminal domain that is positioned on the surface of the needle, while the highly conserved carboxy terminus points towards the lumen. Several methods have been employed in order to identify the array of proteins that comprise

2904-407: A role in the development of depression, and LPS is pro-inflammatory. Inflammation induced by LPS can induce cellular senescence , as has been shown for the lung epithelial cells and microglial cells (the latter leading to neurodegeneration ). Lipopolysaccharides are frequent contaminants in plasmid DNA prepared from bacteria or proteins expressed from bacteria, and must be removed from

3025-458: A secreted chemical substance from a cell or gland . In contrast, excretion is the removal of certain substances or waste products from a cell or organism. The classical mechanism of cell secretion is via secretory portals at the plasma membrane called porosomes . Porosomes are permanent cup-shaped lipoprotein structures embedded in the cell membrane, where secretory vesicles transiently dock and fuse to release intra-vesicular contents from

3146-497: A simple code and this has greatly improved the understanding of how these proteins can alter the transcription of genes in the host plant cells. Hundreds of articles on T3SS have been published since the mid-nineties. However, numerous issues regarding the system remain unresolved: Since the beginning of the 1990s new T3SS proteins are being found in different bacterial species at a steady rate. Abbreviations have been given independently for each series of proteins in each organism, and

3267-607: A type IV secretion system to deliver CagA into gastric epithelial cells, which is associated with gastric carcinogenesis. Bordetella pertussis , the causative agent of whooping cough, secretes the pertussis toxin partly through the type IV system. Legionella pneumophila , the causing agent of legionellosis (Legionnaires' disease) utilizes a type IVB secretion system , known as the icm/dot ( i ntra c ellular m ultiplication / d efect in o rganelle t rafficking genes) system, to translocate numerous effector proteins into its eukaryotic host. The prototypic Type IVA secretion system

3388-435: Is a component of the cytoskeleton and it also participates in motility and in changes in cell shape. Through its T3SS effectors the bacterium is able to utilize the host cell's own machinery for its own benefit. Once the bacterium has entered the cell it is able to secrete other effectors more easily and it can penetrate neighboring cells and quickly infect the whole tissue . T3SS effectors have also been shown to tamper with

3509-888: Is a potent antagonist of LPS in human cells, but is an agonist in hamster and equine cells. It has been speculated that conical lipid A (e.g., from E. coli ) is more agonistic, while less conical lipid A like that of Porphyromonas gingivalis may activate a different signal ( TLR2 instead of TLR4), and completely cylindrical lipid A like that of Rhodobacter sphaeroides is antagonistic to TLRs. In general, LPS gene clusters are highly variable between different strains, subspecies, species of bacterial pathogens of plants and animals. Normal human blood serum contains anti-LOS antibodies that are bactericidal and patients that have infections caused by serotypically distinct strains possess anti-LOS antibodies that differ in their specificity compared with normal serum. These differences in humoral immune response to different LOS types can be attributed to

3630-690: Is a powerful toxin that, when in the body, triggers inflammation by binding to cell receptors. Excessive LPS in the blood, endotoxemia, may cause a highly lethal form of sepsis known as endotoxic septic shock. This condition includes symptoms that fall along a continuum of pathophysiologic states, starting with a systemic inflammatory response syndrome (SIRS) and ending in multiorgan dysfunction syndrome (MODS) before death. Early symptoms include rapid heart rate, quick breathing, temperature changes, and blood clotting issues, resulting in blood vessels widening and reduced blood volume, leading to cellular dysfunction. Recent research indicates that even small LPS exposure

3751-417: Is a summary of the most common protein-series names in several T3SS-containing species. Note that these names include proteins that form the T3SS machinery as well as the secreted effector proteins : Following those abbreviations is a letter or a number. Letters usually denote a "serial number", either the chronological order of discovery or the physical order of appearance of the gene in an operon . Numbers,

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3872-416: Is a very conserved component of the LPS. However Lipid A structure varies among bacterial species. Lipid A structure largely defines the degree and nature of the overall host immune activation. The "rough form" of LPS has a lower molecular weight due to the absence of the O polysaccharide. In its place is a short oligosaccharide: this form is known as Lipooligosaccharide (LOS), and is a glycolipid found in

3993-552: Is a widely used extracorporeal endotoxin removal therapy through direct hemoadsorption (also referred to as hemoperfusion). It is a polystyrene-derived cartridge with molecules of polymyxin B (PMX-B) covalently bound to mesh fibers contained within it. Polymyxins are cyclic cationic polypeptide antibiotics derived from Bacillus polymyxa with an effective antimicrobial activity against Gram-negative bacteria, but their intravenous clinical use has been limited due to their nephrotoxicity and neurotoxicity side effects. The extracorporeal use of

4114-510: Is able to prevent these changes in animals that lack AOAH. Dephosphorylation of LPS by intestinal alkaline phosphatase can reduce the severity of Salmonella tryphimurium and Clostridioides difficile infection restoring normal gut microbiota. Alkaline phosphatase prevents intestinal inflammation (and " leaky gut ") from bacteria by dephosphorylating the Lipid ;A portion of LPS. The entire process of making LPS starts with

4235-411: Is also required for the functioning of omptins , a class of bacterial protease. LPS are amphipathic and composed of three parts: the O antigen (or O polysaccharide) which is hydrophilic, the core oligosaccharide (also hydrophilic), and Lipid A , the hydrophobic domain. The repetitive glycan polymer contained within an LPS is referred to as the O antigen , O polysaccharide , or O side-chain of

4356-463: Is associated with autoimmune diseases and allergies. High levels of LPS in the blood can lead to metabolic syndrome, increasing the risk of conditions like diabetes, heart disease, and liver problems. LPS also plays a crucial role in symptoms caused by infections from harmful bacteria, including severe conditions like Waterhouse-Friderichsen syndrome, meningococcemia, and meningitis. Certain bacteria can adapt their LPS to cause long-lasting infections in

4477-412: Is crucial for promptly managing and treating infections. Since LPS is the main trigger for the immune response in our cells, it acts as an early signal of an acute infection. Therefore, LPS testing is more specific and meaningful than many other serological tests. The current methods for testing LPS are quite sensitive, but many of them struggle to differentiate between different LPS groups. Additionally,

4598-447: Is encoded on Gram-negative conjugative elements in bacteria . T4SS are cell envelope-spanning complexes, or, in other words, 11–13 core proteins that form a channel through which DNA and proteins can travel from the cytoplasm of the donor cell to the cytoplasm of the recipient cell. T4SS also secrete virulence factor proteins directly into host cells as well as taking up DNA from the medium during natural transformation . As shown in

4719-575: Is excreted outside of the outer membrane via a long-tunnel protein channel. Type I secretion system transports various molecules, from ions, drugs, to proteins of various sizes (20 – 900 kDa). The molecules secreted vary in size from the small Escherichia coli peptide colicin V, (10 kDa) to the Pseudomonas fluorescens cell adhesion protein LapA of 520 kDa. The best characterized are the RTX toxins and

4840-422: Is less variable in its structure and composition, a given core structure being common to large groups of bacteria. The LPS cores of many bacteria also contain non-carbohydrate components, such as phosphate, amino acids, and ethanolamine substituents. Lipid A is, in normal circumstances, a phosphorylated glucosamine disaccharide decorated with multiple fatty acids . These hydrophobic fatty acid chains anchor

4961-410: Is like a molecular syringe through which a bacterium (e.g. certain types of Salmonella , Shigella , Yersinia , Vibrio ) can inject proteins into eukaryotic cells. The low Ca concentration in the cytosol opens the gate that regulates T3SS. One such mechanism to detect low calcium concentration has been illustrated by the lcrV (Low Calcium Response) antigen utilized by Yersinia pestis , which

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5082-476: Is presumed to be built from bottom to top; units of needle monomer protein pile upon each other, so that the unit at the tip of the needle is the last one added. The needle subunit is one of the smallest T3SS proteins, measuring at around 9 k Da . 100−150 subunits comprise each needle. The T3SS needle measures around 60−80 nm in length and 8 nm in external width. It needs to have a minimal length so that other extracellular bacterial structures ( adhesins and

5203-643: Is primarily mediated by Toll-like receptor 4 which is responsible for immune system cell activation. Damage to the endothelial layer of blood vessels caused by these inflammatory mediators can lead to capillary leak syndrome , dilation of blood vessels and a decrease in cardiac function and can further worsen shock. LPS is also a potent activator of complemen. Uncontrolled complement activation may trigger destructive endothelial damage leading to disseminated intravascular coagulation (DIC), or atypical hemolytic uremic syndrome (aHUS) with injury to various organs such as including kidneys and lungs. The skin can show

5324-410: Is resuspended in a lysis buffer typically containing lysozyme and sometimes a detergent such as LDAO or Triton X-100 . This buffer disintegrates the cell wall . After several rounds of lysis and washing, the opened bacteria are subjected to a series of ultracentrifugations . This treatment enriches large macromolecular structures and discards smaller cell components. Optionally, the final lysate

5445-416: Is subjected to further purification by CsCl density gradient . An additional approach for further purification uses affinity chromatography . Recombinant T3SS proteins that carry a protein tag (a histidine tag , for instance) are produced by molecular cloning and then introduced ( transformed ) into the researched bacteria. After initial NC isolation, as described above, the lysate is passed through

5566-614: Is the Limulus Amebocyte Lysate (LAL) assay, utilizing blood from the Horseshoe crab ( Limulus polyphemus ). Very low levels of LPS can cause coagulation of the limulus lysate due to a powerful amplification through an enzymatic cascade. However, due to the dwindling population of horseshoe crabs, and the fact that there are factors that interfere with the LAL assay, efforts have been made to develop alternative assays, with

5687-501: Is the VirB complex of Agrobacterium tumefaciens . Protein members of this family are components of the type IV secretion system. They mediate intracellular transfer of macromolecules via a mechanism ancestrally related to that of bacterial conjugation machineries. The Type IV secretion system (T4SS) is the general mechanism by which bacterial cells secrete or take up macromolecules. Their precise mechanism remains unknown. T4SS

5808-401: Is the promoting of uptake of the bacterium by the host cell. Many bacteria possessing T3SSs must enter host cells in order to replicate and propagate infection. The effectors they inject into the host cell induce the host to engulf the bacterium and to practically "eat" it. In order for this to happen the bacterial effectors manipulate the actin polymerization machinery of the host cell. Actin

5929-463: Is then moved to the outer membrane through three different systems: one is Wzy-dependent, another relies on ABC transporters, and the third involves a synthase-dependent process. Ultimately, LPS is transported to the outer membrane by a membrane-to-membrane bridge of lipolysaccharide transport (Lpt) proteins. This transporter is a potential antibiotic target. The human body carries endogenous stores on LPS. The epithelial surfaces are colonized by

6050-467: Is used to detect low calcium concentrations and elicits T3SS attachment. The Hrp system in plant pathogens inject harpins and pathogen effector proteins through similar mechanisms into plants. This secretion system was first discovered in Yersinia pestis and showed that toxins could be injected directly from the bacterial cytoplasm into the cytoplasm of its host's cells rather than simply be secreted into

6171-513: The CD14 / TLR4 / MD2 receptor complex in many cell types, but especially in monocytes , dendritic cells , macrophages and B cells , which promotes the secretion of pro- inflammatory cytokines , nitric oxide , and eicosanoids . Bruce Beutler was awarded a portion of the 2011 Nobel Prize in Physiology or Medicine for his work demonstrating that TLR4 is the LPS receptor. As part of

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6292-485: The Sec system for crossing the inner membrane. Proteins which use this pathway have the capability to form a beta-barrel with their C-terminus which inserts into the outer membrane, allowing the rest of the peptide (the passenger domain) to reach the outside of the cell. Often, autotransporters are cleaved, leaving the beta-barrel domain in the outer membrane and freeing the passenger domain. Some researchers believe remnants of

6413-517: The cytosol , where they are degraded by a proteasome . The vesicles containing the properly folded proteins then enter the Golgi apparatus . In the Golgi apparatus, the glycosylation of the proteins is modified and further post-translational modifications , including cleavage and functionalization, may occur. The proteins are then moved into secretory vesicles which travel along the cytoskeleton to

6534-443: The host-pathogen interface , is considered to be an important factor in the development of alcoholic hepatitis, which is likely to develop on the basis of the small bowel bacterial overgrowth syndrome and an increased intestinal permeability . Lipid A may cause uncontrolled activation of mammalian immune systems with production of inflammatory mediators that may lead to endotoxic septic shock . This inflammatory reaction

6655-421: The ileum rather than in the cecum of animal intestine . The bacteria are able to know where they are thanks to the different ions present in these regions; the ileum contains formate and acetate , while the cecum does not. The bacteria sense these molecules, determine that they are at the ileum and activate their secretion machinery. Molecules present in the cecum, such as propionate and butyrate , provide

6776-523: The lipopolysaccharide layer, for instance) do not interfere with secretion. The hole of the needle has a 3 nm diameter. Most folded effector proteins are too large to pass through the needle opening, so most secreted proteins must pass through the needle unfolded , a task carried out by the ATPase at the base of the structure. The T3SS proteins can be grouped into three categories: Most T3SS genes are laid out in operons . These operons are located on

6897-506: The 1990s, but the way in which they are delivered into host cells was a complete mystery. The homology between many flagellar and T3SS proteins led researchers to suspects the existence of an outer T3SS structure similar to flagella. The identification and subsequent isolation of the needle structure enabled researchers to: As with almost all proteins, the visualization of T3SS NCs is only possible with electron microscopy . The first images of NCs (1998) showed needle structures protruding from

7018-516: The DNA or protein to avoid contaminating experiments and to avoid toxicity of products manufactured using industrial fermentation . Ovalbumin is frequently contaminated with endotoxins. Ovalbumin is one of the extensively studied proteins in animal models and also an established model allergen for airway hyper-responsiveness (AHR). Commercially available ovalbumin that is contaminated with LPS can falsify research results, as it does not accurately reflect

7139-528: The Hcp and VrgG genes in Vibrio cholerae led to decreased virulence and pathogenicity. Since then, Type VI secretion systems have been found in a quarter of all proteobacterial genomes, including animal, plant, human pathogens, as well as soil, environmental or marine bacteria. While most of the early studies of Type VI secretion focused on its role in the pathogenesis of higher organisms, more recent studies suggested

7260-519: The Hly and Tol gene clusters. The process begins as a leader sequence on the protein to be secreted is recognized by HlyA and binds HlyB on the membrane. This signal sequence is extremely specific for the ABC transporter. The HlyAB complex stimulates HlyD which begins to uncoil and reaches the outer membrane where TolC recognizes a terminal molecule or signal on HlyD. HlyD recruits TolC to the inner membrane and HlyA

7381-453: The LPS into the bacterial membrane, and the rest of the LPS projects from the cell surface. The lipid A domain is the most bioactive and responsible for much of the toxicity of Gram-negative bacteria . When bacterial cells are lysed by the immune system , fragments of membrane containing lipid A may be released into the circulation, causing fever, diarrhea, and possible fatal endotoxic septic shock (a form of septic shock ). The Lipid A moiety

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7502-707: The LPS is considered "rough" or "smooth". Full-length O-chains would render the LPS smooth, whereas the absence or reduction of O-chains would make the LPS rough. Bacteria with rough LPS usually have more penetrable cell membranes to hydrophobic antibiotics, since a rough LPS is more hydrophobic . O antigen is exposed on the very outer surface of the bacterial cell, and, as a consequence, is a target for recognition by host antibodies . The core domain always contains an oligosaccharide component that attaches directly to lipid A and commonly contains sugars such as heptose and 3-Deoxy-D-manno-oct-2-ulosonic acid (also known as KDO, keto-deoxyoctulosonate). The core oligosaccharide

7623-590: The LPS is very difficult to measure in whole blood and because most LPS is bound to proteins and complement, the Endotoxin Activity Assay (EAA™) was developed and cleared by the US FDA in 2003. EAA is a rapid in vitro chemiluminescent immunodiagnostic test. It utilizes a specific monoclonal antibody to measure the endotoxin activity in EDTA whole blood specimens. This assay uses the biological response of

7744-452: The T3SS in gram-negative bacteria , including the guadinomines which are naturally produced by Streptomyces species. Monoclonal antibodies have been developed that inhibit the T3SS too. Aurodox , an antibiotic capable of inhibiting the translation of T3SS proteins has been shown to able to prevent T3SS effectors in vitro and in animal models Secretion Secretion is the movement of material from one point to another, such as

7865-403: The T3SS, is made out of many units of a single protein. The majority of the different T3SS proteins are therefore those that build the base and those that are secreted into the host. As mentioned above, the needle complex shares similarities with bacterial flagella. More specifically, the base of the needle complex is structurally very similar to the flagellar base; the needle itself is analogous to

7986-548: The T3SS. Isolated needle complexes can be separated with SDS-PAGE . The bands that appear after staining can be individually excised from the gel and analyzed using protein sequencing and mass spectrometry . The structural components of the NC can be separated from each other (the needle part from the base part, for instance), and by analyzing those fractions the proteins participating in each one can be deduced. Alternatively, isolated NCs can be directly analyzed by mass spectrometry, without prior electrophoresis , in order to obtain

8107-580: The Toraymyxin cartridge allows PMX-B to bind lipid A with a very stable interaction with its hydrophobic residues thereby neutralizing endotoxins as the blood is filtered through the extracorporeal circuit inside the cartridge, thus reversing endotoxemia and avoiding its toxic systemic effects. The molecular mimicry of some LOS molecules is thought to cause autoimmune-based host responses, such as flareups of multiple sclerosis . Other examples of bacterial mimicry of host structures via LOS are found with

8228-485: The ability of some bacterial strains to display molecular mimicry and antigenic diversity , aiding in the evasion of host immune defenses and thus contributing to the virulence of these bacterial strains . In the case of Neisseria meningitidis , the lipid A portion of the molecule has a symmetrical structure and the inner core is composed of 3-deoxy-D-manno-2-octulosonic acid (KDO) and heptose (Hep) moieties. The outer core oligosaccharide chain varies depending on

8349-537: The abolishment of immune tolerance when infecting hosts with certain human leukocyte antigen (HLA) genotypes, such as HLA-B35 . LPS can be sensed directly by hematopoietic stem cells (HSCs) through the bonding with TLR4, causing them to proliferate in reaction to a systemic infection. This response activate the TLR4-TRIF-ROS-p38 signaling within the HSCs and through a sustained TLR4 activation can cause

8470-434: The above figure, TraC, in particular consists of a three helix bundle and a loose globular appendage. T4SS has two effector proteins: firstly, ATS-1, which stands for Anaplasma translocated substrate 1, and secondly AnkA , which stands for ankyrin repeat domain-containing protein A. Additionally, T4SS coupling proteins are VirD4, which bind to VirE2. Also called the autotransporter system, type V secretion involves use of

8591-631: The autotransporters gave rise to the porins which form similar beta-barrel structures. A common example of an autotransporter that uses this secretion system is the Trimeric Autotransporter Adhesins . Type VI secretion systems were originally identified in 2006 by the group of John Mekalanos at the Harvard Medical School (Boston, USA) in two bacterial pathogens, Vibrio cholerae and Pseudomonas aeruginosa . These were identified when mutations in

8712-504: The bacteria Helicobacter pylori and Campylobacter jejuni , organisms which cause gastrointestinal disease in humans, and Haemophilus ducreyi which causes chancroid . Certain C. jejuni LPS serotypes (attributed to certain tetra- and pentasaccharide moieties of the core oligosaccharide) have also been implicated with Guillain–Barré syndrome and a variant of Guillain–Barré called Miller-Fisher syndrome . Epidemiological studies have shown that increased endotoxin load, which can be

8833-410: The bacteria possessing a T3SS have flagella as well and are motile ( Salmonella , for instance), and some do not ( Shigella , for instance). Technically speaking, type III secretion is used both for secreting infection-related proteins and flagellar components. However, the term "type III secretion" is used mainly in relation to the infection apparatus. The bacterial flagellum shares a common ancestor with

8954-433: The bacteria. The O antigen is attached to the core oligosaccharide, and comprises the outermost domain of the LPS molecule. The structure and composition of the O chain is highly variable from strain to strain, determining the serological specificity of the parent bacterial strain; there are over 160 different O antigen structures produced by different E. coli strains. The presence or absence of O chains determines whether

9075-514: The bacterial cytoplasm through the needle directly into the host cytoplasm. Three membranes separate the two cytoplasms: the double membranes (inner and outer membranes) of the Gram-negative bacterium and the eukaryotic membrane. The needle provides a smooth passage through those highly selective and almost impermeable membranes. A single bacterium can have several hundred needle complexes spread across its membrane. It has been proposed that

9196-758: The bacterial strain . A highly conserved host enzyme called acyloxyacyl hydrolase (AOAH) may detoxify LPS when it enters, or is produced in, animal tissues. It may also convert LPS in the intestine into an LPS inhibitor. Neutrophils, macrophages and dendritic cells produce this lipase, which inactivates LPS by removing the two secondary acyl chains from lipid A to produce tetraacyl LPS. If mice are given LPS parenterally, those that lack AOAH develop high titers of non-specific antibodies, develop prolonged hepatomegaly, and experience prolonged endotoxin tolerance. LPS inactivation may be required for animals to restore homeostasis after parenteral LPS exposure. Although mice have many other mechanisms for inhibiting LPS signaling, none

9317-436: The bacterial cell that are released when the cell disintegrates) that are not related to LPS, such as the so-called delta endotoxin proteins produced by Bacillus thuringiensis . Lipopolysaccharides can have substantial impacts on human health, primarily through interactions with the immune system. LPS is a potent activator of the immune system and is a pyrogen (agent that causes fever). In severe cases, LPS can trigger

9438-471: The bacterial chromosome in some species and on a dedicated plasmid in other species. Salmonella , for instance, has a chromosomal region in which most T3SS genes are gathered, the so-called Salmonella pathogenicity island ( SPI ). Shigella , on the other hand, has a large virulence plasmid on which all T3SS genes reside. It is important to note that many pathogenicity islands and plasmids contain elements that allow for frequent horizontal gene transfer of

9559-674: The bacterium does not secrete, its effector proteins are bound to chaperones and float in the cytoplasm. When secretion starts, the chaperones detach from the effectors and the latter are secreted and leave the cell. The lone chaperones then act as transcription factors, binding to the genes encoding their effectors and inducing their transcription and thereby the production of more effectors. Structures similar to Type3SS injectisomes have been proposed to rivet gram negative bacterial outer and inner membranes to help release outer membrane vesicles targeted to deliver bacterial secretions to eukaryotic host or other target cells in vivo. T3SS effectors enter

9680-423: The blood is called endotoxemia. High level of endotoxemia can lead to septic shock , or more specifically endotoxic septic shock, while lower concentration of endotoxins in the bloodstream is called metabolic endotoxemia. Endotoxemia is associated with obesity, diet, cardiovascular diseases, and diabetes, while also host genetics might have an effect. Moreover, endotoxemia of intestinal origin, especially, at

9801-414: The cell wall of live bacteria and flat, two-dimensional isolated NCs. In 2001 images of NCs from Shigella flexneri were digitally analyzed and averaged to obtain a first semi-3D structure of the NC. The helical structure of NCs from Shigella flexneri was resolved at a resolution of 16 Å using X-ray fiber diffraction in 2003, and a year later a 17- Å 3D structure of NCs from Salmonella typhimurium

9922-519: The cell. Secretion in bacterial species means the transport or translocation of effector molecules. For example: proteins , enzymes or toxins (such as cholera toxin in pathogenic bacteria e.g. Vibrio cholerae ) from across the interior ( cytoplasm or cytosol ) of a bacterial cell to its exterior. Secretion is a very important mechanism in bacterial functioning and operation in their natural surrounding environment for adaptation and survival. Eukaryotic cells , including human cells , have

10043-616: The cellular stress response , superoxide is one of the major reactive oxygen species induced by LPS in various cell types that express TLR ( toll-like receptor ). LPS is also an exogenous pyrogen (fever-inducing substance). LPS function has been under experimental research for several years due to its role in activating many transcription factors . LPS also produces many types of mediators involved in septic shock . Of mammals, humans are much more sensitive to LPS than other primates, and other animals as well (e.g., mice). A dose of 1 μg/kg induces shock in humans, but mice will tolerate

10164-458: The crystal structure of MixH from Shigella flexneri , which were both resolved in 2006. In 2012, a combination of recombinant wild-type needle production, solid-state NMR , electron microscopy and Rosetta modeling revealed the supramolecular interfaces and ultimately the complete atomic structure of the Salmonella typhimurium T3SS needle. It was shown that the 80-residue PrgI subunits form

10285-504: The dramatic clinical manifestations of infections with pathogenic Gram-negative bacteria, such as Neisseria meningitidis , the pathogens that causes meningococcal disease , including meningococcemia , Waterhouse–Friderichsen syndrome , and meningitis . Portions of the LPS from several bacterial strains have been shown to be chemically similar to human host cell surface molecules; the ability of some bacteria to present molecules on their surface which are chemically identical or similar to

10406-414: The edge of the cell. More modification can occur in the secretory vesicles (for example insulin is cleaved from proinsulin in the secretory vesicles). Eventually, there is vesicle fusion with the cell membrane at porosomes, by a process called exocytosis , dumping its contents out of the cell's environment. Strict biochemical control is maintained over this sequence by usage of a pH gradient:

10527-421: The effect of the protein antigen on animal physiology. In pharmaceutical production, it is necessary to remove all traces of endotoxin from drug product containers, as even small amounts of endotoxin will cause illness in humans. A depyrogenation oven is used for this purpose. Temperatures in excess of 300 °C are required to fully break down LPS. The standard assay for detecting presence of endotoxin

10648-595: The effects of vascular damage often coupled with depletion of coagulation factors in the form of petechiae , purpura and ecchymoses . The limbs can also be affected, sometimes with devastating consequences such as the development of gangrene , requiring subsequent amputation . Loss of function of the adrenal glands can cause adrenal insufficiency and additional hemorrhage into the adrenals causes Waterhouse-Friderichsen syndrome , both of which can be life-threatening. It has also been reported that gonococcal LOS can cause damage to human fallopian tubes . Toraymyxin

10769-573: The extracellular medium. It is homologous to conjugation machinery of bacteria, the conjugative pili . It is capable of transporting both DNA and proteins. It was discovered in Agrobacterium tumefaciens , which uses this system to introduce the T-DNA portion of the Ti plasmid into the plant host, which in turn causes the affected area to develop into a crown gall (tumor). Helicobacter pylori uses

10890-399: The family of the transient receptor potential ion channels recognize LPS. LPS-mediated activation of TRPA1 was shown in mice and Drosophila melanogaster flies. At higher concentrations, LPS activates other members of the sensory TRP channel family as well, such as TRPV1 , TRPM3 and to some extent TRPM8 . LPS is recognized by TRPV4 on epithelial cells. TRPV4 activation by LPS

11011-431: The flagellar hook, a structure connecting the base to the flagellar filament. The base is composed of several circular rings and is the first structure that is built in a new needle complex. Once the base is completed, it serves as a secretion machine for the outer proteins (the needle). Once the whole complex is completed the system switches to secreting proteins that are intended to be delivered into host cells. The needle

11132-448: The form of bacterial outer membrane vesicles (OMVs) , which may also contain other virulence factors and proteins. LPS is a major component of the outer cell membrane of gram-negative bacteria , contributing greatly to the structural integrity of the bacteria and protecting the membrane from certain kinds of chemical attack. LPS is the most abundant antigen on the cell surface of most gram-negative bacteria, contributing up to 80% of

11253-458: The host's cell cycle and some of them are able to induce apoptosis . One of the most researched T3SS effector is IpaB from Shigella flexneri . It serves a double role, both as a translocator, creating a pore in the host cell membrane, and as an effector, exerting multiple detrimental effects on the host cell. It had been demonstrated that IpaB induces apoptosis in macrophages —cells of the animal immune system —after being engulfed by them. It

11374-459: The island/plasmid to a new species. Effector proteins that are to be secreted through the needle need to be recognized by the system, since they float in the cytoplasm together with thousands of other proteins. Recognition is done through a secretion signal —a short sequence of amino acids located at the beginning (the N-terminus ) of the protein (usually within the first 20 amino acids), that

11495-503: The lipases. Type I secretion is also involved in export of non-proteinaceous substrates like cyclic β-glucans and polysaccharides. Proteins secreted through the type II system, or main terminal branch of the general secretory pathway, depend on the Sec or Tat system for initial transport into the periplasm . Once there, they pass through the outer membrane via a multimeric (12–14 subunits) complex of pore forming secretin proteins. In addition to

11616-399: The most researched T3SSs are from species of: The T3SS is composed of approximately 30 different proteins, making it one of the most complex secretion systems. Its structure shows many similarities with bacterial flagella (long, rigid, extracellular structures used for motility ). Some of the proteins participating in T3SS share amino-acid sequence homology to flagellar proteins. Some of

11737-461: The names usually do not reveal much about the protein's function. Some proteins discovered independently in different bacteria have later been shown to be homologous ; the historical names, however, have mostly been kept, a fact that might cause confusion. For example, the proteins SicA, IpgC and SycD are homologs from Salmonella , Shigella and Yersinia , respectively, but the last letter (the "serial number") in their name does not show that. Below

11858-499: The nature of LPS, which has both water-attracting and water-repelling properties (amphiphilic), makes it challenging to develop sensitive and user-friendly tests. The typical detection methods rely on identifying the lipid A part of LPS because Lipid A is very similar among different bacterial species and serotypes. LPS testing techniques fall into six categories, and they often overlap: in vivo tests, in vitro tests, modified immunoassays, biological assays, and chemical assays. Because

11979-406: The needle complex at the base and make their way inside the needle towards the host cell. The exact way in which effectors enter the host is mostly unknown. It has been previously suggested that the needle itself is capable of puncturing a hole in the host cell membrane; this theory has been refuted. It is now clear that some effectors, collectively named translocators , are secreted first and produce

12100-437: The needle complex is a universal feature of all T3SSs of pathogenic bacteria. The needle complex starts at the cytoplasm of the bacterium, crosses the two membranes and protrudes from the cell. The part anchored in the membrane is the base (or basal body ) of the T3SS. The extracellular part is the needle. A so-called inner rod connects the needle to the base. The needle itself, although the biggest and most prominent part of

12221-436: The needle complex is able to recognize. Unlike other secretion systems, the secretion signal of T3SS proteins is never cleaved off the protein. Contact of the needle with a host cell triggers the T3SS to start secreting; not much is known about this trigger mechanism (see below). Secretion can also be induced by lowering the concentration of calcium ions in the growth medium (for Yersinia and Pseudomonas ; done by adding

12342-580: The neutrophils in a patient’s blood to an immunological complex of endotoxin and exogenous antibody – the chemiluminescent reaction formed creates an emission of light. The amount of chemiluminescence is proportional to the logarithmic concentration of LPS in the sample and is a measure of the endotoxin activity in the blood. The assay reacts specifically with the Lipid A moiety of LPS of Gram-negative bacteria and does not cross-react with cell wall constituents of Gram-positive bacteria and other microorganisms. LPS

12463-405: The oligosaccharide from pathogenic Neisseria spp. LOS is also found in lactoneoseries glycosphingolipids from human cells. Most meningococci from groups B and C, as well as gonococci , have been shown to have this trisaccharide as part of their LOS structure. The presence of these human cell surface 'mimics' may, in addition to acting as a 'camouflage' from the immune system, play a role in

12584-646: The outer membrane of E. coli and Salmonella . LPS increases the negative charge of the cell membrane and helps stabilize the overall membrane structure. It is of crucial importance to many gram-negative bacteria, which die if the genes coding for it are mutated or removed. However, it appears that LPS is nonessential in at least some gram-negative bacteria, such as Neisseria meningitidis , Moraxella catarrhalis , and Acinetobacter baumannii . It has also been implicated in non-pathogenic aspects of bacterial ecology, including surface adhesion, bacteriophage sensitivity, and interactions with predators such as amoebae . LPS

12705-505: The outer membrane of some types of Gram-negative bacteria , such as Neisseria spp. and Haemophilus spp. LOS plays a central role in maintaining the integrity and functionality of the outer membrane of the Gram negative cell envelope. LOS play an important role in the pathogenesis of certain bacterial infections because they are capable of acting as immunostimulators and immunomodulators. Furthermore, LOS molecules are responsible for

12826-699: The pH of the cytosol is 7.4, the ER's pH is 7.0, and the cis-golgi has a pH of 6.5. Secretory vesicles have pHs ranging between 5.0 and 6.0; some secretory vesicles evolve into lysosomes , which have a pH of 4.8. There are many proteins like FGF1 (aFGF), FGF2 (bFGF), interleukin-1 (IL1) etc. which do not have a signal sequence. They do not use the classical ER-Golgi pathway. These are secreted through various nonclassical pathways. At least four nonclassical (unconventional) protein secretion pathways have been described. They include: In addition, proteins can be released from cells by mechanical or physiological wounding and through non-lethal, transient oncotic pores in

12947-525: The past, antibiotic-resistant strains constantly emerge. Understanding the way the T3SS works and developing drugs targeting it specifically have become an important goal of many research groups around the world since the late 1990s. The hallmark of T3SS is the needle (more generally, the needle complex ( NC ) or the T3SS apparatus ( T3SA ); also called injectisome when the ATPase is excluded; see below). Bacterial proteins that need to be secreted pass from

13068-470: The plasma membrane induced by washing cells with serum-free media or buffers. Many human cell types have the ability to be secretory cells. They have a well-developed endoplasmic reticulum , and Golgi apparatus to fulfill this function. Tissues that produce secretions include the gastrointestinal tract which secretes digestive enzymes and gastric acid , the lungs which secrete surfactants , and sebaceous glands which secrete sebum to lubricate

13189-425: The presence of an N-terminal signal peptide on the secreted protein. Others are translocated across the cytoplasmic membrane by the twin-arginine translocation pathway (Tat). Gram-negative bacteria have two membranes, thus making secretion topologically more complex. There are at least six specialized secretion systems in Gram-negative bacteria. Type I secretion is a chaperone dependent secretion system employing

13310-605: The rarer case, denote the molecular weight of the protein in kDa . Examples: IpaA, IpaB, IpaC; MxiH, MxiG, MxiM; Spa9, Spa47. Several key elements appear in all T3SSs: the needle monomer, the inner rod of the needle, the ring proteins, the two translocators, the needle-tip protein, the ruler protein (which is thought to determine the needle's length; see above) and the ATPase , which supplies energy for secretion. The following table shows some of these key proteins in four T3SS-containing bacteria: The isolation of large, fragile, hydrophobic membrane structures from cells has constituted

13431-421: The respiratory and digestive systems. Recent studies have shown that LPS disrupts cell membrane lipids, affecting cholesterol and metabolism, potentially leading to high cholesterol, abnormal blood lipid levels, and non-alcoholic fatty liver disease. In some cases, LPS can interfere with toxin clearance, which may be linked to neurological issues. In general the health effects of LPS are due to its abilities as

13552-423: The secretin protein, 10–15 other inner and outer membrane proteins compose the full secretion apparatus, many with as yet unknown function. Gram-negative type IV pili use a modified version of the type II system for their biogenesis, and in some cases certain proteins are shared between a pilus complex and type II system within a single bacterial species. It is homologous to the basal body in bacterial flagella. It

13673-489: The skin and hair. Meibomian glands in the eyelid secrete meibum to lubricate and protect the eye. Secretion is not unique to eukaryotes – it is also present in bacteria and archaea as well. ATP binding cassette (ABC) type transporters are common to the three domains of life. Some secreted proteins are translocated across the cytoplasmic membrane by the SecYEG translocon , one of two translocation systems, which requires

13794-665: The structure of the LOS molecule, primarily within the structure of the oligosaccharide portion of the LOS molecule. In Neisseria gonorrhoeae it has been demonstrated that the antigenicity of LOS molecules can change during an infection due to the ability of these bacteria to synthesize more than one type of LOS, a characteristic known as phase variation . Additionally, Neisseria gonorrhoeae , as well as Neisseria meningitidis and Haemophilus influenzae , are capable of further modifying their LOS in vitro , for example through sialylation (modification with sialic acid residues), and as

13915-485: The surface molecules of some types of host cells is termed molecular mimicry . For example, in Neisseria meningitidis L2,3,5,7,9, the terminal tetrasaccharide portion of the oligosaccharide (lacto-N-neotetraose) is the same tetrasaccharide as that found in paragloboside , a precursor for ABH glycolipid antigens found on human erythrocytes . In another example, the terminal trisaccharide portion (lactotriaose) of

14036-415: The surrounding environment, and endotoxins, which are toxins "within" the bacterial cell and released only after destruction of the bacterial outer membrane. Subsequent work showed that release of LPS from Gram negative microbes does not necessarily require the destruction of the bacterial cell wall, but rather, LPS is secreted as part of the normal physiological activity of membrane vesicle trafficking in

14157-449: The tail spike of the T4 phage, and the activity of the system is thought to functionally resemble phage infection. In addition to the use of the multiprotein complexes listed above, Gram-negative bacteria possess another method for release of material: the formation of bacterial outer membrane vesicles . Portions of the outer membrane pinch off, forming nano-scale spherical structures made of

14278-421: The type III secretion system. T3SSs are essential for the pathogenicity (the ability to infect) of many pathogenic bacteria. Defects in the T3SS may render a bacterium non-pathogenic. It has been suggested that some non-invasive strains of gram-negative bacteria have lost the T3SS because the energetically costly system is no longer of use. Although traditional antibiotics were effective against these bacteria in

14399-517: Was later shown that IpaB achieves this by interacting with caspase 1 , a major regulatory protein in eukaryotic cells. Another well characterized class of T3SS effectors are Transcription Activator-like effectors ( TAL effectors ) from Xanthomonas . When injected into plants, these proteins can enter the nucleus of the plant cell, bind plant promoter sequences, and activate transcription of plant genes that aid in bacterial infection. TAL effector-DNA recognition has recently been demonstrated to comprise

14520-406: Was necessary and sufficient to induce nitric oxide production with a bactericidal effect. Lipopolysaccharide is a significant factor that makes bacteria harmful, and it helps categorize them into different groups based on their structure and function. This makes LPS a useful marker for telling apart various Gram-negative bacteria. Swiftly identifying and understanding the types of pathogens involved

14641-399: Was published. Recent advances and approaches have allowed high-resolution 3D images of the NC, further clarifying the complex structure of the NC. Numerous T3SS proteins have been crystallized over the years. These include structural proteins of the NC, effectors and chaperones. The first structure of a needle-complex monomer was NMR structure of BsaL from "Burkholderia pseudomallei" and later

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