Nikiya Anton Bettey 2021

Tuesday, April 19, 2022

04-19-2022-0504 - Membrane fusion proteins

Membrane fusion proteins (not to be confused with chimeric or fusion proteins) are proteins that cause fusion of biological membranes. Membrane fusion is critical for many biological processes, especially in eukaryotic development and viral entry. Fusion proteins can originate from genes encoded by infectious enveloped viruses, ancient retroviruses integrated into the host genome,^[1] or solely by the host genome.^[2] Post-transcriptional modifications made to the fusion proteins by the host, namely addition and modification of glycans and acetyl groups, can drastically affect fusogenicity (the ability to fuse).^[3]

https://en.wikipedia.org/wiki/Membrane_fusion_protein

04-19-2022-0502 - ATP-binding domain of ABC transporters

In molecular biology, ATP-binding domain of ABC transporters is a water-soluble domain of transmembrane ABC transporters.

ABC transporters belong to the ATP-Binding Cassette superfamily, which uses the hydrolysis of ATP to translocate a variety of compounds across biological membranes. ABC transporters are minimally constituted of two conserved regions: a highly conserved ATP binding cassette (ABC) and a less conserved transmembrane domain (TMD). These regions can be found on the same protein or on two different ones. Most ABC transporters function as a dimer and therefore are constituted of four domains, two ABC modules and two TMDs.

https://en.wikipedia.org/wiki/ATP-binding_domain_of_ABC_transporters

04-19-2022-0500 - 6065/6-7,8-9

04-19-2022-0459 - ATP-binding cassette transporters (ABC transporters)

The ATP-binding cassette transporters (ABC transporters) are a transport system superfamily that is one of the largest and possibly one of the oldest gene families. It is represented in all extant phyla, from prokaryotes to humans.^[1]^[2]^[3]

ABC transporters often consist of multiple subunits, one or two of which are transmembrane proteins and one or two of which are membrane-associated AAA ATPases. The ATPase subunits utilize the energy of adenosine triphosphate (ATP) binding and hydrolysis to provide the energy needed for the translocation of substrates across membranes, either for uptake or for export of the substrate.

Most of the uptake systems also have an extracytoplasmic receptor, a solute binding protein. Some homologous ATPases function in non-transport-related processes such as translation of RNA and DNA repair.^[4]^[5] ABC transporters are considered to be an ABC superfamily based on the similarities of the sequence and organization of their ATP-binding cassette (ABC) domains, even though the integral membrane proteins appear to have evolved independently several times, and thus comprise different protein families.^[6] Like the ABC exporters, it is possible that the integral membrane proteins of ABC uptake systems also evolved at least 3 times independently, based on their high resolution 3-dimensional structures.^[7] ABC uptake porters take up a large variety of nutrients, biosynthetic precursors, trace metals and vitamins, while exporters transport lipids, sterols, drugs, and a large variety of primary and secondary metabolites. Some of these exporters in humans are involved in tumor resistance, cystic fibrosis and a range of other inherited human diseases. High level expression of the genes encoding some of these exporters in both prokaryotic and eukaryotic organisms (including human) result in the development of resistance to multiple drugs such as antibiotics and anti-cancer agents.

Hundreds of ABC transporters have been characterized from both prokaryotes and eukaryotes.^[8] ABC genes are essential for many processes in the cell, and mutations in human genes cause or contribute to several human genetic diseases.^[9] Forty eight ABC genes have been reported in humans. Among these, many have been characterized and shown to be causally related to diseases present in humans such as cystic fibrosis, adrenoleukodystrophy, Stargardt disease, drug-resistant tumors, Dubin–Johnson syndrome, Byler's disease, progressive familiar intrahepatic cholestasis, X-linked sideroblastic anemia, ataxia, and persistent and hyperinsulimenic hypoglycemia.^[8] ABC transporters are also involved in multiple drug resistance, and this is how some of them were first identified. When the ABC transport proteins are overexpressed in cancer cells, they can export anticancer drugs and render tumors resistant.^[10]

https://en.wikipedia.org/wiki/ATP-binding_cassette_transporter

04-19-2022-0457 - Pyocyanin

Pyocyanin (PCN⁻) is one of the many toxins produced and secreted by the Gram negative bacterium Pseudomonas aeruginosa. Pyocyanin is a blue, secondary metabolite with the ability to oxidise and reduce other molecules^[2] and therefore kill microbes competing against P. aeruginosa as well as mammalian cells of the lungs which P. aeruginosa has infected during cystic fibrosis. Since pyocyanin is a zwitterion at blood pH, it is easily able to cross the cell membrane. There are three different states in which pyocyanin can exist: oxidized, monovalently reduced or divalently reduced. Mitochondria play an important role in the cycling of pyocyanin between its redox states. Due to its redox-active properties, pyocyanin generates reactive oxygen species.

Synthesis[edit]

In order for pyocyanin to be synthesized by P. aeruginosa, two specific genes must be functional. MvfR is a gene which produces a transcription factor which activates phnAB genes. These genes produce the molecule quinolone which then regulates operons 1 and 2 of phzRABCDEFG which are key to the synthesis of phenazine.^[3] The synthesis of pyocyanin is primarily controlled by the quorum sensing process. P.aeruginosa strains which are unable to synthesise pyocyanin can still benefit from its effects if the strain has co-infected the lung with wild-type strains which can produce pyocyanin.^[4] Biosynthesis can be impaired by disrupting the aro pathway which is responsible for the synthesis of chorismic acid from shikimate.^[5] Chorismic acid is the precursor of pyocyanin.

shikimic acid → chorismic acid → phenazine-1-carboxylic acid → 5-methylphenazine-1-carboxylic acid betaine → pyocyanin^[6]

The complete virulence of P. aeruginosa can only be experienced when pyocyanin is produced.^[7]

Redox warfare[edit]

Pyocyanin inactivates catalase by reducing its gene’s transcription as well as directly targeting the enzyme itself. Glutathione is an important antioxidant modulated by pyocyanin.^[8] In particular the pool of the reduced form is depleted while the oxidised form is promoted by hydrogen peroxide which is not dismutated by catalase. In the cystic fibrosis lung, intracellular pyocyanin converts molecular oxygen to the superoxide free radical by oxidizing NADPH to NADP⁺. This has a doubly negative effect on the lungs. Firstly, the NADPH used by pyocyanin depletes the available substrate for the reaction catalysed by the NADPH oxidase enzyme. Secondly, the superoxide radical generated can inhibit cytokines, such as IL-4, IL-13 and IFN-γ, which usually upregulate NADPH oxidase. When the lung is confronted with pyocyanin, an increased concentration of catalase and superoxide dismutase is seen in order to deal with the barrage of radicals being produced.^[9]

Targets[edit]

Pyocyanin is able to target a wide range of cellular components and pathways. Pathways that are affected by pyocyanin include the electron transport chain, vesicular transport, and cell growth. An enhanced susceptibility to pyocyanin is seen in cells with certain mutant proteins or complexes. Mutations in genes affecting V-ATPase synthesis and assembly,^[10] vesicle transport machinery, and protein sorting machinery all confer an increased sensitivity to pyocyanin which further enhances the effects on cystic fibrosis on the patient. Vacuolar- ATPase in yeast cells is a particularly potent target as it is the main non-mitochondrial producer of ATP but also has numerous other functions such as calcium homeostatic control, the facilitation of receptor-mediated endocytosis and the degradation of proteins. Therefore, the inactivation of vacuolar-ATPase by hydrogen peroxide produced by pyocyanin has huge consequences for the lung. Additional to these effects, another target of pyocyanin is caspase 3-like proteases which can then go on to initiate apoptosis and necrosis. Mitochondrial electron carriers ubiquinone and nicotinic acid are also susceptible to pyocyanin.^[11] The cell cycle can be disturbed by the action of pyocyanin, and it can hinder the proliferation of lymphocytes.^[12] This is done by the generation of reactive oxygen intermediates, such as hydrogen peroxide and superoxide, which cause oxidative stress by directly damaging DNA or by targeting other constituents of the cell cycle such as DNA recombination and repair machinery. Pyocyanin contributes to the disproportion of protease and antiprotease activity by disabling α₁- protease inhibitor.

Cystic fibrosis[edit]

Many studies have concluded that pyocyanin has a derogatory effect in cystic fibrosis which enables P. aeruginosa to persist in the cystic fibrosis lung; it is often detected in the sputum of cystic fibrosis patients. Pyocyanin in vitro has the ability to interfere with functions such as ciliary beating and therefore cause epithelial dysfunction as the ciliary are needed to sweep mucus up the throat.^[13] Additionally, neutrophil apoptosis,^[14] immunoglobulin release from B-lymphocytes, and interleukin release (e.g. IL-8^[15] and CCL5) are all impaired by pyocyanin, weakening the immune system of the lung. In vivo studies have shown that the growth of fungus is inhibited in the presence of pyocyanin.^[16] The fungicidal mechanism is the activation of NAD(P)H to induce a redox-active cascade producing reactive oxygen intermediates. This allows P. aeruginosa to have a competitive advantage as it may dominate over other microorganisms in the cystic fibrosis lung. The intracellular concentration of ATP is also diminished by pyocyanin causing further damage to CFTR which are already impaired in cystic fibrosis. CFTR channels rely on ATP for two main purposes. Firstly, the binding and hydrolysis of ATP has to occur at two nucleotide binding domains for the channel to move between its open and closed conformation.^[17] Secondly, phosphorylation of CFTR by Protein kinase A II should occur in order for the channel to be operational. PKA II is activated by cAMP which is produced from ATP. Both these processes are impaired when ATP is depleted by pyocyanin.

Defence against pyocyanin[edit]

Caenorhabditis elegans possesses two specific ABC transporters called pgp-1 and pgp-2 which are effectively able to extrude intracellular pyocyanin in an energy dependent manner.^[18]

Biosynthesis[edit]

Biosynthesis of pyocyanin from Pseudomonas aeruginosa. Hydrogens abstracted during next enzymatic step colored red.

Pyocyanin biosynthesis begins with the synthesis of the phenazine-1-carboxylic acid (PCA) core.^[19] In this reaction the enzyme PhzE catalyzes the loss of the hydroxyl group from C4 of Chorismic Acid as well as the transfer of an amine group from glutamine to form glutamic acid and 2-amino-2-desoxyisochorismic acid (ADIC).^[20] Following this, PhzD catalyzes the hydrolytic removal the pyruvate moiety from ADIC to form (5S,6S)-6-amino-5-hydroxy-1,3-cyclohexadieve-1-carboxylic acid (DHHA).^[20] In the next step, PhzF catalyzes two steps: the abstraction of a hydrogen from C3 of DHHA, delocalization of the double bond system and reprotonation at C1 as well as enol tautomerization to form the highly unstable 6-amino-5-oxocyclohex-2-ene-1-carboxylic acid (AOCHC).^[20] From here two molecules of AOCHC are condensed by PhzB to form the tricyclic compound, hexahydrophenazine-1,6-dicarboxylic acid (HHPDC).^[20] The product of this reaction, HHPDC, is unstable and spontaneously undergoes oxidative decarboxylation in an uncatalyzed reaction to form tetrahydrophenazine-1,6-carboxylic acid (THPCA).^[20] In the final step of phenazine-1-carboxylic acid synthesis the enzyme PhzG catalyzes the oxidation of THPCA to dihydro-phenazine-1-carboxylic acid.^[20] This is the last catalyzed step in the production of PCA, the last step is an uncatalyzed oxidation of DHPCA to PCA.^[20] The conversion of PCA to Pyocyanin is achieved in two enzymatic steps: firstly, PCA is methylated on N5 to 5-methylphenazine-1-carboxylate betaine by the enzyme PhzM using the cofactor S-adenosyl-L-methionine and secondly, PhzS catalyzes the hydroxylative decarboxylation of this substrate to form the final product, Pyocyanin.^[19]

Genomics[edit]

In 2000, the complete genome sequence of a Pseudomonas species was determined; more recently, the sequence of other strains has been determined, including P. aeruginosa strains PAO1 (2000), P. putida KT2440 (2002), P. protegens Pf-5 (2005), P. syringae pathovar tomato DC3000 (2003), P. syringae pathovar syringae B728a (2005), P. syringae pathovar phaseolica 1448A (2005), P. fluorescens Pf0-1, and P. entomophila L48.^[9]

By 2016, more than 400 strains of Pseudomonas had been sequenced.^[17] Sequencing the genomes of hundreds of strains revealed highly divergent species within the genus. In fact, many genomes of Pseudomonas share only 50-60% of their genes, e.g. P. aeruginosa and P. putida share only 2971 proteins out of 5350 (or ~55%).^[17]

By 2020, more than 500 complete Pseudomonas genomes were available in Genebank. A phylogenomic analysis utilized 494 complete proteomes and identified 297 core orthologues, shared by all strains.^[14] This set of core orthologues at the genus level was enriched for proteins involved in metabolism, translation, and transcription and was utilized for generating a phylogenomic tree of the entire genus, to delineate the relationships among the Pseudomonas major evolutionary groups.^[14] In addition, group-specific core proteins were identified for most evolutionary groups, meaning that they were present in all members of the specific group, but absent in other pseudomonads. For example, several P. aeruginosa-specific core proteins were identified that are known to play an important role in this species' pathogenicity, such as CntL, CntM, PlcB, Acp1, MucE, SrfA, Tse1, Tsi2, Tse3, and EsrC.^[14]

Characteristics[edit]

Members of the genus display these defining characteristics:^[18]

Rod-shaped
Gram-negative
Flagellum one or more, providing motility
Aerobic
Non-spore forming
Catalase-positive
Oxidase-positive

Other characteristics that tend to be associated with Pseudomonas species (with some exceptions) include secretion of pyoverdine, a fluorescent yellow-green siderophore^[19] under iron-limiting conditions. Certain Pseudomonas species may also produce additional types of siderophore, such as pyocyanin by Pseudomonas aeruginosa^[20] and thioquinolobactin by Pseudomonas fluorescens,.^[21] Pseudomonas species also typically give a positive result to the oxidase test, the absence of gas formation from glucose, glucose is oxidised in oxidation/fermentation test using Hugh and Leifson O/F test, beta hemolytic (on blood agar), indole negative, methyl red negative, Voges–Proskauer test negative, and citrate positive.

Pseudomonas may be the most common nucleator of ice crystals in clouds, thereby being of utmost importance to the formation of snow and rain around the world.^[22]

Biofilm formation[edit]

All species and strains of Pseudomonas have historically been classified as strict aerobes. Exceptions to this classification have recently been discovered in Pseudomonas biofilms.^[23] A significant number of cells can produce exopolysaccharides associated with biofilm formation. Secretion of exopolysaccharides such as alginate makes it difficult for pseudomonads to be phagocytosed by mammalian white blood cells.^[24] Exopolysaccharide production also contributes to surface-colonising biofilms that are difficult to remove from food preparation surfaces. Growth of pseudomonads on spoiling foods can generate a "fruity" odor.

Antibiotic resistance[edit]

Most Pseudomonas spp. are naturally resistant to penicillin and the majority of related beta-lactam antibiotics, but a number are sensitive to piperacillin, imipenem, ticarcillin, or ciprofloxacin.^[24] Aminoglycosides such as tobramycin, gentamicin, and amikacin are other choices for therapy.

This ability to thrive in harsh conditions is a result of their hardy cell walls that contain porins. Their resistance to most antibiotics is attributed to efflux pumps, which pump out some antibiotics before they are able to act.

Pseudomonas aeruginosa is increasingly recognized as an emerging opportunistic pathogen of clinical relevance. One of its most worrying characteristics is its low antibiotic susceptibility.^[25] This low susceptibility is attributable to a concerted action of multidrug efflux pumps with chromosomally encoded antibiotic resistance genes (e.g., mexAB-oprM, mexXY, etc.,^[26]) and the low permeability of the bacterial cellular envelopes. Besides intrinsic resistance, P. aeruginosa easily develops acquired resistance either by mutation in chromosomally encoded genes or by the horizontal gene transfer of antibiotic resistance determinants. Development of multidrug resistance by P. aeruginosa isolates requires several different genetic events that include acquisition of different mutations and/or horizontal transfer of antibiotic resistance genes. Hypermutation favours the selection of mutation-driven antibiotic resistance in P. aeruginosa strains producing chronic infections, whereas the clustering of several different antibiotic resistance genes in integrons favours the concerted acquisition of antibiotic resistance determinants. Some recent studies have shown phenotypic resistance associated to biofilm formation or to the emergence of small-colony-variants, which may be important in the response of P. aeruginosa populations to antibiotic treatment.^[9]

Sensitivity to Gallium[edit]

Although gallium has no natural function in biology, gallium ions interact with cellular processes in a manner similar to iron(III). When gallium ions are mistakenly taken up in place of iron(III) by bacteria such as Pseudomonas, the ions interfere with respiration, and the bacteria die. This happens because iron is redox-active, allowing the transfer of electrons during respiration, while gallium is redox-inactive.^[27]^[28]

Pathogenicity[edit]

Animal Pathogens[edit]

Infectious species include P. aeruginosa, P. oryzihabitans, and P. plecoglossicida. P. aeruginosa flourishes in hospital environments, and is a particular problem in this environment, since it is the second-most common infection in hospitalized patients (nosocomial infections).^[29] This pathogenesis may in part be due to the proteins secreted by P. aeruginosa. The bacterium possesses a wide range of secretion systems, which export numerous proteins relevant to the pathogenesis of clinical strains.^[30] Intriguingly, several genes involved in the pathogenesis of P.aeruginosa, such as CntL, CntM, PlcB, Acp1, MucE, SrfA, Tse1, Tsi2, Tse3, and EsrC are core group-specific,^[14] meaning that they are shared by the vast majority of P. aeruginosa strains, but they are not present in other Pseudomonads.

Plant Pathogens[edit]

P. syringae is a prolific plant pathogen. It exists as over 50 different pathovars, many of which demonstrate a high degree of host-plant specificity. Numerous other Pseudomonas species can act as plant pathogens, notably all of the other members of the P. syringae subgroup, but P. syringae is the most widespread and best-studied.

Although not strictly a plant pathogen, P. tolaasii can be a major agricultural problem, as it can cause bacterial blotch of cultivated mushrooms.^[31] Similarly, P. agarici can cause drippy gill in cultivated mushrooms.^[32]

Use as Biocontrol Agents[edit]

Since the mid-1980s, certain members of the genus Pseudomonas have been applied to cereal seeds or applied directly to soils as a way of preventing the growth or establishment of crop pathogens. This practice is generically referred to as biocontrol. The biocontrol properties of P. fluorescens and P. protegens strains (CHA0 or Pf-5 for example) are currently best-understood, although it is not clear exactly how the plant growth-promoting properties of P. fluorescens are achieved. Theories include: the bacteria might induce systemic resistance in the host plant, so it can better resist attack by a true pathogen; the bacteria might outcompete other (pathogenic) soil microbes, e.g. by siderophores giving a competitive advantage at scavenging for iron; the bacteria might produce compounds antagonistic to other soil microbes, such as phenazine-type antibiotics or hydrogen cyanide. Experimental evidence supports all of these theories.^[33]

Other notable Pseudomonas species with biocontrol properties include P. chlororaphis, which produces a phenazine-type antibiotic active agent against certain fungal plant pathogens,^[34] and the closely related species P. aurantiaca, which produces di-2,4-diacetylfluoroglucylmethane, a compound antibiotically active against Gram-positive organisms.^[35]

Use as Bioremediation Agents[edit]

Some members of the genus are able to metabolise chemical pollutants in the environment, and as a result, can be used for bioremediation. Notable species demonstrated as suitable for use as bioremediation agents include:

P. alcaligenes, which can degrade polycyclic aromatic hydrocarbons.^[36]
P. mendocina, which is able to degrade toluene.^[37]
P. pseudoalcaligenes, which is able to use cyanide as a nitrogen source.^[38]
P. resinovorans, which can degrade carbazole.^[39]
P. aeruginosa, P. putida, P. desmolyticum, and P. nitroreducens can degrade chlorpyrifos.^[40]
P. veronii, which has been shown to degrade a variety of simple aromatic organic compounds.^[41]^[42]
P. putida, which has the ability to degrade organic solvents such as toluene.^[43] At least one strain of this bacterium is able to convert morphine in aqueous solution into the stronger and somewhat expensive to manufacture drug hydromorphone (Dilaudid).
Strain KC of P. stutzeri, which is able to degrade carbon tetrachloride.^[44]

https://en.wikipedia.org/wiki/Pseudomonas