ANTHRAX. Aim To select a disease and to do a complete study on it.

Transcription

1 Aim To select a disease and to do a complete study on it. ANTHRAX Description Anthrax is an acute disease in humans and animals caused by the bacterium Bacillus anthraces which is highly lethal in some forms, there are effective vaccines against anthrax and some forms of the disease respond well to the antibiotic treatment. Bacillus anthraces is a gram positive, facultatively anaerobic, rod shaped bacterium of the genus bacillus, an endospore forming bacterium, Bacillus anthraces is a natural soil dwelling organism, as well as the causative agent of anthrax, under conditions of environmental stress, Bacillus anthraces bacteria naturally produce endospores which rest in the soil and can survive for thousands of years in this state. Bacillus anthraces may be inoculated into a wound, inhaled or ingested. In ruminants, the bacterium causes sudden death from septicaemia. For this reason any ruminants found to have died suddenly and without obvious reason should be treated as a suspected anthrax case. In these events, a blood sample is taken, by a qualified veterinary surgeon, from a superficial vein and subjected to the MacFaydean polychrome methylene blue staining procedure which screens for Bacillus anthraces. Confirmational diagnosis is achieved through PCR and Immunofluorescence Horses respond variably to Bacillus anthraces depending on the site of entry. Ingestion tends to lead to a severe enteritis and septicaemia. Inoculation in the skin tends to result in a local swelling and associated lymphadenitis. Bacillus anthraces again causes an acute necrotising tonsillitis, or a subacute pharyngeal swelling, or the intestinal disease described in horses. The intestinal disease carries a higher mortality. Dogs and cats seem less susceptible to Bacillus anthraces and require a relative large dose of infectious agent before they begin to show clinical signs. Anthrax is an acute infectious disease caused by the spore-forming bacterium Bacillus anthraces. Anthrax most commonly occurs in wild and domestic lower vertebrates (cattle, sheep, goats, camels, antelopes, and other herbivores), but it can also occur in humans when they are exposed to infected animals or tissue from infected animals Transmission of Anthrax Anthrax infection can occur in three forms: cutaneous (skin), inhalation, and gastrointestinal. Bacillus anthraces spores can live in the soil for many years, and humans can become infected with anthrax by handling products from infected animals or by inhaling anthrax spores from contaminated animal products. Anthrax can also be

2 spread by eating undercooked meat from infected animals. It is rare to find infected animals in the United States. Symptoms of disease vary depending on how the disease was contracted, but symptoms usually occur within 7 days. Cutaneous: Most (about 95%) anthrax infections occur, when the bacterial spore enters a cut or abrasion on the skin, such as when handling contaminated wool, hides, leather or hair products of infected animals. Skin infection begins as a raised itchy bump that resembles an insect bite but within 1-2 days develops into a vesicle and then a painless ulcer, usually 1-3 cm in diameter, with a characteristic black necrotic (dying) area in the center. Edema or swelling of the surrounding tissues may develop and lymph glands in the adjacent area may swell. About 20% of untreated cases of cutaneous anthrax will result in death. Deaths are rare with appropriate antimicrobial therapy. Inhalation: Initial symptoms may resemble a common cold. After several days, the symptoms may progress to severe breathing problems and shock. Inhalation anthrax is usually fatal, and even with aggressive antibiotic and supportive therapy 45% of inhalation anthrax cases were fatal in the bioterrorist attack in the fall of Intestinal: The intestinal disease form of anthrax may follow the consumption of contaminated meat and is characterized by an acute inflammation of the intestinal tract. Initial signs include nausea, loss of appetite, vomiting, fever are followed by abdominal pain, vomiting of blood, and severe diarrhea. Symptoms may also include lesions and soreness in the throat, difficulty swallowing, marked swelling of the neck and regional lymph glands. Intestinal anthrax results in death in 25% to 60% of cases. Structure and Targets of the Anthrax The identification of [the receptor for the anthrax protein] now allows for a more detailed investigation of the mechanism of uptake by cells of anthrax toxin, write John A. T. Young, of the University of Wisconsin-Madison, and colleagues. In the second paper, researchers report the three-dimensional structure of another part of the toxin, called lethal factor. This enzyme disrupts communication within immune cells, inhibiting a response. Cells eventually rupture, which causes shock, a loss of blood pressure, and death. Anthrax toxin has three parts: protective antigen (PA), a protein that binds to a receptor on the target cell surface; and two enzymes that must be transported into the cell to cause damage. The enzymatic portions of the toxin enter the cell through a pore created for them by PA after it binds to the cell's outer surface. PA can be seen as a bundle of seven cigar-shaped parts, a molecular arrangement referred to as "polyvalent," meaning it displays multiple binding sites. The inhibitor designed by Dr. Kane and his colleagues is also polyvalent. Just as a glove matches the shape of a hand more closely than a mitten, and so fits more snugly, the polyvalent inhibitor binds the toxin at multiple sites and is orders of magnitude more potent than an inhibitor that binds at a single site. The multiple peptides on the

3 functionalized liposome are arranged with the same average spacing as the binding sites of the PA molecule, which permits a firmer bond between the two, explains Dr. Kane. When the inhibitor is bound tightly to PA, the subsequent steps of enzyme entry cannot occur and the toxin is effectively neutralized. Anthrax Bacterium Genome Genes are the instructions for making proteins, which in turn build components of the cell or carry out its biochemical processes. The instructions that dictate how a microbe works are encoded within its genes. Bacteria keep most of their genes in a chromosome, a very long stretch of DNA. Smaller circular pieces of DNA called plasmids also carry genes that bacteria may exchange with each other. Because plasmids often contain genes for toxins and antibiotic resistance, knowing the DNA sequence of such plasmids is important. Scientists have sequenced plasmids carrying the toxin genes of B. anthraces. In addition, researchers have sequenced the complete chromosomal DNA sequence of several Bacillus anthraces strains, including one that killed a Florida man in the 2001 anthrax Bioterrorism attack by comparing the DNA blueprints of different Bacillus anthraces strains, researchers are learning why some strains are more virulent than others. Variations between strains might also point to differences in antibiotic susceptibility, permitting doctors to immediately determine the appropriate treatment. Scientists are now analyzing the Bacillus anthraces genome sequence to determine the function of each of its genes and to learn how those genes interact with each other or with host-cell components to cause disease. Knowing the sequence of Bacillus anthraces genes will help scientists discover key bacterial proteins that can then be targeted by new drugs or vaccines. Spore Biology Bacillus anthraces spores are essentially dormant and must wake up, or germinate, to become reproductive, disease-causing bacteria. Researchers are studying the germination process to learn more about the signals that cause spores to become active once inside an animal or person. Efforts are under way to develop models of spore germination in laboratory animals. Scientists hope those models will enable discoveries leading to drugs that block the germination process in Bacillus anthraces spores. Host Immunity People who contract anthrax produce antibodies to protective antigen protein. Similar antibodies appear to block infection in animals. Recent studies also suggest that some animals can produce antibodies to components of Bacillus anthraces spores. Those antibodies, when studied in a test tube, prevent spores from germinating and increase their uptake by the immune system s microbe-eating cells. These discoveries suggest that scientists might be able to develop a vaccine to fight both Bacillus anthraces cells and spores. Researchers also are studying

4 how the immune system responds to Bacillus anthraces infection. Part of the immune system response, known as adaptive immunity, consists of B and T cells that specifically recognize components of the anthrax bacterium. The other type of immune response,innate immunity-aims more generally to combat a wide range of microbial invaders and likely plays a key role in the body s front-line defenses. Scientists are conducting studies of how those two arms of the immune system act to counter infection, including how Bacillus anthraces spore germination affects individual immune responses. In another study, NIAID-supported scientists have discovered a potential target for developing new measures to prevent and treat anthrax toxicity. Their study shows that a human gene called LRP6 plays a role in the delivery of anthrax toxins into cells. Antibodies directed against LRP6 protected cell cultures from anthrax lethal toxin. These results suggest that targeting LRP6 may prove useful in developing ways to protect against the effects of accumulated toxin. Result The disease anthrax was selected as of the interest and their characteristics, symptoms, gene information, treatments were determined.

5 Aim Bacillus Anthraces To study the organism responsible for the disease anthrax Bacillus anthraces. Description Anthrax is an acute disease in humans and animals caused by the bacterium bacillus anthraces which is highly lethal in some forms, there are effective vaccines against anthrax and some forms of the disease respond well to the antibiotic treatment. Classification Kingdom: Phylum: Class: Order: Family: Genus: Species: Bacteria Firmicutes Bacilli Bacillales Bacillaceae Bacillus B. anthraces Table: Classification of Bacillus anthraces Bacillus anthraces is a gram positive, facultatively anaerobic, rod shaped bacterium of the genus bacillus, an endospore forming bacterium, Bacillus anthraces is a natural soil dwelling organism, as well as the causative agent of anthrax, under conditions of environmental stress, Bacillus anthraces bacteria naturally produce endospores which rest in the soil and can survive for thousands of years in this state. Bacillus anthraces may be inoculated into a wound, inhaled or ingested. In ruminants, the bacterium causes sudden death from septicemia. For this reason any ruminants found to have died suddenly and without obvious reason should be treated as a suspected anthrax case. In these events, a blood sample is taken, by a qualified veterinary surgeon, from a superficial vein and subjected to the MacFaydean polychrome methylene blue staining procedure which screens for Bacillus anthraces. Conformational diagnosis is achieved through PCR and Immunofluorescence. Horses respond variably to Bacillus anthraces depending on the site of entry. Ingestion tends to lead to a severe enteritis

6 and septicemia. Inoculation in the skin tends to result in a local swelling and associated lymphadenitis. In pigs, Bacillus anthraces again causes acute necrotising tonsillitis, or a subacute pharyngeal swelling, or the intestinal disease described in horses. The intestinal disease carries a higher mortality. Dogs and cats seem less susceptible to Bacillus anthraces and require a relative large dose of infectious agent before they begin to show clinical signs Bacillus anthraces Bacillus anthraces is typically a disease of herbivores (plant-eating mammals), although it can affect other animals as well. Among domestic animals, cattle, sheep, and goats have been the most frequent victims. In most industrialized countries, livestock are routinely vaccinated, and cases of anthrax are rare. In developing countries, however, where animal vaccination is not regularly practiced, anthrax in animals is a problem. This is especially so in tropical and sub-tropical environments. In the USA, anthrax cases among animals have been generally limited to the western plains. Endospores can survive in the soil for years. Animals consume the spores along with grass as they graze. After the spores enter an animal, they germinate, changing from the resistant form into the growing and dividing vegetative form. The sporangium lyses, the spore germinates, and the bacilli multiply rapidly. Anthrax is a very serious disease in animals, culminating in a fatal septicemia. The carcass of the animal should be burned where it lies. The carcass should never be opened, since doing this will cause the vegetative forms, which can be destroyed relatively easily, to form into the resistant spores that can survive for years. Infection in humans traditionally has been much rarer than infection in animals. Anthrax occurred in people who came in contact with animals or animal products. It was frequently an occupational disease, affecting veterinarians, people who raised livestock, and people who prepared products from wool, hide, and hair of animals. The ordinary citizen in the USA is most likely to encounter anthrax from imported products that have not been treated sufficiently to destroy spores. In humans there are three possible forms of the disease anthrax. Historically, the most common form has been cutaneous anthrax, in which the organism enters through a break in the skin. The cutaneous form begins as a papule at the entry site that progresses over several days to a vesicle and then ulcerates. Edema, sometimes massive, surrounds the lesions, which then develop a characteristic black eschar. The patient may have fever, malaise and headache. A small percentage of cutaneous infections become systemic, and these can be fatal. A more serious form is inhalation anthrax. Here the victim breathes in the organism and develops a severe respiratory disease. Systemic infection resulting from inhalation of Bacillus anthraces has a mortality rate approaching 100%. Initial symptoms are vague and flu-like, progressing to hypotension, shock and massive

7 bacteremia and toxemia. The severe symptoms are believed to be the result of the bacillis exotoxins. Early antibiotic treatment is an absolute necessity and should be started during the incubation period if a person has been exposed. The third form, intestinal anthrax, is contracted from the consumption of contaminated meat. In industrialized countries this is not usually a risk, although rare exceptions have been described. In August 2000, the Minnesota Department of Health was notified that Bacillus anthraces had been isolated from a steer on a farm in Roseau County. The infected steer was one of five dead cattle found in a pasture. On the basis of identification of the bacteria by phage typing of isolates cultured from tissues and blood samples by the North Dakota State University Veterinary Diagnostic Laboratory, anthrax was confirmed. A report of this incident described the management of and public health response to human exposure to meat contaminated with anthrax. Genes Involved In order to cause the disease anthrax, Bacillus anthraces requires two plasmids, px01 and px02, which carry toxin and capsule genes, respectively, that are used as genetic targets in the laboratory detection of the bacterium presence of 10 genes (acpa, capa, capb, CapC, capr, capd, IS1627, ORF 48, ORF 61, and repa) Diagonisation Anthrax is diagnosed by culture and isolation of the causative bacterium, B. anthraces- by detecting the bacterial DNA or antigens; or by measuring specific antibodies in the blood of persons with suspected cases. The bacteria can be cultured from the blood, skin lesions, fluid from the lungs or respiratory secretions, spinal fluid, or other affected tissues prior to the start of antibiotic treatment. Detection of the DNA or antigens of the bacteria, and detection of antibodies in the blood of suspected cases, is important tools for diagnosis because positive culture is unlikely after antibiotic treatment has been started. Treatment The antibiotics ciprofloxacin, doxycycline and penicillin can be given in high doses to treat the condition. Ciprofloxacin and doxycyline are also used as prophylaxis (prevention) for people who have been exposed. Early treatment is needed if inhalational anthrax is suspected. There is an immunisation against anthrax but it takes five doses of vaccine over the course of a year to get immunity. This makes immunisation too slow to deal with accidental or deliberate exposure. It is normally offered to those who handle infected animals, and laboratory staff who work with the bacteria. It would be recommended for a person who had been exposed, in conjunction with antibiotics, because of uncertainty about when the spores may germinate. The armed forces in the USA are currently being given immunisation, but concerns have been expressed about how safe, and effective, it is for the

8 general public. In the UK, the Public Health Laboratory Service controls supply of the vaccine. Their advice, as of 15 October 2001, is that immunisation is not recommended pre-exposure to anyone except people who are in the relevant occupational groups mentioned above. PHLS is monitoring the situation and will act according to any outbreaks or events Result The organism responsible for anthrax was found to be Bacillus anthraces. The structure was described and also the causes, diagnosis, and the treatment of the disease were determined.

9 Aim DOXYCYCLINE To find the protein responsible for the anthrax disease this is the binding sites for the human. Description Doxycycline is a tetracycline antibiotic. It works by slowing the growth of bacteria in the body. Doxycycline is used to treat many different bacterial infections, such as urinary tract infections, acne, gonorrhea, and chlamydia, periodontitis (gum disease), and others. It may be used in combination with other medicines to treat certain amoeba infections. Doxycycline comes as a regular and a coated capsule, a tablet, syrup, and a suspension (liquid), all to take by mouth. Doxycycline is usually taken once or twice a day. Drink a full glass of water with each dose of the capsule or tablet. CAS Number (2Z,4S,4aR,5S,5aS,6R,12aS)-2-(amino-hydroxymethylidene)-4-dimethylamino-5, Chemical IUPAC 10,11,12a-tetrahydroxy-6- methyl-4a,5,5a,6-tetrahydro-4h-tetracene-1,3,12-t rione PubChem Drug Bank KEGG APRD00597 C06973 Table: Details of Doxycycline The structure of doxycycline has positive changes represented in the SAR. The north western portion of Doxycycline has subsistent on R2 thru R4 which increases activity and pharmacokinetic properties compared to older tetracycline agents. Improvements include longer half life due to slower elimination, better stability and tissue penetration, and good oral availability due to higher lipophilicity. There is no R1 substitute, R2 is H, R3 is a methyl, and R4 is a hydroxyl subsistent. Typical tetracycline form nephrotoxic anhydrotetracycline in prolonged acidic medium. Also, epimerization at C4 amine from beta position to alpha causes inactivation. In basic medium, ring opening inactivates the antibiotic. However, the removal of C6 hydroxyl group improves acid and basic

10 stability. Doxycycline is less susceptible to chelation thus has less GI disturbance, such as cramps which is usually most common. Fig.: Structure of Doxycycline Interactions Doxycycline interacts with many types of medications. It can increase the absorption of digoxin, which may lead to digoxin toxicity. The gastrointestinal side effects (nausea, vomiting, stomach upset) of theophylline may be increased by doxycycline. The dosage of oral anticoagulants (blood thinners, such as warfarin) may need to be adjusted when this medication is started. Doxycycline may decrease the effectiveness of oral contraceptives (birth control pills), and pregnancy could result. Barbiturates, carbamazepine, phenytoin, and antacids can lower the levels of doxycycline in the blood, thus decreasing its effectiveness. Iron and antacid containing aluminum, calcium and magnesium can chelate doxycycline in the gastrointestinal tract and form an insoluble complex, which can decrease its absorption and, therefore, its effectiveness. Resistance Doxycycline resistance occurs via two mechanisms. First, bacteria can produce proteins that bind to ribosome and can inhibit drug binding or allow protein biosynthesis to continue in the presence of bound doxycycline. The second mechanism is through active efflux of doxycycline to the outside of the cell. Because doxycycline was once very widely used, the popularity has decreased considerably due to wide spread resistance and the introduction of newer broad spectrum agents. Mechanism of Action Doxycycline works by inhibiting protein synthesis by reversibly binding to bacterial ribosome (30s subunit) at the A site, preventing the attachment of amino acyl t-rna and leading to the termination of translation. It is more selective for the bacterial 70s ribosome versus mammalian 80s ribosome. Doxycycline also has selective toxicity

11 against bacteria because it has high efficiently transport into bacterial cells. They enter Gram negative bacteria through porins due to its hydrophilicity, and through their lipophilicity in Gram positive bacteria. The bacteria mistakes doxycycline for food thus passes through the cytoplasmic membrane by an energy requiring active transport. Divalent ions may be significant in Doxycycline effectiveness. Magnesium ions attached to the phosphates on RNA seem to aid in their initial binding to the ribosome, while magnesium in the cytoplasm will limit their ability to interact with the ribosome. Result The ligand Doxycycline plays a significant role in curing the disease anthrax. The description, interaction, resistance, mechanism of action determines the effect of drug with respect to anthrax.

12 INTERACTION STUDIES USING SWISS-PDB VIEWER Aim To analyze the interaction between the docked protein-ligand complex. Swiss-PDB Viewer Swiss-Pdb Viewer is an application that provides a user friendly interface allowing analyzing several proteins at the same time. The proteins can be superimposed in order to deduce structural alignments and compare their active sites or any other relevant parts. Amino acid mutations, H-bonds, angles and distances between atoms are easy to obtain thanks to the intuitive graphic and menu interface. Swiss-Pdb Viewer has been developed since 1994 by Nicolas Guex. Swiss-Pdb Viewer is tightly linked to SWISS-MODEL, an automated homology modeling server developed within the Swiss Institute of Bioinformatics (SIB) at the Structural Bioinformatics Group at the Biozentrum in Basel. Working with these two programs greatly reduces the amount of work necessary to generate models, as it is possible to thread a protein primary sequence onto a 3D template and get an immediate feedback of how well the threaded protein will be accepted by the reference structure before submitting a request to build missing loops and refine side chain packing. Swiss Pdb Viewer can also read electron density maps, and provides various tools to build into the density. In addition, various modeling tools are integrated and command files for popular energy minimization packages can be generated. Procedure 1. Open the Swiss-PDB Viewer. 2. Load the docked complex obtained from HEX in SPDBV. 3. Go to Windows Control panel. 4. Make the whole molecule invisible and make only the ligand molecule visible using control panel window. 5. Select the ligand using select menu select the neighbouring amino acid from the dialog box appearing a distance of 3.00A 0 click ok. 6. The amino acids around 3.00A distance with respect to the ligand appear in the graphics window. Make the labels visible using the control panel window. 7. Color by secondary structure the amino acid is colored based on the secondary structure. 8. Tools Compute H bond the H bond interaction between the ligand and the molecules within the distance of 3.00A 0 is made visible.

13 9. Tools show H bond distances the distance of the hbonds are made visible. Output Fig.: Interaction between Doxycycline with Anthrax Toxin Result The interaction study is done on the docked model of the anthrax toxin receptor protein with the inhibitor doxycycline.

14 Aim CHEMSKETCH To generate a 2D structure and SMILES notations of polar amino acids using ChemSketch. Procedure 1. Open the ChemSketch tool. 2. Select the structure mode to draw a structure. 3. To start drawing click on C, CH4 appears on the screen, the same way select any desired atom from the structure tool bar to draw, for example Cl, Br, H, N, O, S etc. 4. The atom name can be edited in the structure mode by selecting the corresponding atom and clicking the edit text button. 5. To add a double or triple bond, select the bond position with the cursor. A box will highlight it as seen to the right and then click it once for double bond and same for triple bond and again to go back to single bond. 6. To optimize the structure, especially the rings for standardizing all bond lengths and angles, trigonal and linear for 2D by using the clean structure. 7. To generate the name for a structure click generate name from structure button. 8. To generate SMILES (Simplified Molecular Input Line Entry System) for a specified molecule first select the molecule and then go to tool and choose generate SMILES notation. 9. To save files got to file in the menu and select save as option. It automatically saves it in.sk2 file format. To save it in.mol file format select export from the file menu and give the name.

15 Output Table: Chemical Structure of Non-Polar Amino Acids Generated using ChemSketch Result The chemical structure and SMILES notation of polar amino acids were generated using ChemSketch 11.

16 Aim HEX To predict the interactive molecular docking using Hex 4.2. Description Docking Docking is a method which predicts the preferred orientation of one molecule to a second when bound to each other to form a stable complex. Knowledge of the preferred orientation in turn may be used to predict the strength of association or binding affinity between two molecules using for example scoring functions. Docking is frequently used to predict the binding orientation of small molecule drug candidates to their protein targets in order to in turn predict the affinity and activity of the small molecule. Hex 4.2 Hex 4.2 is an interactive protein docking and molecular superimposition program. Currently, Hex 4.2 understands protein and DNA structures in PDB format. Hex 4.2 was written by Davie Ritchie at the University of Aberdeen. This is the main thing which distinguishes Hex 4.2 from other macro molecular (i.e. protein and DNA) docking programs and molecular calculations. The graphical nature of Hex 4.2 came about largely to visualize the results of such docking calculations in a natural and seamless way, without having to export unmanageably many and usually quite big coordinate files to one of the many existing molecular graphics packages. The graphical capabilities in Hex 4.2 are relatively primitive, although these days user can do quite a lot with a few calls to OpenGL. Procedure Step1: Open HEX.exe file. Step2: Go to file-> select open and select receptor to load the protein molecule. Step3: Again go to open and select ligand to load the ligand molecule. Step4: After loading the protein and ligand select the controls and click Docking. Step5: Docking control box will display, set default option and click Activate. Step6: Save the HEX messages and the docked structure. Output

17 Fig.: Anthrax Receptor Toxin with Doxycycline Fig.: Average Energy Values of Anthrax Receptor Toxin Docked with Doxycycline Result The Anthrax Toxin receptor was docked with Doxycyline ligand and the RMS values, the energy values and the BMP values were observed using HEX4.2