Novel Protein of the Animal Parasite, Toxoplasma Gondii - Lab Report Example

NAME : TOPIC : TUTOR : Question 1 Exon/ Intron structure of the toxoplasma gondii is that it consists of 35 kb genome that segregates and replicates with an organelle during proliferation of the parasite. The presence of apicoplast that segregate with the daughter cells. It has other organelles that function differently polar rings, conoid, apical rings micropore, pellicle,golgi complex, smooth endoplasmic reticula, micropore, subpellicular, mitochondrian, amylopectin granules, golgi adjunct and the rhoptries. It is located in the Y-chromosome that is present in all warm-blooded animals. It is located in the actin, keratin, myosin and the tubulin genes. This is because the parasite is a protein and precisely a cytoskeletal protein that is includes the presence of the actin, coronin, FtsZ, keratin, tubulin and myosin genes. Toxoplasmic gondii can be classified as a apicomplexa parasite since they have the same similarities in terms of unique organelles such as apicoplast as well as an apical complex. In both cases their dependent on hosts and they invade the host through penetration. To a greater extent the Toxoplasma gondii can be classified as a eukaryotic parasite as in both cases they invade the warm-blooded animals and have complex life cycles as stated clearly in the report. The expression profile of Toxoplasma gondii is divided into two actively division and inactive division. The life cycle of Toxolasma gondii is divided into three as discussed in the report that is bradyzoites, tachyzoites and sporozoites. Tachyzoites as the first involve active division of the cells. Bradyzoites involves division of the cells to a smaller extent as compared to the first stage. Question 2 Title Characterization of a novel protein of the animal parasite, Toxoplasma gondii Introduction So as to identify novel proteins and their characterizations I used two approaches. My first approach is known as the radiolabeling approach that uses the photoactivatable compound, 5-[125] iodonaphthalene 1- azide (INA) and hydrophobic. So as to be able to activate iodonaphthalene 1- azide I used photosentisitizing fluorochromes through restriction of fluorochromes to the pellicle. The labeling of iodonaphthalene 1- azide aimed at targeting non – GPI – anchored membrane- embedded proteins of the pellicle. INA can be said to be highly hydrophobic, it can be located in the lipid bilayer membranes of the cellular part of toxoplasma gondii. When INA is activated its azide part is converted into a reactive nitrene. Though covalent bonds it attaches to the near protein domain that is found in the lipid bilayer. Results IMC membranes and plasma membranes of the parasites pellicle acted as the photosensitized labels with INA that needed restrictions through photosensitizing fluorochrome of the membrane. Through fluorescence microscopy c16-eosin can be concentrated in the parasite pellicle1. The pattern of the INA labeled proteins after photosensitization (direct activation) in c16-eosin was recorded. Analysis showed that it is possible to label large numbers of proteins through direct activation. In the absentia of c16-eosin INA cannot be activated in high wavelength of light as used in photosensitization and no proteins can be labeled as well. Eosin – labeled parasites was not a distinct method of labeling since the photosensitizing fluorochrome is concentrated in the parasite pellicle. Other substances that showed peripheral labeling included fluorochromes such as DiOC16 (3,3’ – dihexadecyloxacarbocyanine) and c18- fluorescein( 5- octadecanoylaminofluorescein perchlorate). So as to successfully isolate and characterize novel INA-labeled parasite proteins, labeled extracts were separated through 2-D gel electrophoresis. Broad- pH- range gels (pH 3 to 10) resolved most of the parasite proteins the method was demonstrated through silver straining. While identifying INA – labeled proteins 2-D gels, octylglucoside was used as a major detergent whereby prominent photosensitized proteins were identified in the pH 4 to 7. In the first protein that resembles a charge reain with 4 to 5 spots was defined as an isoform of GAP45 by Western blotting. In the second protein that had been purified with 2-D gels as well as trypsin then analyses done by mass spectrometry, the results were tryptic peptides on the single EST that is then found in T. In the experiment involved 246 specimens. Substance used Number of parasites activated C16-eosin 158 DIOC16 50 C18- FLUORESCEIN 38 Discussion In the experiment it was clear that INA activation can be done in two methods direct and indirect activation. Direct activation occurs when there is UV light exposure, the membrane-embedded proteins that are in multiple membrane compartments. In this process, 370 nm wavelengths are negligible while in indirect process the same wavelengths can activate it through photosensitization. Photosensitization is the transfer of energy from a fluorochrome (excited) to a molecule of INA nature that is within the collisional distance. Hence it true to claim that photosensitizing fluorochromes that have been confined to the parasite pellicle, proteins of the pellicle that membrane – embedded can be selectively labeled with INA. Toxoplasma gondii has a structure of triple bilayer that consists of membranes that are found in the inner membrane complex and the plasma membrane. The roles of the integral protein membranes in a host are to provide invasion, attachment and cell recognition. Despite the glycosylphosphatidylinositol (GPI) – anchored proteins making it difficult to identify non – GPI – linked proteins, the above approach made it possible to analyze it. Through photosensitization, it is possible to label proteins of the membrane in pellicle. In my findings using the approach discussed above I was able to identify a novel 22 –kDa protein known as Photosensitized INA – labeled protein 1( PhOL1). This parasite doesn’t have a sits of lipid modification nor transmembrane domain. PhIL1 is basically found in apicomplexan parasites. In the apicomplexan parasite it is highly concentrated towards the apical end that is on the basal to the conoid. It is true to state that Phil1 is in association with parasite cytoskeleton. In accordance to my findings, gondii can be said to be a parasite of obligate intracellular nature. It has a tendency of repetition of its cycle that is invasion, replication and finally host cells’ lysis, this happens in the tachyzoite stage. This stage is a critical part of the life cycle of the parasite and it can cause damage of tissue during acute infection. Under the pellicle there is a filamentous cytoskeletal structure that is called subpellicular network. The subpellicular network has characterized proteins such as IMC1 and IMCI2. These two proteins (IMC1 and IMC2) has a similar structure as compared to the mammalian intermediate filament proteins and it is also homologous to the articulins of ciliate protozoa. Findings prove that subpellicular network has IMC on its outer face and 22 subpellicular microtubules in the inner face. The subpellicular microtubules have an estimated extension of two-thirds of the parasites’ length, located at the apical end of the basal polar ring that is just at the posterior end to the tubulin – based conoid. The functions of the integral membrane proteins are to provide appropriate extracellular substrate interaction, signal transduction, interaction maintenance in subpellicular network, microtubules and pellicle as well as force transduction Conclusion Toxoplasma gondii has three infectious stages that includes bradyzoites, tachyzoites and sporozoites. Tachyzoites stage It is a stage of rapid multiplication of a cell between an intermediate host and non – intentestinal epithelial cells of a host. The aggregation of many tachyzoites is known as clones, groups or terminal colonies. A tachyzoite has a cresent shape that measures 2 by 6 UM having the conoidal in the anterior end and a round shaped posterior end. Techyzoite has several organelles such as polar rings, conoid, apical rings micropore, pellicle,golgi complex, smooth endoplasmic reticula, micropore, subpellicular, mitochondrian, amylopectin granules, golgi adjunct and the rhoptries. Tachyzoites move through flexing, undulating, rotating and gliding. Bradyzoites stage It is a stage that is symbolized by slow multiplication of organisms within a tissue cyst that grow and remain intracellular. They have a spheroidal shape with a diameter of 70UM, they are mostly found in visceral organs. The only structure difference between bradyzoites and tachyzoites is in the rhoptries contents whereby tachyzoites’ are labyrinthine. Sporozoites These is the last stage of the cycle and it is defined by the residence of the parasites in the oocyts. When consumed by a warm-blooded host the oocyst releases sporozoites. Toxoplasma gondii is a protozoan pathogen is an opportunistic human pathogen that mostly affect immunocompromised to either the fetus or an adult. Generally it is true to claim that this pathogen is abundant in warm-blooded mammals. Other parasites that have the same behavior as toxoplasma gondii are of the phylum Apicomplexa, T. References Tenter, A. M., Heckeroth, A. R., & Weiss, L. M. (2000). Toxoplasma gondii: from animals to humans. International journal for parasitology, 30(12), 1217-1258. Frenkel, J. K., Dubey, J. P., & Miller, N. L. (1970). Toxoplasma gondii in cats: fecal stages identified as coccidian oocysts. Science, 167(3919), 893-896. Howe, D. K., & Sibley, L. D. (1995). Toxoplasma gondii comprises three clonal lineages: correlation of parasite genotype with human disease. Journal of infectious diseases, 172(6), 1561-1566. Suzuki, Y., Orellana, M. A., Schreiber, R. D., & Remington, J. S. (1988). Interferon-gamma: the major mediator of resistance against Toxoplasma gondii. Science, 240(4851), 516. Sibley, L. D., & Boothroyd, J. C. (1992). Virulent strains of Toxoplasma gondii comprise a single clonal lineage. Nature, 359(6390), 82-85. Read More