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Purification and characterization of cysteine protease of Sarcocystis fusiformis from infected Egyptian water buffaloes.

ABSTRACT: Sarcocystis spp. infects water buffaloes (Bubalus bubalis) causing sarcocystosis. In the present study, Sarcocystis fusiformis was recognized in Egyptian water buffaloes based on histological observation and molecular analysis of internal transcribed spacer 1 (ITS1), 18S ribosomal RNA (18S rRNA) and cytochrome c oxidase subunit I (COX-1) gene fragments. Chemotherapy and vaccines against Sarcocystis spp. could potentially target proteases because they may play a crucial role in the infection. Cysteine proteases are multifunctional enzymes involved in vital metabolic processes. However, the involvement of proteases in S. fusiform infection has not yet been characterized. Here, the purification and study on some biochemical properties of protease isolated from cysts of S. fusiform were carried out. Protease with a molecular weight of 100 kDa was purified. LC-MS/MS analyzed the protein sequence of purified protease and the data suggested that the enzyme might be related to the cysteine protease. The purified protease exhibited maximum activity at pH 6 and a temperature of 50 °C. The Michaelis-Menten constant (K_m), the maximum velocity (V_max), and the turnover number (K_cat) were determined. The complete inhibition effect of cysteine inhibitors indicated that the purified enzyme is a cysteine protease. The results suggested that S. fusiform proteolytic enzyme may be necessary for parasite survival in water buffaloes by digesting host tissues. Therefore, cysteine protease could be a suitable target for vaccinations.

SUBMITTER: Barakat AZ

PROVIDER: S-EPMC10522634 | biostudies-literature | 2023 Sep

REPOSITORIES: biostudies-literature

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Purification and characterization of cysteine protease of Sarcocystis fusiformis from infected Egyptian water buffaloes.

Barakat Amal Z AZ Abdel-Aty Azza M AM Ibrahim Marwa K MK Salah Hala A HA Hegazy Usama M UM Azouz Rasha A M RAM Bassuiny Roqaya I RI Shaapan Raafat M RM Mohamed Saleh A SA

Scientific reports 20230926 1

Sarcocystis spp. infects water buffaloes (Bubalus bubalis) causing sarcocystosis. In the present study, Sarcocystis fusiformis was recognized in Egyptian water buffaloes based on histological observation and molecular analysis of internal transcribed spacer 1 (ITS1), 18S ribosomal RNA (18S rRNA) and cytochrome c oxidase subunit I (COX-1) gene fragments. Chemotherapy and vaccines against Sarcocystis spp. could potentially target proteases because they may play a crucial role in the infection. Cys ...[more]

PMID: 37752241

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Project description:Traumatic pericarditis (TP) remains a serious problem facing bovine producers; particularly in the developing countries; causing severe economic losses. This study was carried out on 47 buffaloes including; control buffaloes (n = 20) and buffaloes with TP (n = 27) at Assiut governorate, Egypt. All animals were subjected to thorough clinical examination, whole blood profiling, blood serum biochemical assays, and radiographic and ultrasonographic examinations. The recorded ultrasonographic findings included; the contractility, contour and shape of the reticulum, shape and size of the heart and spleen involvement. All data were statistically analysed. Buffaloes with TP showed lymphocytic leucocytosis, increase in serum activities of AST, hyperproteinaemia, hypoalbuminaemia and hyperglobulinaemia. Radio-opaque metal foreign bodies, cardiomegaly and loss of the normal cardiac shape and contour were the most common radiographic findings in the diseased buffaloes. Ultrasonographically, the diseased buffaloes showed either acute pericarditis (n = 10) or chronic suppurative pericarditis (n = 17). Complete cessation of reticular contractions (0/3 min) and displacement of the reticulum from the diaphragm by a distance about 2.6-4.5 cm were the most common ultrasonographic findings of both types of pericarditis. In addition, the reticulum had uneven contour and the heart showed characteristic changes in its size, shape and contractility that was either accelerated or reduced. Reticular abscesses and peritoneal effusions were also imaged in all diseased buffaloes. In acute TP; the affected heart was enlarged with strong and clear cardiac contractions. Accumulation of hypoechoic fluids interspersed with echogenic deposits of fibrin within heart tissue and pericardium was observed. In chronic suppurative pericarditis, cardiomegaly, thickening of the cardiac wall and loss of the characteristic recognisable heart shape were the common ultrasonographic findings. Accumulation of hypoechoic fluids (usually pus) interspersing with echogenic deposits of fibrin within the pericardium, ventricles, atria and valves was also observed. In conclusion, ultrasonography with the aids of other diagnostic tools such as clinical findings, laboratory analysis and radiography has a high efficacy in evaluation of diseased buffaloes with TP.

Project description:BackgroundSchistosoma japonicum (S. japonicum) is an important zoonotic parasite that is prevalent in China and parts of Southeast Asia. Water buffaloes are an important reservoir and the main transmission sources of S. japonicum. However, self-curing and resistance to re-infection have been observed in water buffaloes.ResultsIn this study, we compared the morphometry and differences in transcriptional expression of adult S. japonicum worms recovered from primary-infected and re-infected water buffaloes using Illumina RNA-sequencing (RNA-Seq) technology. Results of morphometry analysis revealed that adult S. japonicum worms recovered from re-infected water buffaloes were runtish with smaller organs. The ventral length of male worms was shorter in re-infected buffaloes (328 ± 13 vs 273 ± 8 µm, P < 0.05), and in female worms the oral sucker length (44 ± 3 vs 33 ± 5 µm, P < 0.05), ovary length (578 ± 23 vs 297 ± 27 µm, P < 0.05) and width (150 ± 8 vs 104 ± 9 µm, P < 0.05) were shorter, with fewer eggs in the uteri (41 ± 2 vs 12 ± 1, P < 0.05). Of 13,605 identified genes, 112 were differentially expressed, including 51 upregulated and 61 downregulated genes, in worms from re-infected compared with primary-infected water buffaloes. Gene ontology (GO) enrichment analysis revealed that GO terms such as "oxidation-reduction process", "calcium-dependent phospholipid binding", "lipid binding" and "calcium ion binding" were significantly enriched in downregulated genes, whereas GO terms related to metabolism and biosynthesis were significantly enriched in upregulated genes. The results revealed that the downregulation of some important genes might contribute to a reduction in worm numbers and maldevelopment of surviving worms in re-infected water buffaloes. Furthermore, upregulation of genes related to metabolic processes and biosynthesis might be a compensatory mechanism of worms in disadvantageous environments.ConclusionsTo our knowledge, our results present the first large-scale transcriptional expression study identifying the differences between adult S. japonicum worms from primary-infected and re-infected water buffaloes, and particularly emphasize differential expression that may affect the survival and growth of worms in re-infected water buffalo. This will provide new insight into screening for anti-schistosome targets and vaccine candidates.

Dataset Information

Purification and characterization of cysteine protease of Sarcocystis fusiformis from infected Egyptian water buffaloes.

Publications

Purification and characterization of cysteine protease of Sarcocystis fusiformis from infected Egyptian water buffaloes.

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