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.

Figure 1

Macroscopical: oesophagus from water buffalo heavily infected with Sarcocystis fusiformis. The infection appeared as macroscopic spindle-shaped and milky white sarcocysts embedded in the esophagus muscles (A,B). Histological: Section of sarcocyst stained with hematoxylin and eosin (C, × 40; D,E × 100; F × 400). Thin sarcocyst wall (opposing arrowheads), septa (S) groups of bradyzoites (br), and faint staining metrocytes (me).

Figure 2

A phylogenetic tree for Sarcocystis spp. generated by a Fast Tree analysis using an NCBI alignment of (A) 18SrRNA, ITS1 (B), and COX1 (C) nucleotides. (*) Sarcocystis isolate.

Figure 3

A phylogenetic tree form Sarcocystis spp. from water buffaloes generated by a FastTree analysis using an NCBI alignment of18SrRNA showing 50 clones.

Figure 4

A typical chromatography profile of ammonium sulfate fraction of Sarcocystis spp. protease from infected water buffaloes on DEAE-Sepharose (15 × 1.6 cm) pre-equilibrated with 50 mM Tris–HCl buffer, pH 7.0. Three ml fractions were collected at a flow rate of 60 ml/h and 4 °C.

Figure 5

SDS-PAGE for homogeneity and molecular weight determination of protease from Sarcocystis fusiformis. (M) Protein markers; (1) Crude extract, (2) Ammonium sulfate fraction, (3) Purified enzyme of 0.2 M NaCl DEAE-Sepharose fraction.

Figure 6

(A) Optimum-pH of the purified protease, (B) Optimum-temperature of the protease activity, (C) Relative activity % of the purified protease over different time periods (15–90 min) at 50 °C using the caseinolytic standard assay, (D) Thermal stability of the purified enzyme. The enzyme was incubated for 30 min at various temperatures (25–70 °C) before adding the substrate. After that, it was cooled in an ice bath. The residual activity was assessed using the caseinolytic standard assay. The values represent mean ± SD (n = 3).

Figure 7

Michaelis–Menten plot of the purified protease using azocasein as a substrate at a concentration range of 0.025–0.20 mg/ml and reaction rate (velocity) has been measured under standard assay conditions.