Abstract
BackgroundEnolase is an essential multifunctional glycolytic enzyme that is involved in many biological processes of apicomplexan protozoa, such as adhesion and invasion. However, the characteristics of enolase in Cryptosporidium parvum, including the location on the oocyst and the enzyme activity, remain unclear.MethodsThe C. parvum enolase gene (cpeno) was amplified by RT-PCR and sequenced. The deduced amino acid sequence was analysed by bioinformatics software. The gene was expressed in Escherichia coli BL21 (DE3) and purified recombinant protein was used for enzyme activity analysis, binding experiments and antibody preparation. The localisation of enolase on oocysts was examined via immunofluorescence techniques.ResultsA 1,350 bp DNA sequence was amplified from cDNA taken from C. parvum oocysts. The deduced amino acids sequence of C. parvum enolase (CpEno) had 82.1% homology with Cryptosporidium muris enolase, and 54.7–68.0% homology with others selected species. Western blot analysis indicated that recombinant C. parvum enolase (rCpEno) could be recognised by C. parvum-infected cattle sera. Immunolocalization testing showed that CpEno was found to locate mainly on the surface of oocysts. The enzyme activity was 33.5 U/mg, and the Michaelis constant (Km) was 0.571 mM/l. Kinetic measurements revealed that the most suitable pH value was 7.0–7.5, and there were only minor effects on the activity of rCpEno with a change in the reaction temperature. The enzyme activity decreased when the Ca2+, K+, Mg2+ and Na+ concentrations of the reaction solution increased. The binding assays demonstrated that rCpEno could bind to human plasminogen.ConclusionThis study is the first report of immunolocation, binding activity and enzyme characteristics of CpEno. The results of this study suggest that the surface-associated CpEno not only functions as a glycolytic enzyme but may also participate in attachment and invasion process of the parasite.
Highlights
Enolase is an essential multifunctional glycolytic enzyme that is involved in many biological processes of apicomplexan protozoa, such as adhesion and invasion
BLAST analysis demonstrated that the sequence had 100% identity with the reported C. parvum Iowa II enolase gene sequence (GenBank XM_626138), and encoded a 449 amino acid protein with a theoretical molecular weight of 48.6 kDa
The highest homology of amino acid sequences mainly came from apicomplexan protozoa, such as Eimeria tenella, Theileria annulata, T. gondii, Neospora caninum and P. falciparum, which exhibited 68.0, 67.9, 67.3, 66.9 and 66.8% similarities, respectively (Fig. 1)
Summary
Enolase is an essential multifunctional glycolytic enzyme that is involved in many biological processes of apicomplexan protozoa, such as adhesion and invasion. The characteristics of enolase in Cryptosporidium parvum, including the location on the oocyst and the enzyme activity, remain unclear. Cryptosporidium is an important zoonotic protozoan with a wide range of hosts, including humans and various animals. No effective vaccine or drug has been found to prevent this disease, so it is important to identify specific target antigens and better understand the host immune response to the parasite. Many moonlighting proteins in Cryptosporidium have been found to play an important role in parasite adhesion and invasion. C. parvum elongation factor 1α (EF-1α), a novel protein associated with host cell invasion could be a candidate vaccine antigen against cryptosporidiosis [4]. Cryptosporidium parvum Clec (CpClec), a novel mucin-like glycoprotein, contains a C-type lectin domain (CTLD) and localised to the surface of
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