Potential Differential Roles Of The Two Phosphoglucomutase Isoforms In The Protozoan Parasite Cryptosporidium Parvum

Cryptosporidium parvum is an important zoonotic protozoa that can cause severe or fatal diarrhea in the hosts.It is one of the two pathogens responsible for moderate to severe diarrhea in infants and young children in developing countries.This parasite lacks the tricarboxylic acid(TCA)cycle and β-oxidation,so that its energy metabolism mainly depends on glycolysis.Therefore,key glycolysis enzymes are potential targets for drug development.Glycolysis is usually carried out in the cytoplasm,where phosphoglucomutase(PGM)catalyzes the conversion of glucose 1-phosphate(G1P)and glucose 6-phosphate(G6P).Cryptosporidium parvum contains two evolutionarily duplicated PGM1 isoforms,namely CpPGM1A(cgd2＿3260)and CpPGM1B(cgd2＿3270).The former has a classic PGM structure,while the latter contains an N-terminal signal peptide and a linker sequence.The metabolic pathway of C.parvum is extremely simplified.Therefore,why the parasite retains two PGM isoenzymes is an intriguing biological question.In this study,a prokaryotic system was used to express maltose-binding protein(MBP)-fused recombinant CpPGM1 A and CpPGM1 B proteins for determining their enzymatic activity.Both CpPGM1 isoforms could catalyze the conversion from G1 P to G6 P.The Vmax and Km values of CpPGM1 A was 7.30 U(U = μmol/mg/min)and0.17 m M,respectively.The Vmax and Km values of CpPGM1 B was 2.76 U and 0.13 m M,respectively.Two polyclonal antibodies specific to CpPGM1 A and CpPGM1 B were also raised in rabbits.Western blot analysis suggested some levels of protein aggregation of CpPGM1 A and CpPGM1 B from the parasite.Immunofluorescence assay(IFA)showed that CpPGM1 A was mainly distributed in the cytoplasm of sporozoites with two enriched spots immediately before and after the nucleus.CpPGM1 B was mainly localized on the surface of the anterior half of the sporozoites with some granular dots.In the intracellular parasites,CpPGM1 A was mainly located in the merozoite membrane,while CpPGM1 B was located in the parasitophorous vacuole membrane(PVM).Quantitative RT-PCR analysis showed that both CpPGM1 A and CpPGM1 B genes were expressed during the parasite life cycles,but the relatively levels of CpPGM1 A were much higher than those of CpPGM1 B.In sporozoites,the protein level of CpPGM1 A was also higher than that of CpPGM1 B.These observations suggested that these two CpPGM1 isoenzymes might play differential roles in the parasite.CpPGM1 A might be one of the housekeeping proteins in the glycolytic pathway.While CpPGM1 B were enzymatically active,its distribution along the plasma membrane and PVM implied that this protein might possess additional roles.