Fractionation Of Sporogonial Stages Of Nosema Bombycis And Study On Spore Phagocytosis

Microsporidia are spore-forming fungal eukaryotes which develop as obligate intracellular parasites. They can infect an extremely wide range of hosts in the animal kingdom. So far, more than1200microsporidia species belonging to150genera have been discovered. Since the first report of microsporidia infecting human beings in1959, fourteen microsporidia species has been reported as the cause of chronic and life-threatening diseases in human, which drew great medical and scientific attentions. Microsporidia possess a highly specialized infection mechanism thus having a potential use as biocontrol agents. N. bombycis, the first described microsporidium, is the pathogen causing silkworm pebrine which prevailed in Europe, America and Asia during the mid19th century and is still epidemic and causes heavy economic losses in silk producing countries such as China. However, molecular biology research of N. bombycis is still in its infancy due to a serious shortage of basic data accumulation. In the present study, we purified the sporogonial stages of N. bombycis, built a platform for N. bombycis proteomics studies, spore phagocytosis mechanism, and the possible role of spore wall protein during the infection stage. Main results are as follows:(1) This study established a Percoll-gradient based procedure to separate the stages of N. bombycis life-cycle from its natural host. Utilization of continuous gradient consisting of a Percoll-sucrose mixture provides each spore fraction with satisfying degrees of purity. Four cell fractions representative of different life stages of N. bombycis were successfully isolated and purified:empty germinated spore husks, early sporoblasts, late sporoblasts, mature spores. High-purity sporogony phase of spores can be good materials for further biochemical and molecular biology research.(2) We built a platform for N. bombycis proteomics studies and used this platform to analyze protein expression differences between mature spores and late sporoblasts. Total protein extracted by acid-washed glass beads treatment avoiding protein degradation or pollution can be directly used for two-dimensional electrophoresis. Non-linear large protein gels guaranteed high resolution protein profile. One hundred high abundance protein spots (including spots which are different with late sporoblasts) were dug for MALDI-TOF/TOF MS analysis. Eighty-seven protein spots got good mass spectra, and15proteins were identified with high credibility by database research. Protein spot1, the most abundant protein expressed in mature spores but lack in late sporoblasts, matched a186-amino acid protein which was previously described as NbSWP5(3) NbSWP5possesses a20-amino-acid signal peptides and is predicted to be an extracellular (e.g., cell wall) protein. No N-glycosylation sites are predicted in NbSWP5, however, eight O-glycosylation sites are predicted. Neither protein homologies nor protein domains are predicted by using the BLAST program and the Pfam database. To further characterize NbSWP5, we produced a recombinant protein in E. coli NbSWP5-specific polyclonal antibody generated by immunizing rabbit with purified protein was used in Western blot analysis. A single-20kDa band was detected from the N. bombycis spore protein lysate. Immunoelectron microscopy study confirmed NbSWP5to be a high expressed exosporal protein.(4) KOH-treated spores and cold-storaged spores with spore wall defected can be more easily internalized by host cells than freshly recovered intact spores. Indeed, freshly recovered spores seemed to be resistant in host cell phagocytosis. Moreover spore precursor cells lacking NbSWP5is more likely to be phagocytized by host cells than mature spores.(5) Exogenous recombinant NbSWP5can reduce the rate of phagocytosis of spores in a dose-dependent manner. We therefore infer that NbSWP5is a candidate protective protein like RodAp to avoid spore pathogen-associated molecular patterns being recognized by responsive cells. As NbSWP5protein shares no homology with RodAp or any other identified protective proteins, its function remains to be further verified.