| Malaria, the deadliest of the human infectious disease with high incidence and fatality rate, is transmitted by Anopheles mosquito vectors. Control of malaria will be confronted with formidable challenge because of the rapid spread of multi-drug resistant parasite, the emergence of insecticide-resistant Anopheles mosquito and the lack of effective malaria control strategies and it is difficult to control malaria by traditional methods. It is urgent to search alternative approaches and techniques to fight against the disease. Malaria transmission occurred by mosquito bite. When Plasmodium sporozoites located in the salivary glands of Anopheles mosquito enter the bloodstream, on reaching the liver through the KC (Kuppfer cells) of liver sinusoidal cell layer in several minutes, sporozoites invade hepatocytes and develop into EEF (exo-erythrocytic forms). After multiple cycle of replication, the parasite initiate the erythrocytic cycle that causes the disease malaria. Therefore, it is possible to prevent malaria infection by interrupting the invasive Plasmodium, because the key point is the progress of hepatocytes invasion. Preerythrocytic-stage vaccines that are aimed at suppressing the invasive sporozoites, thereby preventing the development of the pathogenic blood stage, is one of the important malaria vaccines. The maturation of Plasmodium sporozoite infectivity follows a developmental program. Upon disruption of the mature midgut oocysts, sporozoites egress and access to hemolymph in the hemocoel, and then reside in salivary glands. After two days development, sporozoites have the ability to invade hepatocytes. Therfore, it is important to research the differential molecules between virulence salivary gland sporozoites and non-virulence oocyst sporozoites. A major limitation for the molecular analysis of the development of Plasmodium sporozoites in the mosquites has been the lack of methods to obtain and purify enough parasite from mosquito oocysts and salivary glands at this stage. With the completion of the genomic sequence of the human malaria parasite, Plasmodium falciparum, and the availability of large amounts of genomic sequence data from other Plasmodium species (Plasmodium yoelii, Plasmodium vivax, Plasmodium berghei, Plasmodium knowlesi, and Plasmodium gallinaceum), researchers will soon have data that many believe will provide the foundation of malaria research for the foreseeable future, and it make the analysis of function genes and the research of proteome most important in the post-genomic phase. Large data that target identification of the differential invasive genes and proteins of Plasmodium sporozoites help us to recognize the molecular mechanisms of the Plasmodium life cycle stages, offer the theory foundation for the protection and therapy of malaria, and lead to identify the novel targets of drugs and vaccines and improve strategies to control malaria in the world. Base on the above reasons, two basic researches on the model of Anopheles stephensi-Plasmodium yoelii for the invasive molecules will be made from genes and proteins. Firstly, the differential cDNA sequences of salivary gland sporozoites versus oocyst sporozoites of the Plasmodium yoelii will be obtained by DD-PCR (differential display RT-PCR) and TA cloning. And the potential virulence genes will be identified by BLAST research. Secondly, the differential invasion-related proteins between the salivary gland sporozoites and oocyst sporozoites of the Plasmodium yoelii will be obtained by 2-DE (two-dimensional gel electrophoresis) and MALDI-TOF-MS (matrix-assisted laser desorption/inoization time of flying mass spectrometry), thus the calculated molecular mass and the potential structure of the proteins will be identified. The potential function of peptides will be initially identified by the obtained PMF (peptide mass fingerprint) by bioinformatics tools, and the invasive proteins will be obtained. The experimental contents and main results are shown as follows: 1. Data from complete gene sequences of Plasmodium have revealed an overall A+T composition of 80% and even more, while it is only 40～50% in mosquito genome. Thus, we utilized a special 60% A+T primer matching the AT rich Plasmodium genome by DD-PCR. This modification of the DD protocol that resulted in the amplification of parasite-specific messages, avoided the barrier of the difficulty to obtain and purify enough sporozoites. Thus, the differential cDNA of salivary gland sporozoites versus oocyst sporozoites were obtained. Because sequencing of the DD-PCR yielded 3 cDNA segments including PyDg1 ～3(Plasmodium yoelii differential gene 1～3) which all match the cDNA sequences to the Plasmodium genome, this suggest the adjusted DD-PCR protocol can amplified parasite-specific gene products from the mosquito genome infected sample. The ESTs wereused to retrieve the GenBank database by BLAST research. The PyDg1(263bp) segment is shared with the homology of 86%(186/215) as compared with uis16-1 of Plasmodium berghei; The PyDg2(517bp) segment is shared with the homology of 74%1(241/325) as compared with Plasmodium berghei spect cDNA for sporozoite microneme protein essential for cell traversal;The PyDg3(244bp) segment is shared with the homology of 92%(226/244) as compared with Plasmodium berghei spect2 cDNA for sporozoite protein with MACPF related domain. We propose that the three differential genes expressed in invasive sporozoites may participate in the invasion proceeds and the developing into EEF, and it establish the foundation of further research for the molecule mechanisms in the mutation of invasive sporozoites. 2. The virulence sporozoites can invade host hepatocytes mediated by receptor and ligand after Plasmodium oocyst sporozoites have developed to salivary gland sporozoites. There are many proteins expressed specifically during the maturation of sporozoite. And change of this process is occurred in the interaction with host hepatocytes and on the cellular level. However, it is possible to elucidate invasive mechanisms of the sporozoites by the systematic study and entire research of gene or protein level. Thus, we utilized the method of 2-DE to research the differential protein of virulence salivary gland sporozoites versus non-virulence oocyst sporozoites of the Plasmodium yoelii, and the potential invasive proteins were identified by MALDI-TOF-MS and PMF. The differential cDNA of salivary gland sporozoites versus oocyst sporozoites were obtained. There are six visible differentially expressed proteins, including PyDp1～6(Plasmodium yoelii differential protein 1～6). The PyDp1～5 are parasite-specific proteins, and they are some potential membrane proteins, surface proteins and motion-related proteins. The PyDp1(pI:pH4, MW:24×103) matches the 235 kDa rhoptry protein(Plasmodium yoelii); The PyDp2(pI:pH9.5, MW:16.5 ×103) matches Ser/Thr protein phosphatase (Plasmodium yoelii); The PyDp3(pI:pH8, MW:30×103) matches cAMP-dependent protein kinase; The PyDp4(pI:pH8, MW:29 ×103) matches dynamin-like protein(Plasmodium chabaudi); The PyDp5(pI:pH4.5, MW:37×103) matches variant surface protein(Plasmodium falciparum). We made a conclusion that these proteins may have the function to participate the progress of Plasmodium sporozoites invading hepatocytes, and they can also be the target of novel transmission-blocking strategies and multi-subunit vaccins. In addition, the mosquitoorigin PyDp6(pI:pH3.5, MW:36.5 ×103) matches ENSANGP00000022306 (Anopheles gambiae). Our finding suggest that after the ingression of sporozoites, the organism of mosquito salivary gland has some adaptable changes and express new proteins to suit the development of parasite. In conclusion, we found that salivary gland sporozoites up-regulate some genes that are not expressed earlier in development. Among them are potential regulatory proteins, secretory molecules, and metabolic enzymes that probably control sporozoite virulence. We have initially researched the invasive molecules of Plasmodium sporozoites from genes and proteins, and study the molecular mechanisms of hepatocytes invasion systematically. This will provide us a stronger foundation for the development of all potential new anti-malarial drugs and vaccines. |
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