Ximei Zhan

Quantitative Analysis of Replication and Tropisms of Dengue Virus Type 2 in Aedes albopictus

Dengue virus serotype 2 (DENV-2) RNA replication profiles and tropisms were studied by using quantitative RT-PCR (q-RTPCR) in intrathoracically infected Aedes albopictus. The virus RNA replication profiles were diverse in mosquito organs. In fat body, brain, salivary gland, and malpighian tubes, it peaked at 8, 23, 23, and 27 days post-infection, respectively, and then, all declined. In midgut, it increased all the time and had no trend of decline. In ovary, it had no apparent increase. Subsequent Western blotting of DENV-2 E protein had similar results. Using ribosomal protein 7 (rpS7) as an internal control, we found that, in salivary gland, brain, fat body, and midgut, the average DENV-2 RNA levels (DENV-2 RNA/rpS7 mRNA) were 1,028, 464, 5.6, and 6.2, respectively; in malpighian tubes, it was 1, and in ovary, it was far less than 1. These results suggest that infection profiles and tropism of DENV-2 RNA in Ae. albopictus organs are significantly different.

Preliminary molecular characterization of the human pathogen Angiostrongylus cantonensis

BackgroundHuman angiostrongyliasis is an emerging food-borne public health problem, with the number of cases increasing worldwide, especially in mainland China. Angiostrongylus cantonensis is the causative agent of this severe disease. However, little is known about the genetics and basic biology of A. cantonensis.ResultsA cDNA library of A. cantonensis fourth-stage larvae was constructed, and ~1,200 clones were sequenced. Bioinformatic analyses revealed 378 cDNA clusters, 54.2% of which matched known genes at a cutoff expectation value of 10-20. Of these 378 unique cDNAs, 168 contained open reading frames encoding proteins containing an average of 238 amino acids. Characterization of the functions of these encoded proteins by Gene Ontology analysis showed enrichment in proteins with binding and catalytic activity. The observed pattern of enzymes involved in protein metabolism, lipid metabolism and glycolysis may reflect the central nervous system habitat of this pathogen. Four proteins were tested for their immunogenicity using enzyme-linked immunosorbent assays and histopathological examinations. The specificity of each of the four proteins was superior to that of crude somatic and excretory/secretory antigens of larvae, although their sensitivity was relatively low. We further showed that mice immunized with recombinant cystatin, a product of one of the four cDNA candidate genes, were partially protected from A. cantonensis infection.ConclusionThe data presented here substantially expand the available genetic information about the human pathogen A. cantonensis, and should be a significant resource for angiostrongyliasis researchers. As such, this work serves as a starting point for molecular approaches for diagnosing and controlling human angiostrongyliasis.

MIC6 associates with aldolase in host cell invasion by Toxoplasma gondii.

The transmembrane microneme protein MIC6 and its partner MIC1, MIC4 comprise an adhesive complex that play important roles in host cell attachment by the obligate intracellular parasite Toxoplasma gondii. Successful penetration of host cells by T. gondii depends on coordinated interactions between MICs complex and the parasites cytoskeleton. We have identified that the carboxy-terminal cytoplasmic domain (C domain) of MIC6 interacts with aldolase and the parasite cytoskeleton. Our finding uncovers new features regarding MIC6–aldolase interactions in host cell invasion by T. gondii.

Differential proteomics of Aedes albopictus salivary gland, midgut and C6/36 cell induced by dengue virus infection

The interaction between dengue virus (DENV) and vector mosquitoes are still poorly understood at present. In this study, 2-D DIGE combined with MS was used to analyze the differential proteomes of Aedes albopictus salivary gland, midgut and C6/36 cells induced by DENV-2. Our results indicated that the virus infection regulated several functional classes of proteins. Among them, 26 were successfully analyzed by real-time RT-PCR. The mRNA levels of 15 were the highest in salivary gland, 2 in midgut and none in C6/36 cells, however, 18 were the least in fat body compared to other organs. Interestingly, the changes of differential proteins mRNA were the most obvious in fat body post-infection. Chaperone, cytoskeleton and energy metabolism enzyme were the most down- or up- regulated proteins after DENV-2 infection. The abundant expression of these proteins in salivary gland may relate to its high susceptibility.

Cloning and functional expression of Rh50-like glycoprotein, a putative ammonia channel, in Aedes albopictus mosquitoes.

Evidence has shown that female mosquitoes can deaminate more than 80% of the ingested bloodmeal protein amino acids, and thus lead to a massive amount of ammonia production. Ammonia transport is a critical step for detoxifying ammonia in organisms. Here we characterized a putative ammonia channel gene, Rhesus (Rh) 50 glycoprotein, from Aedes albopictus (AalRh50) and determined the difference of its expression profile in different tissues at both message and protein levels as well as its response to a blood meal. We showed that AalRh50 shares a low identity with E. coli ammonia transporter (EcoAmtB), but higher identities with human RhBG and Drosophila Rh50 genes. The analysis of ammonia-conductance sites indicates that AalRh50 has residue substitutions of S237L (equivalent to S219 in AmtB) in the external vestibule, F127I (equivalent to F107 in AmtB) in the pore entrance, and S281N (equivalent to S263 in AmtB) in the internal vestibule, which could alter or reduce ammonia-conductance activity. The results from quantitative real-time-PCR and immunohistochemistry revealed that AalRh50 is expressed at significantly higher levels in the head, Malpighian tubules, and thorax of the non-blood-fed females, suggesting that AalRh50 might play roles in maintaining normal neurotransmitter metabolism, acid-base balance, and flight energy production in different tissues of mosquitoes at the non-blood-fed condition. A blood meal significantly increases AalRh50 expression in midgut, fat body, and Malpighian tubules from 3 or 6 to 24h post feeding, indicating that AalRh50 plays an important role in detoxification of excess systemic ammonia of female adults during the gonotrophic cycle.

Enzootic angiostrongyliasis, Guangdong, China, 2008-2009.

To the Editor: The nematode Angiostrongylus cantonensis was discovered in pulmonary arteries and hearts of domestic rats in Guangzhou (Canton), China, by Chen in 1935 (1). This parasite has a complex life cycle (2) and causes cerebral angiostrongyliasis after ingestion of infective larvae found in freshwater and terrestrial snails and slugs, paratenic hosts (such as freshwater fish, shrimp, frogs, and crabs), and contaminated vegetables (3). During 2000–2006, a total of 7 outbreaks of angiostrongyliasis were reported in the People’s Republic of China, including an outbreak in Zhaoqing, Guangdong Province (4–6). We conducted a survey of A. cantonensis nematodes in mollusks and rodents in Qingyuan, Guangong Province, during August 2008–October 2009. Qingyuan is located in northern Guangdong Province (23°31′–25°12′N, 111°55′–113°55′E). It is the largest city in the province. Qingyuan borders Zhaoqing on the west and Guangzhou on the south. Its climate is subtropical monsoon, and it has an average annual temperature of 20.7°C. The city has an area of 19,152.89 km2 and a population of 3.87 million. Nematode hosts were obtained in 3 counties in Qingyuan: Qingxin, Fogang, and Lianzhou. During August 2008–October 2009, we captured 288 rats of 7 species (257 Rattus norvegicus, 13 R. flavipectus, 7 R. losea; 6 R. rattus, 3 Bandicota indica, 1 R. rattus alexandrinus, and 1 Mus musculus). Rats were examined for adult A. cantonensis nematodes in pulmonary arteries and right heart cavities. Among the 288 rats examined, 27 (9.4%) from 3 species were infected with A. cantonensis adults in their cardiopulmonary systems (Table). Infected rodents were found in all 3 counties. The 27 infected rats were 25 R. norvegicus, 1 R. losea, and 1 M. musculus. R. norvegicus rats were most frequently captured in the 3 counties, and this rodent had the highest prevalence of infection. Infected B. indica rats in Lianzhou and M. musculus rats in Qingxin were also found, but the total numbers of infected animals and the prevalences are lower than that for R. norvegicus rats. On the basis of these findings, we conclude that R. norvegicus rats are the major definitive host for A. cantonensis nematodes in Qingyuan. Table Prevalence of infection with Angiostrongylus cantonensis in 3 snail species and rodents in 3 counties in Qingyuan, Guangdong Province, China, 2008–2009 Specimens from 510 snails (144 Pomacea canaliculata, 306 Achatina fulica, and 60 Bradybaena despecta) were digested with pepsin for isolation of A. cantonensis larvae (7). Metastrongylid larvae were found in 21 (4.1%) of 510 examined snails. Prevalence rates of A. cantonensis in P. canaliculata, A. fulica, and B. despecta were 8.3%, 2.0% and 5.0%, respectively. Differences between the 3 prevalence rates were significant (χ2 9.604, p<0.05). Prevalence rates in the 3 counties are shown in the Table. All 3 species of infected snails were found in Qingxin and Fogan Counties. P. canaliculata and B. despecta snails were found infected in Qingxin County. However, only A. fulica snails were found infected in Fogang. These findings are similar to those of studies conducted in Guangdong Province (8–10). Distributions of snail species among the 3 sites differed. Although all 3 species were found in Qingxin and Fogang Counties, only A. fulica snails were found in Lianzhou County. Lower temperatures in this county may contribute to this uneven distribution. Our failure to detect infected snail hosts in Lianzhou County was unexpected, and further surveys are needed to identify parasite hosts in this area. Our findings suggest that the 3 species may play a major role as intermediate hosts for A. cantonensis nematodes in human infections. Qingyuan is a natural focus for A. cantonensis nematodes. Residents in the study area frequently eat raw or undercooked snails and slugs, unaware that these animals may contain infective larvae of A. cantonensis that can cause eosinophilic meningitis. Therefore, to protect local residents from parasite infections, inhabitants of this region must be given relevant information about A. cantonensis nematodes. Control measures to control spread of this parasite must also be implemented.

Molecular characterization and immunolocalization of a protein disulfide isomerase from Angiostrongylus cantonensis

Protein disulfide isomerases (PDIs), belonging to the thioredoxin superfamily, are oxidoreductases that catalyze the formation, reduction, and isomerization of disulfide bonds among cysteine residues of proteins. In this study, we report the cloning and characterization of a cDNA encoding a protein disulfide isomerase (AcPDI) from a cDNA library of fourth-stage larvae of Angiostrongylus cantonensis. The deduced amino acid sequence contains two thioredoxin domains and exhibits high identity to the homologues from other species. Quantitative real-time PCR (qRT-PCR) was performed at the third-stage larvae, fourth-stage larvae, and adult stage of A. cantonensis, and the results revealed that the AcPDI mRNA, while expressed at all three stages, is expressed at a significantly higher level in female adult worms. Results of immunohistochemical studies indicated that the AcPDI expression was specifically localized in the tegument and uterus wall of female adult worms. Biochemical analysis showed that recombinant AcPDI was biologically active in vitro and exhibited the typical biochemical functions of PDIs: oxidase/isomerase and reductase activities. Collectively, these results implied that AcPDI may be a female-enriched protein and associated with the reproductive development of A. cantonensis. In addition, considering its biochemical properties, AcPDI may be involved in the formation of the cuticle of A. cantonensis.

Molecular cloning and characterization of a matrix metalloproteinase, from Caenorhabditis elegans : employed to identify homologous protein from Angiostrongylus cantonensis

Matrix metalloproteinases (MMPs) are a class of zinc-binding endopeptidases mainly responsible for degrading extracellular matrix constituent components, which also serve as effectors of leukocyte recruitment, cytotoxicity, cytokine and chemokine processing, and defensin activation involved in multiple mechanisms of immunomodulation. MMPs are a conserved proteolytic enzyme family participating in normal physiological function. In the present study, we have cloned a gene named CEMMP62 from Caenorhabditis elegans, the putative 62-kDa protein that contained 579 residues with MMP-conserved catalytic domain known as ZnMc_MMP and showed high identities with MMPs from other nematodes, and demonstrated that the recombinant CEMMP62 (rCEMMP62) expressed and purified from Escherichia coli could have weak proteolytic activity on swine gelatin; Western blot analysis revealed that sera from BALB/c mice immunized by recombinant protein could recognize excretory–secretary antigens from Angiostrongylus cantonensis third-stage larvae (L3). Also, the antiserum can recognize larval soluble antigens of L4 coming from mice (nonpermissive host) infected with A. cantonensis while it cannot recognize larval soluble antigens of L4 coming from rats (permissive host) infected with A. cantonensis. The results implied that probably CEMMP62 has homologous proteins which exist in A. cantonensis, and the potential MMP may play an important role in A. cantonensis infection and pathogenic process.

Molecular cloning, expression, and characterization of a putative activation-associated secreted protein from Angiostrongylus cantonensis.

Activation-associated secreted protein (ASP) had been found in many helminthes, which was associated with pathogenesis and stage transition. A complementary DNA (cDNA) sequence encoding a putative two-domain ASP was obtained from an Angiostrongylus cantonensis fourth-stage larvae cDNA library, which we designated as AgASP. The cDNA of AgASP contains an open reading frame encoding 424 amino acids, the first 19 residues being a putative secretion signal. The expression pattern of this protein was investigated by real-time polymerase chain reaction and Western blot. We found that this protein expressed most highly in the brain-stage larvae (Lbr) of this parasite and existed in the excretory/secretory products of this stage. Immunofluorescence showed it existed in the lumen of the Lbr. The recombinant protein can be recognized by the infection sera from mice (nonpermissive host), while it cannot be recognized by infection sera from rats (permissive host). The infiltration of neutrophils in infected nonpermissive host can be lessened by immunizing this host with this protein (immunized vs control group, 13.7 ± 10.2 vs 65.5 ± 19.2). These findings suggest that this protein plays a role in the pathogenesis of human angiostrongyliasis and is worthy of further study.

Characterization and immunolocalization of mutated ornithine decarboxylase antizyme from Angiostrongylus cantonensis.

Ornithine decarboxylase antizyme (OAZ), a prominent regulator of cell proliferation, DNA/RNA transformation and tumorigenesis, can bind to ornithine decarboxylase (ODC) and facilitate its degradation. Expression of OAZ requires a unique ribosomal frame shift that is regulated by levels of polyamine in the cell. In this study, we cloned an OAZ gene with the +1 ribosomal frame-shift from a fourth-stage larvae cDNA library of Angiostrongylus cantonensis. We removed one nucleotide to express the gene without polyamine. The sequence analysis showed that the deleted-mutation ornithine decarboxylase antizyme (DM-AcOAZ) contained a conservative domain related to other species OAZ. Quantitative real-time PCR revealed that DM-AcOAZ was expressed in L3 and L4 stages and adult female worms. More notably the expression level is the highest in the adult female stage. Immunohistochemical studies indicated that DM-AcOAZ was specifically localized in the uterus, oocyte and intestine in adult female worms. MTT assays showed that in DM-AcOAZ transfected HeLa cells, cell proliferation is inhibited. In conclusion, DM-AcOAZ may be a female-enriched protein and may involved in the cell proliferation in A. cantonensis.