Chang-Wang Pan

Induction of protective Th1 immune responses in mice by vaccination with recombinant Toxoplasma gondii nucleoside triphosphate hydrolase-II

The Toxoplasma gondii nucleoside triphosphate hydrolase (TgNTPase) has apyrase activity, degrading ATP to the di- and mono-phosphate forms and may be used by the parasite to salvage purines from the host cell for survival and replication. To study the immune-protective value of TgNTPase-II, BALB/c mice were immunized with a recombinant form of the antigen rTgNTPase-II combined with alum. All immunized mice produced specific anti-rTgNTPase-II immunoglobulins, with high IgG antibody titers and a mixed IgG1/IgG2a response, with predominance of IgG2a production. The cellular immune response was associated with the production of IFN-γ and IL-2 cytokines and the increase of the percentage of CD8+ T cells. Vaccinated mice displayed significant protection against acute infection with the virulent RH strain (P<0.05 in survival rate) and also chronic infection with PRU cyst (62.9% and 57.6% reduction in brain parasite load for rTgNTPase-II+alum and rTgNTPase-II alone vaccinated groups) compared to the non-vaccinated control group. In conclusion, rTgNTPase-II elicits a strong specific Th1 immune response providing partial protection against both T. gondii acute and chronic infection.

Macrolide-resistant Mycoplasma pneumoniae in adults in Zhejiang, China

ABSTRACT Mycoplasma pneumoniae is a major pathogen causing community-acquired pneumoniae (CAP), which is generally treated with macrolides. In recent years, however, although macrolide-resistant M. pneumoniae has been reported frequently, particularly in China, very little is known about the prevalence of macrolide-resistant M. pneumoniae infection in adults. In this study, we survey the macrolide-resistant M. pneumoniae in adults in Zhejiang province and characterize the mechanisms of resistance to macrolide. Six hundred fifty throat swab samples were collected from adult patients with CAP from January 2012 to August 2014. These samples were assayed by nested PCR and then cultivated for M. pneumoniae. All isolates were sequenced to determine the mutation in domain V of the 23S rRNA gene. The activities of 10 antibiotics against macrolide-resistant M. pneumoniae isolates were also investigated in vitro. Moreover, restriction fragment length polymorphism (RFLP) analysis of the amplified P1 gene was used to type 50 resistant strains. One hundred percent (71/71) of M. pneumoniae strains isolated from adults with CAP were resistant to erythromycin (MIC = 128 to >256 μg/ml), clarithromycin (MIC = 128 to >256 μg/ml), and azithromycin (MIC = 32 to >64 μg/ml). Furthermore, all macrolide-resistant M. pneumoniae strains identified had an A2063G mutation in domain V of the 23S rRNA gene. Forty-six resistant strains (92.0%) were classified into type I strain on the basis of P1 gene PCR-RFLP analysis. According to these findings, it is suggested that macrolide-resistant M. pneumoniae infection is very prevalence among adults in Zhejiang province. Thus, there is necessary to perform the epidemiological monitoring of macrolide-resistant M. pneumoniae in the future.

Monoclonal antibodies against nucleoside triphosphate hydrolase-II can reduce the replication of Toxoplasma gondii

Nucleoside triphosphate hydrolase (NTPase) is an abundant protein secreted by the obligate protozoan parasite Toxoplasma gondii, which has a wide specificity toward NTP. In the present study, two monoclonal antibodies (mAbs, MNT1 and MNT2) against recombinant T. gondii NTPase-II (rTgNTPase-II) were developed. Western blot analysis displayed that these two mAbs can recognize specifically rTgNTPase-II as well as a 63kDa molecule in tachyzoites soluble antigens that corresponded to native NTPase-II. T. gondii tachyzoites pretreated with two mAbs were observed under Confocal Laser Microscope and a specific reaction was displayed on tachyzoites after indirect fluorescence antibody test (IFAT). When COS-7 cells were co-cultured with tachyzoites pretreated with two mAbs, the number of intracellular parasites per infected cell was significantly decreased compared with the control. Furthermore, incubation of T. gondii tachyzoites with two mAbs can inhibit NTPase activity in the presence of dithiothreitol, which hinted that the reduction of tachyzoite replication might be owing to the inhibition of NTPase-II by the mAbs. The passive immunization test indicated that the transferred mAbs can significantly prolong the survival time of challenge infected mice. Taken together, we concluded that the mAbs against NTPase-II can reduce the replication of T. gondii and have a crucial effect on the protection of host from T. gondii infection.

Development-Specific Differences in the Proteomics of Angiostrongylus cantonensis

Angiostrongyliasis is an emerging communicable disease. Several different hosts are required to complete the life cycle of Angiostrongylus cantonensis. However, we lack a complete understanding of variability of proteins across different developmental stages and their contribution to parasite survival and progression. In this study, we extracted soluble proteins from various stages of the A. cantonensis life cycle [female adults, male adults, the fifth-stage female larvae (FL5), the fifth-stage male larvae (ML5) and third-stage larvae (L3)], separated those proteins using two-dimensional difference gel electrophoresis (2D-DIGE) at pH 4–7, and analyzed the gel images using DeCyder 7.0 software. This proteomic analysis produced a total of 183 different dominant protein spots. Thirty-seven protein spots were found to have high confidence scores (>95%) by matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS). Comparative proteomic analyses revealed that 29 spots represented cytoskeleton-associated proteins and functional proteins. Eight spots were unnamed proteins. Twelve protein spots that were matched to the EST of different-stage larvae of A. cantonensis were identified. Two genes and the internal control 18s were chosen for quantitative real-time PCR (qPCR) and the qPCR results were consistent with those of the DIGE studies. These findings will provide a new basis for understanding the characteristics of growth and development of A. cantonensis and the host–parasite relationship. They may also assist searches for candidate proteins suitable for use in diagnostic assays and as drug targets for the control of eosinophilic meningitis caused by A. cantonensis.

Differential proteomics analysis of female and male adults of Angiostrongylus cantonensis.

In this study, we identified the differentially expressed proteins of female and male adults of Angiostrongylus cantonensis through differential proteomics. We extracted and purified total proteins from male and female adults, separated proteins by two-dimensional difference gel electrophoresis (2D-DIGE) in pH 4-7, analyzed the gel images by DeCyder 7.0 software, and sacrificed the infected rats to count the number of male and female adults. It was found 28 protein spots that were differentially expressed; seven protein spots were then identified by matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS). Five proteins were up-regulated and two proteins down-regulated in male adults compared with female adults. Three of the five up-regulated proteins with known functions ascribed to them were identified as galectin-1, proteasome alpha subunit and peroxiredoxin. The two down-regulated proteins were identified as indoleamine dioxygenase like-myoglobin and galectin. Furthermore, the female was significantly greater than male adults (P<0.01) in the rats. The findings demonstrate the differences in protein expression profiles and the ability to survive in the final host between female and male adults of A. cantonensis, and may provide a theoretical basis to study their developmental biology further.

Comparison of P1 and 16S rRNA genes for detection of Mycoplasma pneumoniae by nested PCR in adults in Zhejiang, China.

INTRODUCTION Mycoplasma pneumoniae (M. pneumoniae) is the most common atypical pathogen that causes respiratory infections in humans. Laboratory tests are important in the diagnosis of M. pneumoniae because of the atypical features in clinical signs and symptoms. Nowadays, both the P1 adhesin gene and 16S ribosomal (r) RNA (rRNA) gene of M. pneumoniae have been widely detected by polymerase chain reaction (PCR). The purpose of the present study was to evaluate the most suitable target in the detection of M. pneumonia via nested PCR. METHODOLOGY Both the P1 adhesin gene and 16S rRNA gene for nested PCR reaction conditions were optimized through an orthogonal test and single-factor experiment. Then, the sensitivity of the two sets of targets was evaluated. Finally, based on the optimal conditions, 55 clinical samples of throat swabs collected from adult patients in 2013 were examined by established nested PCR. RESULTS The results revealed that PCR detection of the 16S rRNA gene was more sensitive than the P1 adhesin gene because the detection limits for both the P1 gene and 16S rRNA gene were at least 100 fg and 10 fg of M. pneumoniae DNA, respectively. Furthermore, the positive rate for detection of the 16S rRNA gene (30/55; 54.5%) was higher than that of the P1 adhesin gene (25/55; 45.5%). CONCLUSION Our results indicate that the 16S rRNA gene is more suitable for diagnosis of M. pneumoniae infection than the P1 adhesin gene due to its higher sensitivity and positive rate in clinical samples.

Protective immune response in mice induced by a suicidal DNA vaccine encoding NTPase-II gene of Toxoplasma gondii

DNA-based alphaviral RNA replicon vectors, also called suicidal DNA vectors, have been employed to alleviate biosafety concerns attribution to its ability to induce apoptotic cell death of the transfected cells. Toxoplasma gondii nucleoside triphosphate hydrolase-II (TgNTPase-II), which facilitates the parasite to salvage purines from the host cell for survival and replication, have been demonstrated to be a potential vaccine candidate for toxoplasmosis. Herein, we evaluated the immunogenic potential of a suicidal DNA vaccine encoding TgNTPase-II gene, pDREP-TgNTPase-II, delivered intramuscularly in combination with electroporation. Immunization of mice with pDREP-TgNTPase-II elicited specific humoral responses, with high IgG antibody titers and a mixed IgG1/IgG2a response. The cellular immune response was associated with high level production of IFN-γ, IL-2, IL-10 cytokines and low level IL-4 production as well as the increase of the percentage of CD8+ T cells, indicating that a Th1 predominant response was elicited. Furthermore, mice vaccinated with this suicidal DNA vaccine displayed partial protection against acute infection with the virulent RH strain as well as chronic infection with PRU cyst, which shows 77.7% and 71.4% reduction in brain cyst burden in comparison to PBS and pDREP-eGFP control group, respectively. Based on the cellular and antibody responses, the suicidal DNA vaccine elicited a Th1-predominant immune response against T. gondii challenge.

Induction of Autophagy interferes the tachyzoite to bradyzoite transformation of Toxoplasma gondii.

Autophagy process in Toxoplasma gondii plays a vital role in regulating parasite survival or death. Thus, once having an understanding of certain effects of autophagy on the transformation of tachyzoite to bradyzoite this will allow us to elucidate the function of autophagy during parasite development. Herein, we used three TgAtg proteins involved in Atg8 conjugation system, TgAtg3, TgAtg7 and TgAtg8 to evaluate the autophagy level in tachyzoite and bradyzoite of Toxoplasma in vitro based on Pru TgAtg7-HA transgenic strains. We showed that both TgAtg3 and TgAtg8 were expressed at a significantly lower level in bradyzoites than in tachyzoites. Importantly, the number of parasites containing fluorescence-labelled TgAtg8 puncta was significantly reduced in bradyzoites than in tachyzoites, suggesting that autophagy is downregulated in Toxoplasma bradyzoite in vitro. Moreover, after treatment with drugs, bradyzoite-specific gene BAG1 levels decreased significantly in rapamycin-treated bradyzoites and increased significantly in 3-MA-treated bradyzoites in comparison with control bradyzoites, indicating that Toxoplasma autophagy is involved in the transformation of tachyzoite to bradyzoite in vitro. Together, it is suggested that autophagy may serve as a potential strategy to regulate the transformation.