Feng Tan

Systematic Identification of the Lysine Succinylation in the Protozoan Parasite Toxoplasma gondii

Lysine succinylation is a new posttranslational modification identified in histone proteins of Toxoplasma gondii, an obligate intracellular parasite of the phylum Apicomplexa. However, very little is known about their scope and cellular distribution. Here, using LC-MS/MS to identify parasite peptides enriched by immunopurification with succinyl lysine antibody, we produced the first lysine succinylome in this parasite. Overall, a total of 425 lysine succinylation sites that occurred on 147 succinylated proteins were identified in extracellular Toxoplasma tachyzoites, which is a proliferative stage that results in acute toxoplasmosis. With the bioinformatics analysis, it is shown that these succinylated proteins are evolutionarily conserved and involved in a wide variety of cellular functions such as metabolism and epigenetic gene regulation and exhibit diverse subcellular localizations. Moreover, we defined five types of definitively conserved succinylation site motifs, and the results imply that lysine residue of a polypeptide with lysine on the +3 position and without lysine at the -1 to +2 position is a preferred substrate of lysine succinyltransferase. In conclusion, our findings suggest that lysine succinylation in Toxoplasma involves a diverse array of cellular functions, although the succinylation occurs at a low level.

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.

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.

Alphavirus Replicon DNA Vectors Expressing Ebola GP and VP40 Antigens Induce Humoral and Cellular Immune Responses in Mice

Ebola virus (EBOV) causes severe hemorrhagic fevers in humans, and no approved therapeutics or vaccine is currently available. Glycoprotein (GP) is the major protective antigen of EBOV, and can generate virus-like particles (VLPs) by co-expression with matrix protein (VP40). In this study, we constructed a recombinant Alphavirus Semliki Forest virus (SFV) replicon vector DREP to express EBOV GP and matrix viral protein (VP40). EBOV VLPs were successfully generated and achieved budding from 293 cells after co-transfection with DREP-based GP and VP40 vectors (DREP-GP+DREP-VP40). Vaccination of BALB/c mice with DREP-GP, DREP-VP40, or DREP-GP+DREP-VP40 vectors, followed by immediate electroporation resulted in a mixed IgG subclass production, which recognized EBOV GP and/or VP40 proteins. This vaccination regimen also led to the generation of both Th1 and Th2 cellular immune responses in mice. Notably, vaccination with DREP-GP and DREP-VP40, which produces both GP and VP40 antigens, induced a significantly higher level of anti-GP IgG2a antibody and increased IFN-γ secreting CD8+ T-cell responses relative to vaccination with DREP-GP or DREP-VP40 vector alone. Our study indicates that co-expression of GP and VP40 antigens based on the SFV replicon vector generates EBOV VLPs in vitro, and vaccination with recombinant DREP vectors containing GP and VP40 antigens induces Ebola antigen-specific humoral and cellular immune responses in mice. This novel approach provides a simple and efficient vaccine platform for Ebola disease prevention.

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.

Identification of TgAtg8–TgAtg3 interaction in Toxoplasma gondii

Autophagy is a catabolic process in eukaryotic cells involved in the targeted degradation of cellular organelles and the cytoplasm. Recent works in Toxoplasma gondii suggest that the autophagy processes may serve as an important pathway in modulating parasite survival or death. As an important modulator of Atg8 lipidation and autophagy, Atg8-Atg3 interaction has been attracting increasing attention. However, there is no direct evidence that TgAtg8-TgAtg3 interaction occurs in the parasite. In this study, we firstly found TgAtg8 partially colocalized with TgAtg3 in GFP-TgAtg8 transgenic strains using IFA. Then, lysates from GFP-TgAtg8 tachyzoites were directly subject to large-scale tandem affinity purification with anti-GFP antibody. Western blot and tandem mass spectrometry (MS/MS) analysis determined the interaction between TgAtg8 and TgAtg3. Additionally, we performed real-time interaction analysis with a surface plasmon resonance biosensor using BIAcore system. As expected, the result demonstrated a concentration-dependent increases in resonance signals and indicated the TgAtg8 could bind directly TgAtg3 in vitro. Noteworthily, A KD of 34.9nM obtained from TgAtg8-TgAtg3 interaction indicate a high-affinity between Atg8-Atg3 in Toxoplasma. Furthermore, homology modeling and sequence alignment showed that TgAtg8 has greatest sequence and structural conservation. Within TgAtg3, this protein possesses the core E2 enzymatic activity structure and a truncated handle region which may contain AIM sequence. Taken together, our findings would help elucidate the formation mechanism of autophagosome in Toxoplasma and provide a possibility for looking into parasitic drug targets.

Induction of Protective Immunity against Toxoplasma gondii in Mice by Nucleoside Triphosphate Hydrolase-II (NTPase-II) Self-amplifying RNA Vaccine Encapsulated in Lipid Nanoparticle (LNP)

RNA-based vaccine represents an irresistible and safe immunization strategy with decreasing theoretical risks of genomic integration and malignant cell transformation. To our knowledge, however, there is no report about development of RNA vaccine against Toxoplasma gondii infection. We have previously demonstrated that the recombinant T. gondii nucleoside triphosphate hydrolase-II (NTPase-II) protein is able to provide protective Th1 cell-mediated immunity against T. gondii. Herein, we evaluated the immunogenic potential of a self-amplifying RNA vaccine-encoding T. gondii NTPase-II gene, RREP-NTPase-II, delivered by a synthetic lipid nanoparticle (LNP). Immunization of mice with naked RREP-NTPase-II induced a strong cellular and humoral immune response with high-IgG antibody titers and IFN-γ production. The immunized mice displayed significantly prolonged survival time and reduction in brain parasite load (46.4%) compared with control group. Furthermore, mice vaccinated with RREP-NTPase-II-encapsulated LNP displayed significantly enhanced protection against acute infection as well as chronic infection with PRU cyst, which shows 62.1% reduction in brain cyst burden in comparison to control group. These results suggest that the combination of self-amplifying RNA and LNP ion would be beneficial to the development of a safe and long-acting vaccine against toxoplasmosis.

Characterisation of two Toxoplasma PROPPINs homologous to Atg18/WIPI suggests they have evolved distinct specialised functions

Toxoplasma gondii is a parasitic protist possessing a limited set of proteins involved in the autophagy pathway, a self-degradative machinery for protein and organelle recycling. This distant eukaryote has even repurposed part of this machinery, centered on protein ATG8, for a non-degradative function related to the maintenance of the apicoplast, a parasite-specific organelle. However, some evidence also suggest Toxoplasma is able to generate autophagic vesicles upon stress, and that some autophagy-related proteins, such as ATG9, might be involved solely in the canonical autophagy function. Here, we have characterised TgPROP1 and TgPROP2, two Toxoplasma proteins containing WD-40 repeat that can bind lipids for their recruitment to vesicular structures upon stress. They belong to the PROPPIN family and are homologues to ATG18/WIPI, which are known to be important for the autophagic process. We conducted a functional analysis of these two Toxoplasma PROPPINs. One of them is dispensable for normal in vitro growth, although it may play a role for parasite survival in specific stress conditions or for parasite fitness in the host, through a canonical autophagy-related function. The other, however, seems important for parasite viability in normal growth conditions and could be primarily involved in a non-canonical function. These divergent roles for two proteins from the same family illustrate the functional versatility of the autophagy-related machinery in Toxoplasma.