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Targeted overexpression of cyclic AMP-dependent protein kinase subunit in Toxoplasma gondii promotes replication and virulence in host cells
Accepted Manuscript Title: Targeted overexpression of cyclic AMP-Dependent Protein Kinase Subunit in Toxoplasma gondii promotes replication and virulence in host cells Authors: Hongchao Sun, Suhua Wang, Xianfeng Zhao, Chaoqun Yao, Haohan Zhuang, Yechuan Huang, Xueqiu Chen, Yi Yang, Aifang Du PII: DOI: Reference:
S0304-4017(17)30258-3 http://dx.doi.org/doi:10.1016/j.vetpar.2017.06.002 VETPAR 8367
To appear in:
Veterinary Parasitology
Received date: Revised date: Accepted date:
29-3-2017 20-5-2017 2-6-2017
Please cite this article as: Sun, Hongchao, Wang, Suhua, Zhao, Xianfeng, Yao, Chaoqun, Zhuang, Haohan, Huang, Yechuan, Chen, Xueqiu, Yang, Yi, Du, Aifang, Targeted overexpression of cyclic AMP-Dependent Protein Kinase Subunit in Toxoplasma gondii promotes replication and virulence in host cells.Veterinary Parasitology http://dx.doi.org/10.1016/j.vetpar.2017.06.002 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
Targeted overexpression of cyclic AMP-Dependent Protein Kinase Subunit in Toxoplasma gondii promotes replication and virulence in host cells
Hongchao Sun1, Suhua Wang2, Xianfeng Zhao3, ChaoqunYao4, Haohan Zhuang1, Yechuan Huang1, Xueqiu Chen1, Yi Yang1 , Aifang Du1*
1. College of Animal Sciences，Zhejiang Provincial Key Laboratory of Preventive Veterinary Medicine, Zhejiang University, Hangzhou 310058, China 2. Wenzhou Entry-exit Inspection and Quarantine Bureau, Wenzhou, Zhejiang 325027, China 3. Shenzhen Entry-exit Inspection and Quarantine Bureau, Shenzhen, Guangdong 518045 4. Department of Biomedical Sciences and One Health Center for Zoonoses and Tropical Veterinary Medicine, Ross University School of Veterinary Medicine, P.O. Box 334, Basseterre, St. Kitts, West Indies
*Correspondence should be addressed to: Dr. Aifang Du, College of Animal Sciences, Zhejiang Provincial Key Laboratory of Preventive Veterinary Medicine, Zhejiang University, Hangzhou, Zhejiang 310058, China Telephone number: +86-571-8898-2583 E-mail: [email protected]
1
Highlights:
1 T.gondii PKAC (TgPKAC) overexpression strain (TgPKAC-OE) was
constructed.
2 Inhibition of TgPKAC could cause autophagy of Toxoplasma gondii and
then influence the replication of the parasite.
3 TgPKAC plays an important role in parasite virulence in vivo.
4 TgPKAC was mainly expressed in the cytoplasm of Vero cell.
Abstract: Toxoplasma gondii (T. gondii) is one of the most common parasite that can infect almost
any
warm-blooded
animals
including
humans.
The
cyclic
nucleotide-dependent protein kinase (PKA) regulates a spectrum of intracellular signal pathways in many organisms. Protein kinase catalytic subunit (PKAC) is the core of the whole protein, and plays an important role in the life cycle of T.gondii. Here, T.gondii PKAC (TgPKAC) overexpression strain (TgPKAC-OE) was constructed. The growth of the TgPKAC-OE, RH△ Ku80, and TgPKAC inhibition strains (TgPKAC-H89) were analysed by SYBR-green real-time PCR, and the ultrastructure was observed by transmission electron microscopy. The survival rate in mice was also recorded to analyse the virulence of the parasites. We also investigated the subcellular localization of TgPKAC in Vero cells by laser scanning microscope. We found that TgPKAC-OE strain exhibited obviously increased growth rate in Vero cells in vitro, and infected mice survived for a shorter time compared to wild type strain. Ultrastructural analysis found more autophagosomes-like structures in TgPKAC-H89 parasite compared to RH△Ku80 strain, and the relative expression level of Toxoplasma gondii autophagy-related protein (ATG8) in TgPKAC-H89 parasite was higher than wild type parasite. Laser confocal results showed that TgPKAC was mainly expressed in the cytoplasm of Vero cells. In conclusion, we 2
hypothesized that inhibition of TgPKAC could cause autophagy of Toxoplasma gondii and then influence the replication of the parasite. TgPKAC plays an important role in parasite virulence in vivo, and the subcellular localization was successfully detected in Vero cells. Our data will provide a basis for further study of TgPKAC function and help screen drug targets of T. gondii.
Key words: Toxoplasma gondii, protein kinase catalytic subunit, growth, autophagy, virulence, subcellular localization.
1 Introduction Toxoplasma gondii (T. gondii) is a specific intracellular protozoan parasite that can infect almost all mammals including humans (Hunter and Sibley, 2012; Montoya and Liesenfeld, 2004). Despite enjoying a broad host range, the feline is the only definitive host where the sexual reproductive stages occur (Dlugonska H, 2014). The asexual stages of T. gondii do not have host specificity, thus the variety of intermediate hosts facilitates the transmission of T. gondii (Gazzinelli et al., 2014). During the acute stage of infection, the parasites replicate as the fast-growing tachyzoite form. Under a variety of stress conditions, such as pH change (Weiss et al., 1995) and heat shock (Soete et al., 1994), the fast-growing tachyzoite can be transformed into slow-growing bradyzoite (Skariah et al., 2010), and the host cells also play an important role in the conversion between tachyzoite and bradyzoite (Skariah et al., 2010; Weilhammer et al., 2012). The complex life stages of T. gondii require a series of regulatory mechanisms, such as phosphorylation and transcriptional modifications (Wei et al., 2013). Among a variety of serine, threonine protein kinase families, the cyclic nucleotide-dependent protein kinase (PKA) is thought to be an important model protein for studying other kinases (Gerlits et al., 2014; Johnson D A, 2001), it was one of the first protein kinase had been found, sequenced and cloned (Shoji et al., 1981; Uhler
et
al.,
1986;
Walsh
et
al.,
1968).
In
eukaryotic
cells,
cyclic
nucleotide-dependent protein kinases are present in two forms, the regulatory subunits 3
(PKARs) and catalytic subunits (PKACs) (Kim C, 2005). Protein kinase catalytic subunit (PKAC) is the core of the whole protein, mainly composed of N-terminal domain and C-terminal domain, and they determine the localization and enzyme active of PKAC protein (Taylor et al., 2008). Among eukaryotes, all protozoa or helminth parasites use protein kinases to maintain the cellular functions (Dissous et al., 2007). PKAC had been reported in Schistosoma (Dissous et al., 2007; Swierczewski and Davies, 2009), Plasmodium falciparum (Beraldo et al., 2005), 2005), Plasmodium chabaudi (Gazarini et al., 2011), Toxoplasma gondii (Sugi et al., 2016), Giardia lamblia (Gibson et al., 2006), Microsporidia (Equinet et al., 2004), Leishmania (Siman-Tov MM, 2002). Matsuyama and his colleagues found that the swimming speed of Schistosoma mansonimiracidia became slowly when the parasites are treated with specific PKAC inhibitor (Matsuyama et al., 2004). In Plasmodium falciparum, the expression level of PKAC varies at different life stages, and PKAC showed higher expression at asexual stage. If the Plasmodium falciparum schizont was inhibited with H89, the parasites exhibited growth defects in infected erythrocytes (Syin et al., 2001). Eaton et al found that PKAC was involved in tachyzoite-bradyzoite transition in T. gondii (Eaton et al., 2006) , and inhibition of PKAC with H89 could slow down the growth of tachyzoites (Kurokawa et al., 2011). In this study, we analysed the function of TgPKAC in proliferation and virulence by the construction of TgPKAC overexpression strains and the ultrastructures of parasites were also observed by transmission electron microscopy. The results showed that TgPKAC plays an important role in parasites virulence in vivo, inhibition of TgPKAC using H89 could cause autophagy of T. gondii and then influence the replication of T. gondii.
2 Materials and methods 2.1 Ethics All mice experiments were performed accoding to the recommendations of the Guide for the regulation for the Administration of Affairs concerning Experimental 4
Animal of the People’s Republic of China. The animal usage was approved by Zhejiang University Experimental Animal Ethics Committee (Permit Number: ZJU201308-1-10-072).
2.2 Host cell and parasite cultures T. gondii RHΔKu80 strain was maintained on monolayers of African green monkey kidney (Vero) cells at 37 ℃ with 5% CO2. The culture medium was Dubelcco’s Modified Eagle Medium (DMEM, Hyclone) supplemented with 10% fetal calf serum (FCS,Hyclone), 2 mM L-glutamine and 100 units penicillin/100 mg streptomycin.
2.3 Construction of overexpression strain 107 of T. gondii tachyzoites were collected, total RNA was extracted and reverse transcribed into cDNA. The opening reading frame of TgPKAC (Gene ID, 7898189) was amplified by PCR with primers p1/p2 (Table 1), the PCR reaction condition was 94℃ 5min, 94℃ 30s, 56℃ 1min30s, 72℃ 5min, 30 cycles and 72℃ 10min. SAG1 promoter and GRA2 polytail were also amplified by PCR to initiate TgPKAC gene. The vector contained chloramphenicol resistance gene CAT. 20-25 μg of the plasmid were linearized with NotI and transfected into RH△Ku80 parasites. Stable clones were selected by 20 μM chloramphenicol (Sigma). PKAC expression was confirmed by PCR and Western blotting.
2.4 Preparation of polyclonal antibody and western blotting analysis The coding sequence of TgPKAC was amplified using the primers p3/p4 (Table 1), according to the gene sequence from GenBank (Gene ID, 7898189), then subcloned into pMD18T vector. TgPKAC-pMD18T plasmid and pET28a vector were digested with restriction enzymes NdeI/HindIII, DNA fragments were purified by DNA purification kit and connected with T4 DNA ligase. The vector of TgPKAC-pET28a was identified by double enzyme digestion and DNA sequencing analysis. The recombinant plasmid was transformed into Escherichia coli BL21 cells. After induction with IPTG (1mM), the cells were collected at 0, 2, 4, 6 and 8 h, centrifuged 5
at 12000 rpm for 1 min. The precipitate was cracked with loading buffer and analysed by SDS-PAGE. SDS-PAGE result showed that large amount of recombinant protein was induced after 2 to 4 h. The recombinant protein was identified by Western blotting analysis. The protein concentration was measured using BCA kit, equal amount of protein and Freund's complete adjuvant were emulsified and injected into the back muscle of the rabbit (0.5mg/kg). For the second and third inoculation, the protein was emulsified with freund's incomplete adjuvant (0.25 mg/kg). After the third immunization, the ear vein blood was collected and serum was prepared at 4℃ overnight. The antibody titer was detected by enzyme-linked immune sorbent assay (ELISA) and the specificity of the antibody was analyzed by Western blotting.
2.5 Construction and expression of the recombinant eukaryotic expression plasmid PKAC gene in Vero cells The primers p5/p6 (Table 1) were designed according to the gene sequence from GenBank (Gene ID, 7898189). The pMD-TgPKAC plasmid was constructed and digested by BglⅡ/HindIII enzymes to construct the eukaryotic expression vector TgPKAC-EGFP-C2. The expression plasmid was transfected into Vero cells by liposome 2000. After inoculated for 24 h, the cell climbing tablets were removed from the 6 well plates and washed with 0.1 M PBS for three times. Then fixed with 4% paraformaldehyde for 10 min. 50 μl DAPI fluorescent dyes were added to the climbing tablets and incubated for 5min. Finally, the localization of PKAC in Vero cells was observed by a laser confocal microscope.
2.6 Transmission electron microscopy analysis Vero cells were cultured in 6 well plates (105/well) for 24h. Equal amounts of T gondii RHΔKu80 tachyzoites (105) were incubated with TgPKAC inhibitor H89 (30 μM) (Selleck Chemicals) or DMSO for 3 h. The parasites were centrifuged at 3000 rpm for 5min, then washed with DMEM to remove the H89 and DMSO. Afterwards the pretreated T. gondii were inoculated to Vero cells. About 48 h, the parasites were 6
collected and filtered with 27G needle in order to obtain purified tachyzoites, then centrifuged at 3000 rpm for 5min. The precipitate was fixed with 4% glutaraldehyde for 3-5 days at 4 ℃, centrifuged at 1500 rpm for 5min, washed with 0.1 M PBS for three times. 1% agar solution was added to the cell pellet, the solidified agar was removed and trimmed into the right size. The cells were fixed with 1% osmic acid for 2 h, followed by washing with PBS. The samples were dehydrated with 50%, 70%, 80%, 90%, 95%, 100% ethanol every 20 min and finally treated with acetone for 20 min. The samples were embedded overnight at 70 ℃, sliced with ultra-thin slicer, stained with 2% uranyl acetate and 0.25% lead citrate for 5 min, and observed by transmission electron microscopy. Then the expression level of TgATG8 was detected by qRT-PCR, equal number of parasites (105) were inoculated to Vero cells. After cultured for 24 h, 48 h, 72 h, the samples were collected and RNAs were extracted by Trizol reagent (Invitrogen) and reverse transcribed into cDNA using transcription kit (Toyobo). qRT-PCR was performed by Applied Biosystems Inc 7500 fluorescence quantitative PCR instrument. The primers for actin and ATG8 in T. gondii were included in Table 1. The data were expressed as means ± SD for three experiments and results were calculated using the 2-△△t method.
2.7 Virulence analysis in BALB/c mice 100 of TgPKAC-OE, RHΔKu80, and H89 inhibitor treatment strains were injected into BALB/c mice by intraperitoneal injection, with 5 mice in each group. The status of infected mice was monitored each day, and the survival rate was recorded. The data represented three independent experiments.
2.8 Plaque assay and intracellular growth For plaque assay, 200 of TgPKAC-OE, RH△Ku80, and TgPKAC inhibition strains were inoculated in the 6-well Vero cells, and cultured at 37 ℃ with 5% CO2 for 14 days. After 14 days, the culture medium was removed and the Vero cells were washed with PBS for three times, and fixed with 4% paraformaldehyde for 10 min. Then 7
stained with 0.1% crystal violet for 30 min, and the plaques were observed using an Olympus microscope. Each sample was at least randomly observed for 50 times, and the result represented three replicates experiments. Vero cells were cultured in 6-well plate for 24 h, with 105 in each well. TgPKAC-OE, RH△Ku80, and TgPKAC inhibition strains were inoculated to Vero cells, the infection ratio was 1:1. After infection for 2 h, the noninvasive tachyzoites were removed and fresh medium was added to the cells. Next the samples were collected after infection for 24 h, 48 h, 72 h, and genome DNA were extracted using the TIANGEN genomic DNA isolation kit by following the manufacturer’s protocol. The parasites numbers were determined by real-time PCR using B1 gene primers (Table 1). Triplicates samples were used for each time point of three independent experiments.
2.9 Statistical analysis Results in the present study represented mean ± S.D., numbers in groups were compared by Student's t-test. P value of <0.05 was considered significant difference.
3 Results
3.1 Preparation of polyclonal antibody and identified by western blotting The coding sequence was obtained and clonal vector of TgPKAC-pMD18T was constructed successfully (Fig 1a, 1b). SDS-PAGE analysis showed that recombinant protein was purified successfully, the molecular mass was 57.2 KDa (Fig 1c). For subsequent experiments, the rabbit polyclonal antibody was prepared. The protein was immunized into the back of rabbit to generate antiserum. ELISA result revealed that the antibody titer was 0.256 million and indirect immunofluorescence assay (IFA) analyzed the reactivity of the polyclonal antibody, Western blotting results showed that the rabbit antibody could specifically recognize T. gondii antigen (Fig 1d), and the TgPKAC was located in the cytoplasm of the intracellular parasites (Fig 1e). 8
3.2 Construction of TgPKAC overexpression strain To characterize the biological role of TgPKAC, we successfully constructed a TgPKAC overexpression strain (TgPKAC-OE), the schematic diagram was showed in Fig 2a, 2b. RT-PCR results showed that the gene expression level of TgPKAC was significantly higher than wild type strain (Fig 2c), and western blotting result also showed that overexpression strain was constructed successfully (Fig 2d).
3.3 Growth rate detection in Vero cells As shown in microscope observation, the growth rate of TgPKAC inhibition
strains (Fig 3b) were slower than wild parasites (Fig 3c), and the TgPKAC-OE (Fig 3a) strains revealed higher growth rate than wild strains. Accordingly, the plaque area of TgPKAC-OE (Fig 3d) was larger than the other two strains (Fig 3e, 3f). To further assess the proliferation role of TgPKAC in T. gondii, we calculated the growth rate at different time points. As showed in Fig 3g, the growth of TgPKAC-OE strains were increased by 2.21-fold (P<0.05), 2.57-fold (P<0.01), 3.41-fold (P<0.01) in comparison with that of the H89 inhibitor parasites, and increased by 1.05-fold, 1.13-fold (P<0.05), 1.30-fold (P<0.05) comparing to RH△Ku80 strains at 24 h, 48 h and 72 h, respectively. These data collectively demonstrated that TgPKAC is required for the normal growth of T. gondii.
3.4 Analysis of ultrastructures by transmission electron microscopy Our results showed TgPKAC was involved in the replication of T. gondii, next we sought to know if the growth defects were related to autophagy of T. gondii. RH△Ku80 and TgPKAC inhibition strains were cultured in Vero cells. After infection for 48 h, the samples were collected and observed by transmission electron microscopy. Ultrastructural analysis showed more structures like autophagosomes were found in TgPKAC inhibition parasites (Fig 4a) compared to RH△Ku80 strains (Fig 4b), suggesting that TgPKAC could induce the autophagy of T. gondii. In order to further confirm the role of TgPKAC in induction of autophagy, the 9
relative expression level of TgATG8 (autophagy-related protein) was detected by qRT-PCR assay, which revealed that inhibition of TgPKAC generated an increased expression of TgATG8 (P<0.05) compared to the RH△Ku80 strains (Fig 4c).
3.5 Survival rate of TgPKAC-OE, RH△Ku80 andTgPKAC inhibition parasites Our results showed that TgPKAC was related to the replication of T. gondii in Vero cells, we therefore investigated the virulence of TgPKAC inhibition strains and TgPKAC-OE. We injected 100 tachyzoites of TgPKAC inhibition strains, RH△ Ku80, and TgPKAC-OE to the BALB/c mice. The survival time was recorded, and we found that the mice began to appear symptoms 4 days after infection. TgPKAC-OE and RH△Ku80 strains showed a significantly shorter life time than the H89 inhibitor parasites (Fig 5), implying that TgPKAC could be a virulence factor of T. gondii.
3.6 Localization of TgPKAC in Vero cell The subcellular localization of proteins in host cells are closely related to their function, thus we analysed the expression and localization of TgPKAC in Vero cells. Laser confocal results showed that TgPKAC was mainly expressed in the cytoplasm of Vero cells, and the expression was gathered around the nuclei (Fig 6d, 6e). EGFP-C2 plasmid was transfected into Vero cells as the control (Fig 6a) and the nucleus DNA was stained with DAPI (blue) (Fig 6c, 6f), EGFP and DAPI were merged in the Fig 6b, 6e.
4 Discussion To understand the role of TgPKAC in T. gondii, we generated a TgPKAC overexpression strain (TgPKAC-OE), the phenotype of the TgPKAC gene was analysed in our study. Firstly, we analysed the replication rate of the TgPKAC-OE, RH△Ku80, and TgPKAC inhibition strains in Vero cells. Although the T. gondii PKAC gene was studied by Kurokawa et al (Kurokawa et al., 2011), the TgPKAC-OE strain had not been reported in previous study. In this study, we found that the TgPKAC-OE strain revealed higher growth rate and larger plaque area than the other 10
two strains (Fig 3). cAMP-dependent protein kinase (PKAC) studies of other parasites such as Plasmodium falciparum showed that when the schizont was inhibited with H89, the parasites exhibited growth defects in infected erythrocytes (Syin et al., 2001). In Giardia Iamblia (Abel et al., 2001), PKAC played an important role in regulating trophozoite motility as well as cellular activation of excystation. Our result suggested that PKAC in T. gondii could be involved into the growth stage, and this process could be controlled by a variety of signaling pathways. Autophagy is considered as an important regulatory mechanism in mammalian cells in recent years (Corcelle et al., 2007; Ozpolat, 2009; Simone, 2007), but little is known in parasites. In Leishmania (Bhattacharya et al., 2012), Bhattacharya et al found that cAMP-binding PKA regulatory subunit plays an important role in regulating parasitic differentiation by modulating induction of autophagy in the parasites. In T. gondii, when the parasites were inhibited with tamoxifen, the autophagy marker light chain 3-green fluorescent protein (LC3-GFP) could be recruited to the membrane of the parasitophorous vacuole and the growth was restricted, this revealed that tomoxifen could restrict the growth of T. gondii by inducing xenophagy or autophagic (Dittmar et al., 2016). In the present study, TgPKAC was suggested to be involved in the replication of T. gondii. We would like to know if the growth defects were related to autophagy of T. gondii. RH△Ku80, and TgPKAC inhibition strains were observed by transmission electron microscopy. Ultrastructural analysis showed that more autophagosomes-like structures were found in TgPKAC inhibition parasite compared to RH△Ku80 strains (Fig 5). The relative expression level of T. gondii autophagy-related protein (ATG8) was detected by qRT-PCR, the result revealed that the expression level of TgATG8 was increased when TgPKAC was inhibited with H89. We hypothesized that inhibition of PKAC gene may cause autophagy of T. gondii, and the growth rate in Vero Cells may be influenced by autophagy. Further studies need to be carried out to investigate the exact mechanism. Many protein kinases in T. gondii are virulence factors, such as the rhoptry proteins (ROPS) (Sibley et al., 2009), casein kinase 1 α (CK1 α) (Wang et al., 2016b), aurora kinase (Ark3) (Berry et al., 2016). The virulence 11
was regulated by a variety of signaling pathways and different members of the same protein family could balance each other to regulate the virulence of T. gondii in mice (Wang et al., 2016a). Because TgPKAC was involved in the replication of T. gondii in Vero cells, we wanted to know if it was related to the virulence in mice. The result showed that TgPKAC-OE strain showed a significantly shorter life time than the RH△Ku80 and H89 inhibitor parasites, suggesting that TgPKAC may be a virulence factor of T. gondii. While it was not clear whether PKARs, the regulatory subunits of cAMP-depdent protein kinase, were coordinated with PKAC to regulate the virulence. In addition to study the gene function of TgPKAC in replication and virulence of T. gondii, the localization of TgPKAC in Vero cells was also detected by laser scanning microscope (Fig 6). Other parasites, such as Theileria annulata (Ma and Baumgartner, 2014), Giadia lamblia (Abdul-Wahid and Faubert, 2004; Chen et al., 2008), the eukaryotic transfection was also used to study the gene function in host cells. Our results showed that TgPKAC could be successfully transfected into Vero cells, and TgPKAC was mainly expressed in the cytoplasm of Vero cells and the expression was gathered around the nuclei. In conclusion, we constructed TgPKAC overexpression strain in this work to investigate the role of PKAC in T. gondii. Our results showed that TgPKAC contributed to replication of T. gondii in host cells and TgPKAC playd an important role in parasite virulence in vivo. Inhibition of TgPKAC resulted in the formation of autophagosomes-like structures in T. gondii, suggesting that the growth defect of TgPKAC inhibition strain was related to the autophagy of T. gondii. These data will provide a basis for further study of TgPKAC function and help screen drug targets of T. gondii. Future studies of TgPKAC are needed to clarify the interaction molecules of PKAC.
Conflict of interest 12
The authors have no conflict of interest. Acknowledgments This project was supported by grant from the National Natural Science Foundation of China (No. 31672543), the Science and Technology Department of Zhejiang (NO. 2012C12009-2), Key Project of Science and Technology Innovation Team of Zhejiang Province (No. 2012R10031-14）
13
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of Toxoplasma-Gondii in-Vitro. Experimental Parasitology 78, 361-370. Sugi, T., Ma, Y.F., Tomita, T., Murakoshi, F., Eaton, M.S., Yakubu, R., Han, B., Tu, V., Kato, K., Kawazu, S., Gupta, N., Suvorova, E.S., White, M.W., Kim, K., Weiss, L.M., 2016. Toxoplasma gondii Cyclic AMP-Dependent Protein Kinase Subunit 3 Is Involved in the Switch from Tachyzoite to Bradyzoite Development. MBio 7. Swierczewski, B.E., Davies, S.J., 2009. A schistosome cAMP-dependent protein kinase catalytic subunit is essential for parasite viability. PLoS Negl Trop Dis 3, e505. Syin, C., Parzy, D., Traincard, F., Boccacio, I., Joshi, M.B., Lin, D.T., Yang, X.M., Assemat, K., Doerig, C., Langsley, G., 2001. The H89 cAMP-dependent protein kinase inhibitor blocks Plasmodium falciparum development in infected erythrocytes. European Journal of Biochemistry 268, 4842-4849. Taylor, S.S., Kim, C., Cheng, C.Y., Brown, S.H., Wu, J., Kannan, N., 2008. Signaling through cAMP and cAMP-dependent protein kinase: diverse strategies for drug design. Biochim Biophys Acta 1784, 16-26. Uhler, M.D., Carmichael, D.F., Lee, D.C., Chrivia, J.C., Krebs, E.G., Mcknight, G.S., 1986. Isolation of Cdna Clones Coding for the Catalytic Subunit of Mouse Camp-Dependent Protein-Kinase. P Natl Acad Sci USA 83, 1300-1304. Walsh,
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Weilhammer, D.R., Iavarone, A.T., Villegas, E.N., Brooks, G.A., Sinai, A.P., Sha, W.C., 2012. Host metabolism regulates growth and differentiation of Toxoplasma gondii. Int J Parasitol 42, 947-959. Weiss, L.M., Laplace, D., Takvorian, P.M., Tanowitz, H.B., Cali, A., Wittner, M., 1995. A Cell-Culture System
for Study of the Development
Toxoplasma-Gondii Bradyzoites. J Eukaryot Microbiol 42, 150-157.
18
of
Legends to figures Fig 1 Preparation of recombination protein and analysis of polyclonal antibody specificity by Western blotting and Indirect immunofluorescence
(a) PCR amplification of the opening reading frame of TgPKAC, lane 1: PCR amplification product. (b) Identification of TgPKAC-pMD18T vector by enzyme digestion, lane 1: Control of TgPKAC-pMD18T plasmid; lane 2 and 3: TgPKAC-pMD18T plasmid was identified by restriction enzymes NdeI/HindIII. (c) Purification of recombinant protein using nickel column, lane 1 and 2: SDS-PAGE analysis of TgPKAC-pET28a-BL21 protein purified by nickel. (d) The specific response of polyclonal antibody was determined by Western
blotting
analysis,
lane
1:
Recombination
protein
of
TgPKAC-pET28a-BL21 was analysed using TgPKAC polyclonal antibody; lane 2: Toxoplama lysate antigens was confirmed by Western blotting. (e) Indirect immunofluorescence analysis to show the cellular localization of TgPKAC.
19
Fig 2 Generation of TgPKAC overexpression strain and identification of the parasite by PCR and Western blotting (a, b) Schematic of the experimental design of the TgPKAC overexpression strain. A PKAC coding sequence and the CAT selection marker，which was flanked by T. gondii Tublin promoter and 3’ poly A signal (a gift from professor Liuqun, College of Veterinary Medicine, China Agricultural University), was transfected into the RH△Ku80 strain to generate the overexpression strain. (c) PCR analysis of (TgPKAC-OE) strain. (d) Western blotting was detected with anti-rTgPKAC antibody on total extracts from RH△Ku80 and TgPKAC-OE strains, and TgActin was used as control.
20
Fig 3 Microscope observation, plaque assay and quantitative real-time PCR to analyse the growth of PKAC overexpression strain (TgPKAC-OE) in vitro (a, b, c) Observation of TgPKAC-OE, TgPKAC inhibition and RH△Ku80 parasites by inverted microscope. (d, e, f) Plaque assay comparing growth of TgPKAC-OE, TgPKAC inhibition and RH△ku80 parasites. Data were collected from three independent experiments. (g) Quantitative real-time PCR was used to detect the number of parasites by T.gondii B1 gene primers. Results showed higher growth rate of TgPKAC-OE strain, compared to the TgPKAC inhibition and RH△Ku80 parasites. The data were means ± SD for three experiments. *, P< 0.05; **, P< 0.01;***,P< 0.001.
21
Fig 4 Analysis of ultrastructure by transmission electron microscopy and detection of expression level of TgATG8 by relative fluorescence quantitative PCR (a, b) Ultrastructural analysis of TgPKAC inhibition and RH △ Ku80 parasites. Arrowheads indicate structures like autophagosomes. (c) The relative expression level of T. gondii autophagy-related protein (TgATG8) was analysed by qRT-PCR assay. The data were means ± SD for three experiments. *, P< 0.05.
22
Fig 5 Virulence of TgPKAC-OE, RH△Ku80 and TgPKAC inhibition parasites Purified tachyzoites from different strains were intraperitoneally injected (100 parasites/mice) into 6-8 weeks of female BALB/c mice, with 5 mice ineach group, the survival rate was monitored. The survival curve of mice infected with TgPKAC-OE, RH△Ku80 and TgPKAC inhibition parasites, showing that overexpression of PKAC increased virulence of T. gondii while inhibition of PKAC decreased virulence of T. gondii. The experiment was performed three times.
23
Fig 6 Subcellular localization analysis of TgPKAC TgPKAC-EGFP-C2 vector was constructed and transfected into Vero cells. (a, b, c) Nuclear DNA was labelled with DAPI (blue). Localization of EGFP-C2 plasmid was analysed by laser confocal microscope. (d, e, f) TgPKAC was mainly expressed in the cytoplasm of Vero cells and was gathered expression around the nuclei.
24
Table 1 Primers used in this study primers P1 P2 P3 P4 P5 P6 Actin-F Actin-R B1-F B1-R ATG8-F ATG8-R
sequences 5’-CCGCTCGAGATGCCGTTGCGTACAATGTCG-3’ 5’-CCCATCGATTCAAAAATTGTCGAAGAAGGCCTGC-3’ 5’-ATTCCATATGCCGTTGCGTACAATGTCG-3’ 5’-CCCAAGCTTTCAAAAATTGTCGAAGAAGGCCTGC-3’ 5’-GGAAGATCTATGCCGTTGCGTACAATGTCG-3’ 5’-CCCAAGCTTAAAATTGTCGAAGAAGGCCTGC-3’ 5’-CACGAGAGAGGATACGGCTTCACCA-3’ 5’-CCATCGGGCAATTCATAGGACTTCTC-3’ 5’-GGAACTGCATCCGTTCATGAG-3’ 5’-TCTTTAAAGCGTTCGTGGTC-3’ 5’-TGATTGACAAGAAGAAGTT-3’ 5’-GAGAAAACGATTTATTTG-3’
25
S0304-4017(17)30258-3 http://dx.doi.org/doi:10.1016/j.vetpar.2017.06.002 VETPAR 8367
To appear in:
Veterinary Parasitology
Received date: Revised date: Accepted date:
29-3-2017 20-5-2017 2-6-2017
Please cite this article as: Sun, Hongchao, Wang, Suhua, Zhao, Xianfeng, Yao, Chaoqun, Zhuang, Haohan, Huang, Yechuan, Chen, Xueqiu, Yang, Yi, Du, Aifang, Targeted overexpression of cyclic AMP-Dependent Protein Kinase Subunit in Toxoplasma gondii promotes replication and virulence in host cells.Veterinary Parasitology http://dx.doi.org/10.1016/j.vetpar.2017.06.002 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
Targeted overexpression of cyclic AMP-Dependent Protein Kinase Subunit in Toxoplasma gondii promotes replication and virulence in host cells
Hongchao Sun1, Suhua Wang2, Xianfeng Zhao3, ChaoqunYao4, Haohan Zhuang1, Yechuan Huang1, Xueqiu Chen1, Yi Yang1 , Aifang Du1*
1. College of Animal Sciences，Zhejiang Provincial Key Laboratory of Preventive Veterinary Medicine, Zhejiang University, Hangzhou 310058, China 2. Wenzhou Entry-exit Inspection and Quarantine Bureau, Wenzhou, Zhejiang 325027, China 3. Shenzhen Entry-exit Inspection and Quarantine Bureau, Shenzhen, Guangdong 518045 4. Department of Biomedical Sciences and One Health Center for Zoonoses and Tropical Veterinary Medicine, Ross University School of Veterinary Medicine, P.O. Box 334, Basseterre, St. Kitts, West Indies
*Correspondence should be addressed to: Dr. Aifang Du, College of Animal Sciences, Zhejiang Provincial Key Laboratory of Preventive Veterinary Medicine, Zhejiang University, Hangzhou, Zhejiang 310058, China Telephone number: +86-571-8898-2583 E-mail: [email protected]
1
Highlights:
1 T.gondii PKAC (TgPKAC) overexpression strain (TgPKAC-OE) was
constructed.
2 Inhibition of TgPKAC could cause autophagy of Toxoplasma gondii and
then influence the replication of the parasite.
3 TgPKAC plays an important role in parasite virulence in vivo.
4 TgPKAC was mainly expressed in the cytoplasm of Vero cell.
Abstract: Toxoplasma gondii (T. gondii) is one of the most common parasite that can infect almost
any
warm-blooded
animals
including
humans.
The
cyclic
nucleotide-dependent protein kinase (PKA) regulates a spectrum of intracellular signal pathways in many organisms. Protein kinase catalytic subunit (PKAC) is the core of the whole protein, and plays an important role in the life cycle of T.gondii. Here, T.gondii PKAC (TgPKAC) overexpression strain (TgPKAC-OE) was constructed. The growth of the TgPKAC-OE, RH△ Ku80, and TgPKAC inhibition strains (TgPKAC-H89) were analysed by SYBR-green real-time PCR, and the ultrastructure was observed by transmission electron microscopy. The survival rate in mice was also recorded to analyse the virulence of the parasites. We also investigated the subcellular localization of TgPKAC in Vero cells by laser scanning microscope. We found that TgPKAC-OE strain exhibited obviously increased growth rate in Vero cells in vitro, and infected mice survived for a shorter time compared to wild type strain. Ultrastructural analysis found more autophagosomes-like structures in TgPKAC-H89 parasite compared to RH△Ku80 strain, and the relative expression level of Toxoplasma gondii autophagy-related protein (ATG8) in TgPKAC-H89 parasite was higher than wild type parasite. Laser confocal results showed that TgPKAC was mainly expressed in the cytoplasm of Vero cells. In conclusion, we 2
hypothesized that inhibition of TgPKAC could cause autophagy of Toxoplasma gondii and then influence the replication of the parasite. TgPKAC plays an important role in parasite virulence in vivo, and the subcellular localization was successfully detected in Vero cells. Our data will provide a basis for further study of TgPKAC function and help screen drug targets of T. gondii.
Key words: Toxoplasma gondii, protein kinase catalytic subunit, growth, autophagy, virulence, subcellular localization.
1 Introduction Toxoplasma gondii (T. gondii) is a specific intracellular protozoan parasite that can infect almost all mammals including humans (Hunter and Sibley, 2012; Montoya and Liesenfeld, 2004). Despite enjoying a broad host range, the feline is the only definitive host where the sexual reproductive stages occur (Dlugonska H, 2014). The asexual stages of T. gondii do not have host specificity, thus the variety of intermediate hosts facilitates the transmission of T. gondii (Gazzinelli et al., 2014). During the acute stage of infection, the parasites replicate as the fast-growing tachyzoite form. Under a variety of stress conditions, such as pH change (Weiss et al., 1995) and heat shock (Soete et al., 1994), the fast-growing tachyzoite can be transformed into slow-growing bradyzoite (Skariah et al., 2010), and the host cells also play an important role in the conversion between tachyzoite and bradyzoite (Skariah et al., 2010; Weilhammer et al., 2012). The complex life stages of T. gondii require a series of regulatory mechanisms, such as phosphorylation and transcriptional modifications (Wei et al., 2013). Among a variety of serine, threonine protein kinase families, the cyclic nucleotide-dependent protein kinase (PKA) is thought to be an important model protein for studying other kinases (Gerlits et al., 2014; Johnson D A, 2001), it was one of the first protein kinase had been found, sequenced and cloned (Shoji et al., 1981; Uhler
et
al.,
1986;
Walsh
et
al.,
1968).
In
eukaryotic
cells,
cyclic
nucleotide-dependent protein kinases are present in two forms, the regulatory subunits 3
(PKARs) and catalytic subunits (PKACs) (Kim C, 2005). Protein kinase catalytic subunit (PKAC) is the core of the whole protein, mainly composed of N-terminal domain and C-terminal domain, and they determine the localization and enzyme active of PKAC protein (Taylor et al., 2008). Among eukaryotes, all protozoa or helminth parasites use protein kinases to maintain the cellular functions (Dissous et al., 2007). PKAC had been reported in Schistosoma (Dissous et al., 2007; Swierczewski and Davies, 2009), Plasmodium falciparum (Beraldo et al., 2005), 2005), Plasmodium chabaudi (Gazarini et al., 2011), Toxoplasma gondii (Sugi et al., 2016), Giardia lamblia (Gibson et al., 2006), Microsporidia (Equinet et al., 2004), Leishmania (Siman-Tov MM, 2002). Matsuyama and his colleagues found that the swimming speed of Schistosoma mansonimiracidia became slowly when the parasites are treated with specific PKAC inhibitor (Matsuyama et al., 2004). In Plasmodium falciparum, the expression level of PKAC varies at different life stages, and PKAC showed higher expression at asexual stage. If the Plasmodium falciparum schizont was inhibited with H89, the parasites exhibited growth defects in infected erythrocytes (Syin et al., 2001). Eaton et al found that PKAC was involved in tachyzoite-bradyzoite transition in T. gondii (Eaton et al., 2006) , and inhibition of PKAC with H89 could slow down the growth of tachyzoites (Kurokawa et al., 2011). In this study, we analysed the function of TgPKAC in proliferation and virulence by the construction of TgPKAC overexpression strains and the ultrastructures of parasites were also observed by transmission electron microscopy. The results showed that TgPKAC plays an important role in parasites virulence in vivo, inhibition of TgPKAC using H89 could cause autophagy of T. gondii and then influence the replication of T. gondii.
2 Materials and methods 2.1 Ethics All mice experiments were performed accoding to the recommendations of the Guide for the regulation for the Administration of Affairs concerning Experimental 4
Animal of the People’s Republic of China. The animal usage was approved by Zhejiang University Experimental Animal Ethics Committee (Permit Number: ZJU201308-1-10-072).
2.2 Host cell and parasite cultures T. gondii RHΔKu80 strain was maintained on monolayers of African green monkey kidney (Vero) cells at 37 ℃ with 5% CO2. The culture medium was Dubelcco’s Modified Eagle Medium (DMEM, Hyclone) supplemented with 10% fetal calf serum (FCS,Hyclone), 2 mM L-glutamine and 100 units penicillin/100 mg streptomycin.
2.3 Construction of overexpression strain 107 of T. gondii tachyzoites were collected, total RNA was extracted and reverse transcribed into cDNA. The opening reading frame of TgPKAC (Gene ID, 7898189) was amplified by PCR with primers p1/p2 (Table 1), the PCR reaction condition was 94℃ 5min, 94℃ 30s, 56℃ 1min30s, 72℃ 5min, 30 cycles and 72℃ 10min. SAG1 promoter and GRA2 polytail were also amplified by PCR to initiate TgPKAC gene. The vector contained chloramphenicol resistance gene CAT. 20-25 μg of the plasmid were linearized with NotI and transfected into RH△Ku80 parasites. Stable clones were selected by 20 μM chloramphenicol (Sigma). PKAC expression was confirmed by PCR and Western blotting.
2.4 Preparation of polyclonal antibody and western blotting analysis The coding sequence of TgPKAC was amplified using the primers p3/p4 (Table 1), according to the gene sequence from GenBank (Gene ID, 7898189), then subcloned into pMD18T vector. TgPKAC-pMD18T plasmid and pET28a vector were digested with restriction enzymes NdeI/HindIII, DNA fragments were purified by DNA purification kit and connected with T4 DNA ligase. The vector of TgPKAC-pET28a was identified by double enzyme digestion and DNA sequencing analysis. The recombinant plasmid was transformed into Escherichia coli BL21 cells. After induction with IPTG (1mM), the cells were collected at 0, 2, 4, 6 and 8 h, centrifuged 5
at 12000 rpm for 1 min. The precipitate was cracked with loading buffer and analysed by SDS-PAGE. SDS-PAGE result showed that large amount of recombinant protein was induced after 2 to 4 h. The recombinant protein was identified by Western blotting analysis. The protein concentration was measured using BCA kit, equal amount of protein and Freund's complete adjuvant were emulsified and injected into the back muscle of the rabbit (0.5mg/kg). For the second and third inoculation, the protein was emulsified with freund's incomplete adjuvant (0.25 mg/kg). After the third immunization, the ear vein blood was collected and serum was prepared at 4℃ overnight. The antibody titer was detected by enzyme-linked immune sorbent assay (ELISA) and the specificity of the antibody was analyzed by Western blotting.
2.5 Construction and expression of the recombinant eukaryotic expression plasmid PKAC gene in Vero cells The primers p5/p6 (Table 1) were designed according to the gene sequence from GenBank (Gene ID, 7898189). The pMD-TgPKAC plasmid was constructed and digested by BglⅡ/HindIII enzymes to construct the eukaryotic expression vector TgPKAC-EGFP-C2. The expression plasmid was transfected into Vero cells by liposome 2000. After inoculated for 24 h, the cell climbing tablets were removed from the 6 well plates and washed with 0.1 M PBS for three times. Then fixed with 4% paraformaldehyde for 10 min. 50 μl DAPI fluorescent dyes were added to the climbing tablets and incubated for 5min. Finally, the localization of PKAC in Vero cells was observed by a laser confocal microscope.
2.6 Transmission electron microscopy analysis Vero cells were cultured in 6 well plates (105/well) for 24h. Equal amounts of T gondii RHΔKu80 tachyzoites (105) were incubated with TgPKAC inhibitor H89 (30 μM) (Selleck Chemicals) or DMSO for 3 h. The parasites were centrifuged at 3000 rpm for 5min, then washed with DMEM to remove the H89 and DMSO. Afterwards the pretreated T. gondii were inoculated to Vero cells. About 48 h, the parasites were 6
collected and filtered with 27G needle in order to obtain purified tachyzoites, then centrifuged at 3000 rpm for 5min. The precipitate was fixed with 4% glutaraldehyde for 3-5 days at 4 ℃, centrifuged at 1500 rpm for 5min, washed with 0.1 M PBS for three times. 1% agar solution was added to the cell pellet, the solidified agar was removed and trimmed into the right size. The cells were fixed with 1% osmic acid for 2 h, followed by washing with PBS. The samples were dehydrated with 50%, 70%, 80%, 90%, 95%, 100% ethanol every 20 min and finally treated with acetone for 20 min. The samples were embedded overnight at 70 ℃, sliced with ultra-thin slicer, stained with 2% uranyl acetate and 0.25% lead citrate for 5 min, and observed by transmission electron microscopy. Then the expression level of TgATG8 was detected by qRT-PCR, equal number of parasites (105) were inoculated to Vero cells. After cultured for 24 h, 48 h, 72 h, the samples were collected and RNAs were extracted by Trizol reagent (Invitrogen) and reverse transcribed into cDNA using transcription kit (Toyobo). qRT-PCR was performed by Applied Biosystems Inc 7500 fluorescence quantitative PCR instrument. The primers for actin and ATG8 in T. gondii were included in Table 1. The data were expressed as means ± SD for three experiments and results were calculated using the 2-△△t method.
2.7 Virulence analysis in BALB/c mice 100 of TgPKAC-OE, RHΔKu80, and H89 inhibitor treatment strains were injected into BALB/c mice by intraperitoneal injection, with 5 mice in each group. The status of infected mice was monitored each day, and the survival rate was recorded. The data represented three independent experiments.
2.8 Plaque assay and intracellular growth For plaque assay, 200 of TgPKAC-OE, RH△Ku80, and TgPKAC inhibition strains were inoculated in the 6-well Vero cells, and cultured at 37 ℃ with 5% CO2 for 14 days. After 14 days, the culture medium was removed and the Vero cells were washed with PBS for three times, and fixed with 4% paraformaldehyde for 10 min. Then 7
stained with 0.1% crystal violet for 30 min, and the plaques were observed using an Olympus microscope. Each sample was at least randomly observed for 50 times, and the result represented three replicates experiments. Vero cells were cultured in 6-well plate for 24 h, with 105 in each well. TgPKAC-OE, RH△Ku80, and TgPKAC inhibition strains were inoculated to Vero cells, the infection ratio was 1:1. After infection for 2 h, the noninvasive tachyzoites were removed and fresh medium was added to the cells. Next the samples were collected after infection for 24 h, 48 h, 72 h, and genome DNA were extracted using the TIANGEN genomic DNA isolation kit by following the manufacturer’s protocol. The parasites numbers were determined by real-time PCR using B1 gene primers (Table 1). Triplicates samples were used for each time point of three independent experiments.
2.9 Statistical analysis Results in the present study represented mean ± S.D., numbers in groups were compared by Student's t-test. P value of <0.05 was considered significant difference.
3 Results
3.1 Preparation of polyclonal antibody and identified by western blotting The coding sequence was obtained and clonal vector of TgPKAC-pMD18T was constructed successfully (Fig 1a, 1b). SDS-PAGE analysis showed that recombinant protein was purified successfully, the molecular mass was 57.2 KDa (Fig 1c). For subsequent experiments, the rabbit polyclonal antibody was prepared. The protein was immunized into the back of rabbit to generate antiserum. ELISA result revealed that the antibody titer was 0.256 million and indirect immunofluorescence assay (IFA) analyzed the reactivity of the polyclonal antibody, Western blotting results showed that the rabbit antibody could specifically recognize T. gondii antigen (Fig 1d), and the TgPKAC was located in the cytoplasm of the intracellular parasites (Fig 1e). 8
3.2 Construction of TgPKAC overexpression strain To characterize the biological role of TgPKAC, we successfully constructed a TgPKAC overexpression strain (TgPKAC-OE), the schematic diagram was showed in Fig 2a, 2b. RT-PCR results showed that the gene expression level of TgPKAC was significantly higher than wild type strain (Fig 2c), and western blotting result also showed that overexpression strain was constructed successfully (Fig 2d).
3.3 Growth rate detection in Vero cells As shown in microscope observation, the growth rate of TgPKAC inhibition
strains (Fig 3b) were slower than wild parasites (Fig 3c), and the TgPKAC-OE (Fig 3a) strains revealed higher growth rate than wild strains. Accordingly, the plaque area of TgPKAC-OE (Fig 3d) was larger than the other two strains (Fig 3e, 3f). To further assess the proliferation role of TgPKAC in T. gondii, we calculated the growth rate at different time points. As showed in Fig 3g, the growth of TgPKAC-OE strains were increased by 2.21-fold (P<0.05), 2.57-fold (P<0.01), 3.41-fold (P<0.01) in comparison with that of the H89 inhibitor parasites, and increased by 1.05-fold, 1.13-fold (P<0.05), 1.30-fold (P<0.05) comparing to RH△Ku80 strains at 24 h, 48 h and 72 h, respectively. These data collectively demonstrated that TgPKAC is required for the normal growth of T. gondii.
3.4 Analysis of ultrastructures by transmission electron microscopy Our results showed TgPKAC was involved in the replication of T. gondii, next we sought to know if the growth defects were related to autophagy of T. gondii. RH△Ku80 and TgPKAC inhibition strains were cultured in Vero cells. After infection for 48 h, the samples were collected and observed by transmission electron microscopy. Ultrastructural analysis showed more structures like autophagosomes were found in TgPKAC inhibition parasites (Fig 4a) compared to RH△Ku80 strains (Fig 4b), suggesting that TgPKAC could induce the autophagy of T. gondii. In order to further confirm the role of TgPKAC in induction of autophagy, the 9
relative expression level of TgATG8 (autophagy-related protein) was detected by qRT-PCR assay, which revealed that inhibition of TgPKAC generated an increased expression of TgATG8 (P<0.05) compared to the RH△Ku80 strains (Fig 4c).
3.5 Survival rate of TgPKAC-OE, RH△Ku80 andTgPKAC inhibition parasites Our results showed that TgPKAC was related to the replication of T. gondii in Vero cells, we therefore investigated the virulence of TgPKAC inhibition strains and TgPKAC-OE. We injected 100 tachyzoites of TgPKAC inhibition strains, RH△ Ku80, and TgPKAC-OE to the BALB/c mice. The survival time was recorded, and we found that the mice began to appear symptoms 4 days after infection. TgPKAC-OE and RH△Ku80 strains showed a significantly shorter life time than the H89 inhibitor parasites (Fig 5), implying that TgPKAC could be a virulence factor of T. gondii.
3.6 Localization of TgPKAC in Vero cell The subcellular localization of proteins in host cells are closely related to their function, thus we analysed the expression and localization of TgPKAC in Vero cells. Laser confocal results showed that TgPKAC was mainly expressed in the cytoplasm of Vero cells, and the expression was gathered around the nuclei (Fig 6d, 6e). EGFP-C2 plasmid was transfected into Vero cells as the control (Fig 6a) and the nucleus DNA was stained with DAPI (blue) (Fig 6c, 6f), EGFP and DAPI were merged in the Fig 6b, 6e.
4 Discussion To understand the role of TgPKAC in T. gondii, we generated a TgPKAC overexpression strain (TgPKAC-OE), the phenotype of the TgPKAC gene was analysed in our study. Firstly, we analysed the replication rate of the TgPKAC-OE, RH△Ku80, and TgPKAC inhibition strains in Vero cells. Although the T. gondii PKAC gene was studied by Kurokawa et al (Kurokawa et al., 2011), the TgPKAC-OE strain had not been reported in previous study. In this study, we found that the TgPKAC-OE strain revealed higher growth rate and larger plaque area than the other 10
two strains (Fig 3). cAMP-dependent protein kinase (PKAC) studies of other parasites such as Plasmodium falciparum showed that when the schizont was inhibited with H89, the parasites exhibited growth defects in infected erythrocytes (Syin et al., 2001). In Giardia Iamblia (Abel et al., 2001), PKAC played an important role in regulating trophozoite motility as well as cellular activation of excystation. Our result suggested that PKAC in T. gondii could be involved into the growth stage, and this process could be controlled by a variety of signaling pathways. Autophagy is considered as an important regulatory mechanism in mammalian cells in recent years (Corcelle et al., 2007; Ozpolat, 2009; Simone, 2007), but little is known in parasites. In Leishmania (Bhattacharya et al., 2012), Bhattacharya et al found that cAMP-binding PKA regulatory subunit plays an important role in regulating parasitic differentiation by modulating induction of autophagy in the parasites. In T. gondii, when the parasites were inhibited with tamoxifen, the autophagy marker light chain 3-green fluorescent protein (LC3-GFP) could be recruited to the membrane of the parasitophorous vacuole and the growth was restricted, this revealed that tomoxifen could restrict the growth of T. gondii by inducing xenophagy or autophagic (Dittmar et al., 2016). In the present study, TgPKAC was suggested to be involved in the replication of T. gondii. We would like to know if the growth defects were related to autophagy of T. gondii. RH△Ku80, and TgPKAC inhibition strains were observed by transmission electron microscopy. Ultrastructural analysis showed that more autophagosomes-like structures were found in TgPKAC inhibition parasite compared to RH△Ku80 strains (Fig 5). The relative expression level of T. gondii autophagy-related protein (ATG8) was detected by qRT-PCR, the result revealed that the expression level of TgATG8 was increased when TgPKAC was inhibited with H89. We hypothesized that inhibition of PKAC gene may cause autophagy of T. gondii, and the growth rate in Vero Cells may be influenced by autophagy. Further studies need to be carried out to investigate the exact mechanism. Many protein kinases in T. gondii are virulence factors, such as the rhoptry proteins (ROPS) (Sibley et al., 2009), casein kinase 1 α (CK1 α) (Wang et al., 2016b), aurora kinase (Ark3) (Berry et al., 2016). The virulence 11
was regulated by a variety of signaling pathways and different members of the same protein family could balance each other to regulate the virulence of T. gondii in mice (Wang et al., 2016a). Because TgPKAC was involved in the replication of T. gondii in Vero cells, we wanted to know if it was related to the virulence in mice. The result showed that TgPKAC-OE strain showed a significantly shorter life time than the RH△Ku80 and H89 inhibitor parasites, suggesting that TgPKAC may be a virulence factor of T. gondii. While it was not clear whether PKARs, the regulatory subunits of cAMP-depdent protein kinase, were coordinated with PKAC to regulate the virulence. In addition to study the gene function of TgPKAC in replication and virulence of T. gondii, the localization of TgPKAC in Vero cells was also detected by laser scanning microscope (Fig 6). Other parasites, such as Theileria annulata (Ma and Baumgartner, 2014), Giadia lamblia (Abdul-Wahid and Faubert, 2004; Chen et al., 2008), the eukaryotic transfection was also used to study the gene function in host cells. Our results showed that TgPKAC could be successfully transfected into Vero cells, and TgPKAC was mainly expressed in the cytoplasm of Vero cells and the expression was gathered around the nuclei. In conclusion, we constructed TgPKAC overexpression strain in this work to investigate the role of PKAC in T. gondii. Our results showed that TgPKAC contributed to replication of T. gondii in host cells and TgPKAC playd an important role in parasite virulence in vivo. Inhibition of TgPKAC resulted in the formation of autophagosomes-like structures in T. gondii, suggesting that the growth defect of TgPKAC inhibition strain was related to the autophagy of T. gondii. These data will provide a basis for further study of TgPKAC function and help screen drug targets of T. gondii. Future studies of TgPKAC are needed to clarify the interaction molecules of PKAC.
Conflict of interest 12
The authors have no conflict of interest. Acknowledgments This project was supported by grant from the National Natural Science Foundation of China (No. 31672543), the Science and Technology Department of Zhejiang (NO. 2012C12009-2), Key Project of Science and Technology Innovation Team of Zhejiang Province (No. 2012R10031-14）
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Legends to figures Fig 1 Preparation of recombination protein and analysis of polyclonal antibody specificity by Western blotting and Indirect immunofluorescence
(a) PCR amplification of the opening reading frame of TgPKAC, lane 1: PCR amplification product. (b) Identification of TgPKAC-pMD18T vector by enzyme digestion, lane 1: Control of TgPKAC-pMD18T plasmid; lane 2 and 3: TgPKAC-pMD18T plasmid was identified by restriction enzymes NdeI/HindIII. (c) Purification of recombinant protein using nickel column, lane 1 and 2: SDS-PAGE analysis of TgPKAC-pET28a-BL21 protein purified by nickel. (d) The specific response of polyclonal antibody was determined by Western
blotting
analysis,
lane
1:
Recombination
protein
of
TgPKAC-pET28a-BL21 was analysed using TgPKAC polyclonal antibody; lane 2: Toxoplama lysate antigens was confirmed by Western blotting. (e) Indirect immunofluorescence analysis to show the cellular localization of TgPKAC.
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Fig 2 Generation of TgPKAC overexpression strain and identification of the parasite by PCR and Western blotting (a, b) Schematic of the experimental design of the TgPKAC overexpression strain. A PKAC coding sequence and the CAT selection marker，which was flanked by T. gondii Tublin promoter and 3’ poly A signal (a gift from professor Liuqun, College of Veterinary Medicine, China Agricultural University), was transfected into the RH△Ku80 strain to generate the overexpression strain. (c) PCR analysis of (TgPKAC-OE) strain. (d) Western blotting was detected with anti-rTgPKAC antibody on total extracts from RH△Ku80 and TgPKAC-OE strains, and TgActin was used as control.
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Fig 3 Microscope observation, plaque assay and quantitative real-time PCR to analyse the growth of PKAC overexpression strain (TgPKAC-OE) in vitro (a, b, c) Observation of TgPKAC-OE, TgPKAC inhibition and RH△Ku80 parasites by inverted microscope. (d, e, f) Plaque assay comparing growth of TgPKAC-OE, TgPKAC inhibition and RH△ku80 parasites. Data were collected from three independent experiments. (g) Quantitative real-time PCR was used to detect the number of parasites by T.gondii B1 gene primers. Results showed higher growth rate of TgPKAC-OE strain, compared to the TgPKAC inhibition and RH△Ku80 parasites. The data were means ± SD for three experiments. *, P< 0.05; **, P< 0.01;***,P< 0.001.
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Fig 4 Analysis of ultrastructure by transmission electron microscopy and detection of expression level of TgATG8 by relative fluorescence quantitative PCR (a, b) Ultrastructural analysis of TgPKAC inhibition and RH △ Ku80 parasites. Arrowheads indicate structures like autophagosomes. (c) The relative expression level of T. gondii autophagy-related protein (TgATG8) was analysed by qRT-PCR assay. The data were means ± SD for three experiments. *, P< 0.05.
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Fig 5 Virulence of TgPKAC-OE, RH△Ku80 and TgPKAC inhibition parasites Purified tachyzoites from different strains were intraperitoneally injected (100 parasites/mice) into 6-8 weeks of female BALB/c mice, with 5 mice ineach group, the survival rate was monitored. The survival curve of mice infected with TgPKAC-OE, RH△Ku80 and TgPKAC inhibition parasites, showing that overexpression of PKAC increased virulence of T. gondii while inhibition of PKAC decreased virulence of T. gondii. The experiment was performed three times.
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Fig 6 Subcellular localization analysis of TgPKAC TgPKAC-EGFP-C2 vector was constructed and transfected into Vero cells. (a, b, c) Nuclear DNA was labelled with DAPI (blue). Localization of EGFP-C2 plasmid was analysed by laser confocal microscope. (d, e, f) TgPKAC was mainly expressed in the cytoplasm of Vero cells and was gathered expression around the nuclei.
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Table 1 Primers used in this study primers P1 P2 P3 P4 P5 P6 Actin-F Actin-R B1-F B1-R ATG8-F ATG8-R
sequences 5’-CCGCTCGAGATGCCGTTGCGTACAATGTCG-3’ 5’-CCCATCGATTCAAAAATTGTCGAAGAAGGCCTGC-3’ 5’-ATTCCATATGCCGTTGCGTACAATGTCG-3’ 5’-CCCAAGCTTTCAAAAATTGTCGAAGAAGGCCTGC-3’ 5’-GGAAGATCTATGCCGTTGCGTACAATGTCG-3’ 5’-CCCAAGCTTAAAATTGTCGAAGAAGGCCTGC-3’ 5’-CACGAGAGAGGATACGGCTTCACCA-3’ 5’-CCATCGGGCAATTCATAGGACTTCTC-3’ 5’-GGAACTGCATCCGTTCATGAG-3’ 5’-TCTTTAAAGCGTTCGTGGTC-3’ 5’-TGATTGACAAGAAGAAGTT-3’ 5’-GAGAAAACGATTTATTTG-3’
25