Isolation and identification of sporozoite membrane protein of Cryptosporidium parvum and evaluation of calmodulin-like protein immune protection
Yan Huang
Key Laboratory of Animal Parasitology of Ministry of Agriculture, Laboratory of Quality and Safety Risk Assessment for Animal Products on Biohazards (Shanghai) of Ministry of Agriculture, Shanghai Veterinary Research Institute, Chinese Academy of Agricultural Sciences, Shanghai, China
Search for more papers by this authorYu Chen
Key Laboratory of Animal Parasitology of Ministry of Agriculture, Laboratory of Quality and Safety Risk Assessment for Animal Products on Biohazards (Shanghai) of Ministry of Agriculture, Shanghai Veterinary Research Institute, Chinese Academy of Agricultural Sciences, Shanghai, China
Search for more papers by this authorYuxuan Liu
Key Laboratory of Animal Parasitology of Ministry of Agriculture, Laboratory of Quality and Safety Risk Assessment for Animal Products on Biohazards (Shanghai) of Ministry of Agriculture, Shanghai Veterinary Research Institute, Chinese Academy of Agricultural Sciences, Shanghai, China
Search for more papers by this authorRongsheng Mi
Key Laboratory of Animal Parasitology of Ministry of Agriculture, Laboratory of Quality and Safety Risk Assessment for Animal Products on Biohazards (Shanghai) of Ministry of Agriculture, Shanghai Veterinary Research Institute, Chinese Academy of Agricultural Sciences, Shanghai, China
Search for more papers by this authorXiangan Han
Key Laboratory of Animal Parasitology of Ministry of Agriculture, Laboratory of Quality and Safety Risk Assessment for Animal Products on Biohazards (Shanghai) of Ministry of Agriculture, Shanghai Veterinary Research Institute, Chinese Academy of Agricultural Sciences, Shanghai, China
Search for more papers by this authorHaiyan Gong
Key Laboratory of Animal Parasitology of Ministry of Agriculture, Laboratory of Quality and Safety Risk Assessment for Animal Products on Biohazards (Shanghai) of Ministry of Agriculture, Shanghai Veterinary Research Institute, Chinese Academy of Agricultural Sciences, Shanghai, China
Search for more papers by this authorLong Cheng
Key Laboratory of Animal Parasitology of Ministry of Agriculture, Laboratory of Quality and Safety Risk Assessment for Animal Products on Biohazards (Shanghai) of Ministry of Agriculture, Shanghai Veterinary Research Institute, Chinese Academy of Agricultural Sciences, Shanghai, China
Search for more papers by this authorCorresponding Author
Zhaoguo Chen
Key Laboratory of Animal Parasitology of Ministry of Agriculture, Laboratory of Quality and Safety Risk Assessment for Animal Products on Biohazards (Shanghai) of Ministry of Agriculture, Shanghai Veterinary Research Institute, Chinese Academy of Agricultural Sciences, Shanghai, China
Correspondence
Zhaoguo Chen, Key Laboratory of Animal Parasitology of Ministry of Agriculture, Laboratory of Quality and Safety Risk Assessment for Animal Products on Biohazards (Shanghai) of Ministry of Agriculture, Shanghai Veterinary Research Institute, Chinese Academy of Agricultural Sciences, Shanghai, China.
Email: [email protected]
Search for more papers by this authorYan Huang
Key Laboratory of Animal Parasitology of Ministry of Agriculture, Laboratory of Quality and Safety Risk Assessment for Animal Products on Biohazards (Shanghai) of Ministry of Agriculture, Shanghai Veterinary Research Institute, Chinese Academy of Agricultural Sciences, Shanghai, China
Search for more papers by this authorYu Chen
Key Laboratory of Animal Parasitology of Ministry of Agriculture, Laboratory of Quality and Safety Risk Assessment for Animal Products on Biohazards (Shanghai) of Ministry of Agriculture, Shanghai Veterinary Research Institute, Chinese Academy of Agricultural Sciences, Shanghai, China
Search for more papers by this authorYuxuan Liu
Key Laboratory of Animal Parasitology of Ministry of Agriculture, Laboratory of Quality and Safety Risk Assessment for Animal Products on Biohazards (Shanghai) of Ministry of Agriculture, Shanghai Veterinary Research Institute, Chinese Academy of Agricultural Sciences, Shanghai, China
Search for more papers by this authorRongsheng Mi
Key Laboratory of Animal Parasitology of Ministry of Agriculture, Laboratory of Quality and Safety Risk Assessment for Animal Products on Biohazards (Shanghai) of Ministry of Agriculture, Shanghai Veterinary Research Institute, Chinese Academy of Agricultural Sciences, Shanghai, China
Search for more papers by this authorXiangan Han
Key Laboratory of Animal Parasitology of Ministry of Agriculture, Laboratory of Quality and Safety Risk Assessment for Animal Products on Biohazards (Shanghai) of Ministry of Agriculture, Shanghai Veterinary Research Institute, Chinese Academy of Agricultural Sciences, Shanghai, China
Search for more papers by this authorHaiyan Gong
Key Laboratory of Animal Parasitology of Ministry of Agriculture, Laboratory of Quality and Safety Risk Assessment for Animal Products on Biohazards (Shanghai) of Ministry of Agriculture, Shanghai Veterinary Research Institute, Chinese Academy of Agricultural Sciences, Shanghai, China
Search for more papers by this authorLong Cheng
Key Laboratory of Animal Parasitology of Ministry of Agriculture, Laboratory of Quality and Safety Risk Assessment for Animal Products on Biohazards (Shanghai) of Ministry of Agriculture, Shanghai Veterinary Research Institute, Chinese Academy of Agricultural Sciences, Shanghai, China
Search for more papers by this authorCorresponding Author
Zhaoguo Chen
Key Laboratory of Animal Parasitology of Ministry of Agriculture, Laboratory of Quality and Safety Risk Assessment for Animal Products on Biohazards (Shanghai) of Ministry of Agriculture, Shanghai Veterinary Research Institute, Chinese Academy of Agricultural Sciences, Shanghai, China
Correspondence
Zhaoguo Chen, Key Laboratory of Animal Parasitology of Ministry of Agriculture, Laboratory of Quality and Safety Risk Assessment for Animal Products on Biohazards (Shanghai) of Ministry of Agriculture, Shanghai Veterinary Research Institute, Chinese Academy of Agricultural Sciences, Shanghai, China.
Email: [email protected]
Search for more papers by this authorYan Huang and Yu Chen contributed equally to this study.
Funding information: Natural Science Foundation of China, Grant/Award Number: 31702025; Shanghai Agriculture Applied Technology Development Program, China, Grant/Award Number: 2019-02-08-00-08-F01151; Shanghai Science and Technology Commission Scientific Research Project, Grant/Award Number: 20140900400; Agricultural Science and Technology Innovation Program of Chinese Academy of Agricultural Sciences, Grant/Award Number: CAAS-ZDRW202203; National Key Research and Development Program of China, Grant/Award Number: 2017YFD0500401
Abstract
Until now, no completely effective parasite-specific drugs or vaccines have been approved for the treatment of cryptosporidiosis. Through the separation and identification of the sporozoite membrane protein of Cryptosporidium parvum (C. parvum), 20 related proteins were obtained. Among them, a calmodulin-like protein (CML) has a similar functional domain-exchange factor hand (EF-hand) motif as calmodulin proteins (CaMs), so it may play a similarly important role in the invasion process. A 663 bp full gene encoding the C. parvum calmodulin-like protein (CpCML) was inserted in pET28a vector and expressed in Escherichia coli. An immunofluorescence assay showed that CpCML was mainly located on the surface of the sporozoites. Three-week-old female BALB/c mice were used for modelling the immunoreactions and immunoprotection of recombinant CpCML (rCpCML) against artificial Cryptosporidium tyzzeri infections. The results indicated a significantly increased in anti-CpCML antibody response, which was induced by the immunized recombinant protein. Compared to rP23 (recombinant P23), GST6P-1 (expressed by pGEX-6P-1 transfected E. coli), GST4T-1 (expressed by pGEX-4T-1 transfected E. coli), glutathione (GSH), adjuvant and blank control groups, rCpCML-immunized mice produced specific spleen cell proliferation in addition to different production levels of IL-2, IFN-γ, TNF-α, IL-4 and IL-5. Additionally, immunization with rCpCML led to 34.08% reduction of oocyst shedding in C. tyzzeri infected mice faeces which was similar to rP23. These results suggest that CpCML may be developed as a potential vaccine candidate antigen against cryptosporidiosis.
CONFLICT OF INTEREST
The authors declare that they have no conflict of interests.
Open Research
DATA AVAILABILITY STATEMENT
The data that support the findings of this study are openly available in NCBI at https://pubmed.ncbi.nlm.nih.gov/.
REFERENCES
- 1Dumaine JE, Tandel J, Striepen B. Cryptosporidium parvum. Trends Parasitol. 2020; 36(5): 485-486. doi:10.1016/j.pt.2019.11.003
- 2Gomez-Puerta LA, Gonzalez AE, Vargas-Calla A, Lopez-Urbina MT, Cama V, Xiao L. Cryptosporidium parvum as a risk factor of diarrhea occurrence in neonatal alpacas in Peru. Parasitol Res. 2020; 119(1): 243-248. doi:10.1007/s00436-019-06468-7
- 3Gerace E, Lo Presti VDM, Biondo C. Cryptosporidium Infection: epidemiology, pathogenesis, and differential diagnosis. Eur J Microbiol Immunol. 2019; 9(4): 119-123. doi:10.1556/1886.2019.00019
- 4Kotloff KL, Nataro JP, Blackwelder WC, et al. Burden and aetiology of diarrhoeal disease in infants and young children in developing countries (the Global Enteric Multicenter Study, GEMS): a prospective, case-control study. Lancet. 2013; 382(9888): 209-222. doi:10.1016/S0140-6736(13)60844-2
- 5Kelly P. Treatment and prevention of cryptosporidiosis: what options are there for a country like Zambia? Parasitology. 2011; 138(12): 1488-1491. doi:10.1017/S0031182011000035
- 6Gargala G, Delaunay A, Li X, Brasseur P, Favennec L, Ballet JJ. Efficacy of nitazoxanide, tizoxanide and tizoxanide glucuronide against Cryptosporidium parvum development in sporozoite-infected HCT-8 enterocytic cells. J Antimicrob Chemother. 2000; 46(1): 57-60. doi:10.1093/jac/46.1.57
- 7Mi R, Yang X, Huang Y, et al. Immunolocation and enzyme activity analysis of Cryptosporidium parvum enolase. Parasit Vectors. 2017; 10(1): 273. doi:10.1186/s13071-017-2200-y
- 8Burton AJ, Nydam DV, Jones G, et al. Antibody responses following administration of a Cryptosporidium parvum rCP15/60 vaccine to pregnant cattle. Vet Parasitol. 2011; 175(1–2): 178-181. doi:10.1016/j.vetpar.2010.09.013
- 9Askari N, Shayan P, Mokhber-Dezfouli MR, et al. Evaluation of recombinant P23 protein as a vaccine for passive immunization of newborn calves against Cryptosporidium parvum. Parasite Immunol. 2016; 38(5): 282-289. doi:10.1111/pim.12317
- 10Sagodira S, Buzoni-Gatel D, Iochmann S, Naciri M, Bout D. Protection of kids against Cryptosporidium parvum infection after immunization of dams with CP15-DNA. Vaccine. 1999; 17(19): 2346-2355. doi:10.1016/s0264-410x(99)00041-9
- 11Ehigiator HN, Romagnoli P, Priest JW, Secor WE, Mead JR. Induction of murine immune responses by DNA encoding a 23-kDa antigen of Cryptosporidium parvum. J Parasitol Res. 2007; 101(4): 943-950. doi:10.1007/s00436-007-0565-0
10.1007/s00436?007?0565?0 Google Scholar
- 12Benitez A, Priest JW, Ehigiator HN, McNair N, Mead JR. Evaluation of DNA encoding acidic ribosomal protein P2 of Cryptosporidium parvum as a potential vaccine candidate for cryptosporidiosis. Vaccine. 2011; 29(49): 9239-9245. doi:10.1016/j.vaccine.2011.09.094
- 13Baum J, Gilberger TW, Frischknecht F, Meissner M. Host-cell invasion by malaria parasites: insights from Plasmodium and Toxoplasma. Trends Parasitol. 2008; 24(12): 557-563. doi:10.1016/j.pt.2008.08.006
- 14Sibley LD. How apicomplexan parasites move in and out of cells. Curr Opin Biotechnol. 2010; 21(5): 592-598. doi:10.1016/j.copbio.2010.05.009
- 15Carruthers VB, Giddings OK, Sibley LD. Secretion of micronemal proteins is associated with toxoplasma invasion of host cells. Cell Microbiol. 1999; 1(3): 225-235. doi:10.1046/j.1462-5822.1999.00023.x
- 16Zhu G, Keithly JS. Molecular analysis of a P-type ATPase from Cryptosporidium parvum. Molecul Biochem Parasitol. 1997; 90(1): 307-316. doi:10.1016/S0166-6851(97)00168-0
- 17Bhandage AK, Barragan A. Calling in the CaValry-Toxoplasma gondii hijacks GABAergic signaling and voltage-dependent calcium channel signaling for Trojan horse-mediated dissemination. Front Cell Infect Microbiol. 2019; 9: 61. doi:10.3389/fcimb.2019.00061
- 18Docampo R, Moreno SN. Calcium signaling in intracellular protist parasites. Curr Opin Microbiol. 2021; 64: 33-40. doi:10.1016/j.mib.2021.09.002
- 19Nagamune K, Sibley LD. Comparative genomic and phylogenetic analyses of calcium ATPases and calcium-regulated proteins in the apicomplexa. Mol Biol Evol. 2006; 23(8): 1613-1627. doi:10.1093/molbev/msl026
- 20Billker O, Lourido S, Sibley LD. Calcium-dependent signaling and kinases in apicomplexan parasites. Cell Host Microbe. 2009; 5(6): 612-622. doi:10.1016/j.chom.2009.05.017
- 21Wernimont AK, Artz JD, Finerty P Jr, et al. Structures of apicomplexan calcium-dependent protein kinases reveal mechanism of activation by calcium. Nat Struct Mol Biol. 2010; 17(5): 596-601. doi:10.1038/nsmb.1795
- 22Arrowood MJ, Sterling CR. Isolation of Cryptosporidium oocysts and sporozoites using discontinuous sucrose and isopycnic Percoll gradients. J Parasitol. 1987; 73(2): 314-319.
- 23Riggs MW, McGuire TC, Mason PH, Perryman LE. Neutralization-sensitive epitopes are exposed on the surface of infectious Cryptosporidium parvum sporozoites. J Immunol. 1989; 143(4): 1340-1345.
- 24Huang Y, Mi RS, Cao W, et al. Isolation and proteomic analysis of rhoptry-enriched fractions from Cryptosporidium parvum. Iran J Public Health. 2015; 44(9): 1187-1195.
- 25Huang Y, Cao W, Shi K, et al. Protective efficacy of recombinant Cryptosporidium parvum CpPRP1 sushi domain against C. tyzzeri infection in mice. Parasite Immunol. 2017; 39(9). doi:10.1111/pim.12449
- 26Clapham DE. Calcium signaling. Cell. 2007; 131(6): 1047-1058. doi:10.1016/j.cell.2007.11.028
- 27Gifford JL, Walsh MP, Vogel HJ. Structures and metal-ion-binding properties of the Ca2+−binding helix-loop-helix EF-hand motifs. Biochem J. 2007; 405(2): 199-221. doi:10.1042/BJ20070255
- 28Astegno A, Bonza MC, Vallone R, et al. Arabidopsis calmodulin-like protein CML36 is a calcium (Ca(2+)) sensor that interacts with the plasma membrane Ca(2+)-ATPase isoform ACA8 and stimulates its activity. J BioloChem. 2017; 292(36): 15049-15061. doi:10.1074/jbc.M117.787796
- 29Mohanta TK, Kumar P, Bae H. Genomics and evolutionary aspect of calcium signaling event in calmodulin and calmodulin-like proteins in plants. BMC Plant Biol. 2017; 17(1): 38. doi:10.1186/s12870-017-0989-3
- 30Popescu SC, Popescu GV, Bachan S, et al. Differential binding of calmodulin-related proteins to their targets revealed through high-density Arabidopsis protein microarrays. Proc Natl Acad Sci U S A. 2007; 104(11): 4730-4735. doi:10.1073/pnas.0611615104
- 31Pezzella-D'Alessandro N, Le Moal H, Bonhomme A, et al. Calmodulin distribution and the actomyosin cytoskeleton in Toxoplasma gondii. J Histochem Cytochem. 2001; 49(4): 445-454. doi:10.1177/002215540104900404
- 32Boulter-Bitzer JI, Lee H, Trevors JT. Molecular targets for detection and immunotherapy in Cryptosporidium parvum. Biotechnol Adv. 2007; 25(1): 13-44. doi:10.1016/j.biotechadv.2006.08.003
- 33Elliot BC, Wisnewski AV, Johnson J, et al. In vitro inhibition of Cryptosporidium parvum infection by human monoclonal antibodies. Infect Immun. 1997; 65(9): 3933-3935. doi:10.1128/iai.65.9.3933-3935.1997
- 34Petry F, Jakobi V, Tessema TS. Host immune response to Cryptosporidium parvum infection. Exp Parasitol. 2010; 126(3): 304-309. doi:10.1016/j.exppara.2010.05.022
- 35Farthing MJG. Clinical aspects of human cryptosporidiosis. In: F Petry, ed. Cryptosporidiosis and Microsporidiosis. Contributions to Microbiology. Vol 6. Karger; 2000: 50-74.
10.1159/000060368 Google Scholar
- 36Kváč M, Kodádková A, Sak B, et al. Activated CD8+ T cells contribute to clearance of gastric Cryptosporidium muris infections. Parasite Immunol. 2011; 33(4): 210-216. doi:10.1111/j.1365-3024.2010.01271.x PMID: 21204850.
- 37Tessema TS, Schwamb B, Lochner M, Förster I, Jakobi V, Petry F. Dynamics of gut mucosal and systemic Th1/Th2 cytokine responses in interferon-gamma and interleukin-12p40 knock out mice during primary and challenge Cryptosporidium parvum infection. Immunobiology. 2009; 214(6): 454-466. doi:10.1016/j.imbio.2008.11.015
- 38Wumba R, Longo-Mbenza B, Menotti J, et al. Epidemiology, clinical, immune, and molecular profiles of microsporidiosis and cryptosporidiosis among HIV/AIDS patients. Int J Gen Med. 2012; 5: 603-611. doi:10.2147/IJGM.S32344
- 39Wiwanitkit V. Intestinal parasitic infections in Thai HIV-infected patients with different immunity status. BMC Gastroenterol. 2001; 1: 3. doi:10.1186/1471-230x-1-3
- 40McDonald V, Korbel DS, Barakat FM, Choudhry N, Petry F. Innate immune responses against Cryptosporidium parvum infection. Parasite Immunol. 2013; 35(2): 55-64. doi:10.1111/pim.12020
- 41Bonafonte MT, Smith LM, Mead JR. A 23-kDa recombinant antigen of Cryptosporidium parvum induces a cellular immune response on in vitro stimulated spleen and mesenteric lymph node cells from infected mice. Exp Parasitol. 2000; 96(1): 32-41. doi:10.1006/expr.2000.4545
- 42Perez-Cordon G, Yang G, Zhou B, et al. Interaction of Cryptosporidium parvum with mouse dendritic cells leads to their activation and parasite transportation to mesenteric lymph nodes. Pathog Dis. 2014; 70(1): 17-27. doi:10.1111/2049-632X.12078
- 43Lacroix S, Mancassola R, Naciri M, Laurent F. Cryptosporidium parvum-specific mucosal immune response in C57BL/6 neonatal and gamma interferon-deficient mice: role of tumor necrosis factor alpha in protection. Infect Immun. 2001; 69(3): 1635-1642. doi:10.1128/IAI.69.3.1635-1642.2001
- 44Lean IS, Lacroix-Lamande S, Laurent F, McDonald V. Role of tumor necrosis factor alpha in development of immunity against Cryptosporidium parvum infection. Infect Immun. 2006; 74(7): 4379-4382. doi:10.1128/IAI.00195-06
- 45Choudhry N, Korbel DS, Edwards LA, Bajaj-Elliott M, McDonald V. Dysregulation of interferon-gamma-mediated signalling pathway in intestinal epithelial cells by Cryptosporidium parvum infection. Cell Microbiol. 2009; 11(9): 1354-1364. doi:10.1111/j.1462-5822.2009.01336.x
- 46Takatsu K, Nakajima H. IL-5 and eosinophilia. Curr Opin Immunol. 2008; 20(3): 288-294. doi:10.1016/j.coi.2008.04.001
- 47Stark JM, Tibbitt CA, Coquet JM. The metabolic requirements of Th2 cell differentiation. Front Immunol. 2019; 10: 2318. doi:10.3389/fimmu.2019.02318
- 48Kaushik K, Khurana S, Wanchu A, Malla N. Lymphoproliferative and cytokine responses to Cryptosporidium parvum in patients coinfected with C. parvum and human immunodeficiency virus. Clin Vaccine Immunol. 2009; 16(1): 116-121. doi:10.1128/CVI.00395-07
- 49Campbell LD, Stewart JN, Mead JR. Susceptibility to Cryptosporidium parvum infections in cytokine- and chemokine-receptor knockout mice. J Parasitol. 2002; 88(5): 1014-1016. doi:10.1645/0022-3395(2002)088[1014:STCPII]2.0.CO;2
- 50Asquith KL, Ramshaw HS, Hansbro PM, Beagley KW, Lopez AF, Foster PS. The IL-3/IL-5/GM-CSF common receptor plays a pivotal role in the regulation of Th2 immunity and allergic airway inflammation. J Immunol. 2008; 180(2): 1199-1206. doi:10.4049/jimmunol.180.2.1199
- 51Ayelign B, Akalu Y, Teferi B, Molla MD, Shibabaw T. Helminth induced immunoregulation and novel therapeutic avenue of allergy. J Asthma Allergy. 2020; 13: 439-451. doi:10.2147/JAA.S273556
- 52Sateriale A, Slapeta J, Baptista R, et al. A genetically tractable, natural mouse model of cryptosporidiosis offers insights into host protective immunity. Cell Host Microbe. 2019; 26(1): 135-146 e135. doi:10.1016/j.chom.2019.05.006
- 53Qin PL, Li Y, Mi RS, et al. Tandem expression of major epitope domain of Cryptosporidium parvum three antigen genes and its antigenicity analysis. Chin J Anim Infect Dis. 2012; 20(3): 36-43.
- 54Liu K, Zai D, Zhang D, et al. Divalent Cp15-23 vaccine enhances immune responses and protection against Cryptosporidium parvum infection. Parasite Immunol. 2010; 32(5): 335-344. doi:10.1111/j.1365-3024.2009.01191.x
