Cryptosporidium parvum infects enterocytes in diverse vertebrates, including humans, and causes diarrheal illness. However, no effective drugs are available for this protozoan infection. The P23 protein of C. parvum is a protective antigen, considered a potential candidate for developing an effective vaccine against cryptosporidiosis. In this study, the complementary DNA (cDNA) of the p23 gene was subcloned to Escherichia coli DH5α, with one nucleotide difference. The constructed plasmid pNZ8149-P23 was transferred by electroporation to Lactococcus lactis NZ3900, and the recombinant L. lactis NZ3900/pNZ8149-P23 strain was screened in Elliker-medium by adding bromocresolpurple indicator. A 23-kDa protein was detected by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) after nisin induction in LM17 broth medium, suggesting that P23 protein was in the form of glycosylation. Simultaneously, an optimal induction time of 9 h was determined, and the density of OD600 = 2.7 was tested. Through western blot and indirect immunofluorescence (IIF) analysis, the immunocompetence of expressed P23 antigen was identified, and its location of release to the cell interior of recombinant L. lactis was manifested. The first report of a food-grade genetically engineered L. lactis strain expressing a P23 antigen of C. parvum is herein presented. This result provides a novel and safe utilization method of P23 against C. parvum infection.

College of Animal Science and Veterinary Medicine Henan Institute of Science and Technology Xinxiang 453003 China

College of Veterinary Medicine Sichuan Agricultural University Chengdu 611130 China

See more in PubMed

Askari N et al (2016) Evaluation of recombinant P23 protein as a vaccine for passive immunization of newborn calves against Cryptosporidium parvum. Parasite Immunol 38:282–289. https://doi.org/10.1111/pim.12317 PubMed DOI

Bonafonte MT, Smith LM, Mead JR (2000) 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 96:32–41. https://doi.org/10.1006/expr.2000.4545 PubMed DOI

Certad G, Viscogliosi E, Chabe M, Caccio SM (2017) Pathogenic mechanisms of Cryptosporidium and Giardia. Trends Parasitol 33:561–576. https://doi.org/10.1016/j.pt.2017.02.006 PubMed DOI

Checkley W et al (2015) A review of the global burden, novel diagnostics, therapeutics, and vaccine targets for Cryptosporidium. Lancet Infect Dis 15:85–94. https://doi.org/10.1016/S1473-3099(14)70772-8 PubMed DOI

Chen S, Zhang R, Duan G, Shi J (2011) Food-grade expression of Helicobacter pylori ureB subunit in Lactococcus lactis and its immunoreactivity. Cur Microbiol 62:1726–1731. https://doi.org/10.1007/s00284-011-9920-6 DOI

Ebrahimzadeh E, Shayan P, Dezfouli MM, Rahbari S (2009) Recombinant Cryptosporidium parvum P23 as a candidate vaccine for cryptosporidiosis. Iran J Parasitol 4:1–7. https://doi.org/10.4269/ajtmh.2012.11-0273 DOI

Ehigiator HN, Romagnoli P, Priest JW, Secor WE, Mead JR (2007) Induction of murine immune responses by DNA encoding a 23-kDa antigen of Cryptosporidium parvum. Parasitol Res 101:943–950. https://doi.org/10.1007/s00436-007-0565-0 PubMed DOI

Geriletu XuR, Jia H, Terkawi MA, Xuan X, Zhang H (2011) Immunogenicity of orally administrated recombinant Lactobacillus casei Zhang expressing Cryptosporidium parvum surface adhesion protein P23 in mice. Cur Microbiol 62:1573–1580. https://doi.org/10.1007/s00284-011-9894-4 DOI

Jakobi V, Petry F (2006) Differential expression of Cryptosporidium parvum genes encoding sporozoite surface antigens in infected HCT-8 host cells. Microbes Infect 8:2186–2194. https://doi.org/10.1016/j.micinf.2006.04.012 PubMed DOI

Lan DTB, Lan TT, Viet LQ, Quyet PV, Quang HT, Loc NH, Long PT (2014) Cloning and expression of gene encoding p23 protein from Cryptosporidium parvum. J BioSci Biotech 3:189–193. http://www.jbb.uni-plovdiv.bg

Leblanc JG et al (2013) Mucosal targeting of therapeutic molecules using genetically modified lactic acid bacteria: an update. Fems Microbiol Lett 344:1–9. https://doi.org/10.1111/1574-6968.12159 PubMed DOI

Nader JL et al (2019) Evolutionary genomics of anthroponosis in Cryptosporidium. Nat Microbiol 4:826–836. https://doi.org/10.1038/s41564-019-0377-x PubMed DOI

Omidian Z, Ebrahimzadeh E, Shahbazi P, Asghari Z, Shayan P (2014) Application of recombinant Cryptosporidium parvum P23 for isolation and prevention. Parasitol Res 113:229–237. https://doi.org/10.1007/s00436-013-3648-0 PubMed DOI

Peng X et al (2018) Production and Delivery of Helicobacter Pylori NapA in Lactococcus Lactis and Its Protective Efficacy and Immune Modulatory Activity Sci Rep 8:6435. https://doi.org/10.1038/s41598-018-24879-x PubMed DOI

Roellig DM, Xiao L (2020) Cryptosporidium genotyping for epidemiology tracking. Methods Mol Biol 2052:103–116. https://doi.org/10.1007/978-1-4939-9748-0_7 PubMed DOI

Ryan U, Zahedi A, Paparini A (2016) Cryptosporidium in humans and animals-a one health approach to prophylaxis. Parasite Immunol 38:535–547. https://doi.org/10.1111/pim.12350 PubMed DOI

Santin M (2020) Cryptosporidium and Giardia in ruminants. Vet Cli N Am-Food A 36:223–238. https://doi.org/10.1016/j.cvfa.2019.11.005 DOI

Shayan P, Ebrahimzadeh E, Mokhber-Dezfouli MR, Rahbari S (2008) Recombinant Cryptosporidium parvum p23 as a target for the detection of Cryptosporidium-specific antibody in calf sera. Parasitol Res 103:1207–1211. https://doi.org/10.1007/s00436-008-1117-y PubMed DOI

Sun N et al (2017) An engineered food-grade Lactococcus lactis strain for production and delivery of heat-labile enterotoxin B subunit to mucosal sites. BMC Biotechnol 17:25. https://doi.org/10.1186/s12896-017-0345-6 PubMed DOI PMC

Sun N, Zhang RG, Duan GC, Peng XY, Wang C, Chen SY, Fan QT (2019) A food-grade engineered Lactococcus lactis strain delivering Helicobacter pylori Lpp20 alleviates bacterial infection in H. pylori-challenged mice. Biotechnol Lett 41:1415–1421. https://doi.org/10.1007/s10529-019-02740-z PubMed DOI

Thompson RC, Ash A (2019) Molecular epidemiology of Giardia and Cryptosporidium infections What’s new? Infect Genet Evol : MEEGID 75:103951. https://doi.org/10.1016/j.meegid.2019.103951 PubMed DOI

Ungar BLP (2018) Cryptosporidiosis in humans (Homo Sapiens). In: Fayer R (ed) Cryptosporidiosis of man and animals. CRC Press, Florida, pp 59–82 DOI

Wang L et al (2020) Oral vaccination with the porcine circovirus type 2 (PCV-2) capsid protein expressed by Lactococcus lactis induces a specific immune response against PCV-2 in mice. J Appl Microbiol 128:74–87. https://doi.org/10.1111/jam.14473 PubMed DOI

Wang R, Zhao G, Gong Y, Zhang L (2017) Advances and perspectives on the epidemiology of bovine Cryptosporidium in China in the Past 30 years. Front Microbiol 8 Artn 1823 https://doi.org/10.3389/Fmicb.2017.01823

Wang S, Liu H, Zhang X, Qian F (2015) Intranasal and oral vaccination with protein-based antigens: advantages, challenges and formulation strategies. Protein Cell 6:480–503. https://doi.org/10.1007/s13238-015-0164-2 PubMed DOI PMC

Wang Z, Cao X, Du X, Feng H, Di W, He S, Zeng X (2014) Mucosal and systemic immunity in mice after intranasal immunization with recombinant Lactococcus lactis expressing ORF6 of PRRSV. Cell Immunol 287:69–73. https://doi.org/10.1016/j.cellimm.2013.12.004 PubMed DOI

Xiao L, Cama VA (2018) Cryptosporidium and cryptosporidiosis. In: Ortega YR, Sterling CR (eds) Foodborne parasites. Springer International Publishing, Cham, pp 73–117. doi: https://doi.org/10.1007/978-3-319-67664-7_5

Zhang Q, Zhong J, Huan L (2011) Expression of hepatitis B virus surface antigen determinants in Lactococcus lactis for oral vaccination. Microbiol Res 166:111–120. https://doi.org/10.1016/j.micres.2010.02.002 PubMed DOI

Zhang XJ, Duan G, Zhang R, Fan Q (2009) Optimized expression of Helicobacter pylori ureB gene in the Lactococcus lactis nisin-controlled gene expression (NICE) system and experimental study of its immunoreactivity. Cur Microbiol 58:308–314. https://doi.org/10.1007/s00284-008-9349-8 DOI