Introduction: Cryptosporidium parvum is a protozoan parasite that causes diarrhoea in many host species worldwide. CpCDPK1 appears to be essential for invasion and a promising drug target.
Aim of the study: The aims of this study were to expand the knowledge of CpCDPK1. To achieve that, attempts were made to inhibit this gene by BKI-1294 in vitro and generate CpCDPK1 KO C. parvum. To maintain the genetically modified parasites, I studied the suitability of infection and propagation in a new animal model RAGgc × IFN-gamma mouse and in vitro model COLO - 680 N cell line.
Animals, materials and methods: 4×106 freshly excysted C. parvum sporozoites were seeded into transfected GFP-MDBK cultures at the confluency of 70 – 80 % and simultaneously exposed to 500 nM of BKI-1294. IFA was applied to observe the invasion and host cell actin accumulation. Guide RNA (gRNA) for CRISPR-mediated transfection was designed and the Nluc-neoR repair cassette was flanked with 50 bp long 5’- and 3’UTR of CpCDPK1 by PCR. Transfection was performed by octaarginine transportation and compared to electroporation. COLO - 680 N cells with the confluency of 70 – 80% were infected with 4×106 non-transfected and transfected sporozoites of C. parvum. To establish a laboratory animal model for propagation of C. parvum and drug screening RAGgc × IFN-gamma mice were infected with 500 (G2), 1000 (G3) and 5000 (G4) of oocysts. BALB/c WT mice were inoculated with 5000 (G1) oocysts as control. Faeces were sampled for C. parvum DNA extraction. Real time PCR was applied to calculate the oocyst yield.
Results: In the presence of 500 nM BKI-1294, parasite-induced host cell actin accumulation was not observed at 24 and 48 h after inoculation in vitro pointing at altered infectivity of CDPK inhibited sporozoites. Extracellular noninvasive sporozoites were found at 24 h p.i., only one meront was observed in a host cell at 72 h p.i. CRISPR-mediated gene editing was applied to C. parvum to knock out CDPK1. Transfected C. parvum were found in COLO-680 N cells through 6 passages. However, no newly generated oocysts were harvested. RAGgc × IFN-gamma mice were tested suitable as an animal model for C. parvum infection studies and oocyst propagation. These crossbred mice are very sensitive to infection at doses as low as 500 oocysts. They displayed emaciation, rough fur and trembling. The survival percentage was 71.4 % (G2), 85.7 % (G3), 57.1 % (G4) and 100 % (G1) at the end of study. Oocyst yield of 108 OPG was calculated in the crossbred mice whereas only 104 OPG were counted in Balb/C mice. Yields did not differ significantly (P > 0.05) in crossbred mice infected with different oocysts doses.
Conclusions: 1.The function of CpCDPK1 is obviously important to the invasion process including attachment and utilization of host cell actin to form PV. This assumption was confirmed by CDPK inhibition and genetic KO. However, methods that increase the transfection efficiency are needed to enhance the generation of KO C. parvum. 2. The transfection method mediated by octargninine is superior to electroporation in consideration of DNA consumption and requirement of device. 3. Due to the low required infection dose and clinical manifestation RAGgc × IFN-gamma mice appear very well suited to serve as an in vivo laboratory model of C. parvum infection and for propagation of particularly transgenic C. parvum strains. 4. COLO – 680 N cells appear suited to be an in vitro model for C. parvum infection and transfection study, however, not qualified for propagation.:Contents
1. Introduction 1
2. Literature Review 2
2.1 Biology 2
2.1.1 Systematics 2
2.1.2 Life cycle 2
2.1.3 Tenacity of oocysts 4
2.1.4 Excystation of oocysts and invasion of host cells 4
2.1.5 Formation of the PV 6
2.1.6 Nutrient supply by the host 7
2.2 Epidemiology 8
2.2.1 Human Cryptosporidiosis 8
2.2.2 Animal Cryptosporidiosis 9
2.3 Detection and Diagnosis 11
2.4 Treatment options 12
2.5 Hygiene 14
2.6 Vaccine 16
2.7 In vitro and vivo Models 16
2.8 Structure and function of Calcium-dependent protein kinases 18
3. Animals, materials and methods 21
3.1 Animals and materials 21
3.1.3 Mice 21
3.1.4 Cells 21
3.1.5 C. parvum oocysts 21
3.1.6 Reagents 21
3.1.7 Plasmids and oligonucleotides 23
3.1.7.1 Plasmids 23
3.1.7.2 Primers and probes 24
3.1.8 Kits 25
3.1.9 Instruments and software 25
3.2 Methods 26
3.2.1 Preparation of reagents 26
3.2.2 C. parvum oocysts maintaince 27
3.2.3 PCR 27
3.2.3.1 Amplification of NdeI and AatII flanked 5’CDPK1 27
3.2.3.2 Annealing of gRNA 27
3.2.3.3 Amplification of repair cassette via Touchdown PCR (TD-PCR) 28
3.2.3.4 Colony PCR 29
3.2.3.5 Real-time PCR for C. parvum hsp70 30
3.2.4 Restriction enzyme digestion 31
3.2.4.1 Restriction enzyme digestion of pA - pD 31
3.2.4.2 Enzyme digestion and dephosphorylation of p185 31
3.2.5 Agarose gel electrophoresis 32
3.2.6 Gel purification 32
3.2.7 Ligation 33
3.2.7.1 Ligation of CDPK1 KO plasmids 33
3.2.7.2 Ligation of gRNA and p185 33
3.2.8 Transformation 34
3.2.9 Plasmid extraction 34
3.2.10 C. parvum oocysts excystation 35
3.2.11 C. parvum infection 35
3.2.11.1 In vitro infection 35
3.2.11.2 C. parvum infection in mice 36
3.2.12 Transfection 36
3.2.12.1 Electroporation for MDBK transfection 36
3.2.12.2 Electroporation for C. parvum transfection 37
3.2.12.3 CpCDPK1 knock out through Cell penetrating peptide (CPP) - octaarginine mediated transfection 38
3.2.13 Geneticin screening for GFP-MDBK cells 39
3.2.14 Indirect immunofluorescent assay (IFA) 39
3.2.15 Animal feeding and body conditioning score (BCS) monitoring 40
3.2.16 Faecal sample collection 43
3.2.17 DNA extraction and oocysts per gram (OPG) determination of fecal samples 43
3.2.18 Statistical analysis 44
4. Results 45
4.1 CDPK1 knockout by REMI 45
4.1.1 Construction of Knockout plasmid 45
4.1.2 Electroporation protocol and in vitro analysis 49
4.2 CDPK1 knockout by CRISPR/Cas 9-mediated gene editing 50
4.2.1 Constructing CRISPR/Cas9_CpCDPK1_7 plasmid 51
4.2.2 Amplification of CDPK1 flanked repair cassette 52
4.2.3 Knockout CDPK1 via CRISPR/cas 9 53
4.2.3.1 Electroporation and in vitro analysis 53
4.2.3.2 CPP transfection and in vitro analysis 55
4.2.3.3 Genetic assay of transfection 57
4.3 In vitro and in vivo model for infection and propagation 58
4.3.1 In vitro model - C. parvum cultivation in COLO - 680 N cells 58
4.3.2 In vivo model Infection pattern of C. parvum in RAGgc x IFN-g KO mice 60
4.3.2.1 Clinical symptoms 60
4.3.2.2 Oocysts excretion 63
4.4 In vitro inhibition of CDPK1 66
4.4.1 Generating bAct-GFP-MDBK cells 67
4.4.2 Influence of CDPK1 inhibition on infection 70
5. Discussion 73
5.1 Sub-cloning 73
5.2 Inhibition of CpCDPK1 delays the host cell actin accumulation in vitro 73
5.3 RAGgc x IFN-gamma KO mice for C. parvum propagation 76
5.4 CpCDPK1 knockout by CRISPR/cas 9 79
5.5 COLO-680 N cells are not suited to propagate C. parvum in vitro 82
6. Summary 85
7. Zusammenfassung 87
8. References 89
| Identifer | oai:union.ndltd.org:DRESDEN/oai:qucosa:de:qucosa:38747 |
| Date | 16 March 2020 |
| Creators | Zheng, Wanpeng |
| Contributors | Universität Leipzig |
| Source Sets | Hochschulschriftenserver (HSSS) der SLUB Dresden |
| Language | English |
| Detected Language | English |
| Type | info:eu-repo/semantics/updatedVersion, doc-type:doctoralThesis, info:eu-repo/semantics/doctoralThesis, doc-type:Text |
| Rights | info:eu-repo/semantics/openAccess |
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