Molecular Basis of Arrested Liver Stage Development of the Gamma-irradiated Plasmodium yoelii exo-erythrocytic form.

by

George Asanga Ndeta

INTRODUCTIONMalaria is one of the deadliest of human parasitic diseases that results in the dead of about 3 million individuals annually in the tropical and sub-tropical parts of the world.

Efforts to check the spread of malaria has recently been complicated by the problem of rise in resistance to chloroquine by the malaria parasite as well as rise in resistance to insecticides by the mosquito vector.

A possible solution to these problems of resistance is to explore alternate forms of therapies; the gamma-irradiated sporozoites as a vaccine candidate need further studies to understand the molecular events that results in its developmental arrest in the liver so as to improve on its efficacy.

-Gamma-irradiated and non-irradiated Plasmodium yoelii (17XNL) sporozoites were isolated from Anopheles stephensi and purifiedon a renografin-60 gradient.-Sporozoites were axenically cultured at 37oC for 24 hours.-Total RNA was isolated with chloroform/isopropanol.-cDNA (1) library representing the gamma-irradiated EEF andcDNA (2) library representing the non-irradiated EEF were synthesized using the PCR-Select cDNA Subtraction Kit.-cDNA were digested with Rsa I restriction enzyme followedby adaptor ligation. -The two tester cDNA libraries were subjected to two roundsof hybridization followed by1ery and 2ndry PCR amplification.-Cloning

Experimental design For SSH

Synthesis Of First cDNA Strand

� Poly A+ RNA (2µg) 2-3 µL

� cDNA Synthesis Primer (10µM) 1.0 µL � (mix content & spin)

� Then incubate at 70oC for 2 min in a thermal cycler

� Cool on ice for 2 min and briefly centrifuge

� Add the following reagents to each reaction:

� 5X First-Strand Buffer 2.0 µL

� dNTP Mix (10mM each) 1.0 µL

� Sterile H20 1.0 µL

� AMV Reverse Transcriptase 1.0 µL

� 420C x 1.5Hrs

� Stop Rxn on ice

2nd Strand cDNA Synthesis

� Sterile H20 48.4 µL

� 5x Second-Strand buffer 16.0 µL

� dNTP Mix (10 mM) 1.6 µL

� 20x Second-Strand Enzyme Cocktail 4.0 µL

� 16oC x 2.0Hrs

� T4 DNA Polymerase 2.0 µL

� 16oC x 30min

� Stop Rxn – 4.0 µL of 20x EDTA/Glycogen

Recover Synthesize cDNA

� 100 µL of Phenol:Chloroform:Isoamyl Alc

� 100 µL of Chloroform:Isoamyl Alcohol 2X

� 40 µL NH4OAc & 300 µl of 95% ETOH

� 500 µL of 80% ETOH

� Air Dry cDNA x 10 min.

� Dilute with 50.0 µL of H2O

Rsa I Digestion

� ds cDNA 43.5 µL

� 10 x Rsa I Restriction Buffer 5.0 µL

� Rsa I Restriction Enzyme 1.5 µL

� Mix & Incubate @ 37oC x 1.5 Hrs

� Stop Rxn ĉ 2.5 µL of 20x EDTA/Glycogen

Recover Blunt End cDNA

� 50 µL Phenol:Chloroform:Isoamyl alcohol

� 50 µL Chloroform:Isoamyl alcohol 2x

� 25 µL NH4OAc & 187.5 ul Etoh

� 200 µL 80% Etoh

� Air dry x 10 min

� Dissolve ĉ 5.5 µL of H2O

Ligation

� Make 1:5 dilution of blunt end cDNA

� Ligation mix:

� Sterile H20 3.0 µL

� 5x Ligation Buffer 2.0 µL

� T4 DNA Ligase 1.0 µL

Adaptor ligation

� Dilute cDNA………

� Adaptor 1………….

� Adaptor 2………….

� Master mix………..

� Final Volume……..

� 16oC overnight

� Stop Rxn ĉ 1ul of EDTA/Glycogen

� Heat at 72oC to inactivate Ligase

2.0uL

2.0uL

6.0uL 6.0uL

2.0uL

2.0uL

10.0uL10.0uL

1-1 2-2

+cDNA2NI

+cDNA1I

2-11-2

+cDNA2NI

+cDNA1I

Experimental (tester) cDNA-1Irradiated cDNA

Experimental (tester) cDNA-2Non-Irradiated-cDNA

Mix withAdaptor 1

Mix withAdaptor 2 Mix with

Adaptor 1Mix withAdaptor 2

First HybridizationDriver with no Adaptors

Second HybridizationDriver with no Adaptors

+cDNA2NI

+cDNA1I

1o and 2o (PCR) 1o and 2o (PCR)

POOLPOOL

-Amplified cDNA was cloned to TOPO vector (plasmid).-Cloned products were used to transform TOP 10 OneShot chemically competent cells.

-Transformed bacteria were incubated at 37oC for 24 hrson plates with x-gal and ampicillin.

Template plasmid, spin column purified with a SV miniprep kit; Sequencing; Dye terminator Method.

Insert check was done on positive clones to ascertain the success of cloning.

Insert check of cloned cDNA from gamma-irradiated exo-erythrocytic form (EEF)

mak

er

500 bp-

Irradiated clones Contig Protein Identified

I 1-4 Malpy000471 Hypothetical proteinDI 2-2 Malpy01825 Hypothetical proteinDI 2-11 Malpy00500 Putative yir1 proteinDI 2-5 Malpy00137 Putative yir4 proteinDI 1-12 Malpy00664 Tubulin-tyrosine ligase familyDI 2-2 Malpy01557 Putative yir4 proteinI 1-6 Malpy01953 Hypothetical proteinDI 1-15 Malpy01115 Uncharacterized protein familyDI 2-15 Malpy00137 Putative yir3 proteinDI 2-9 Malpy00296 hypothetical proteinDI 2-13 Malpy00664 CCAAT-box DNA binding protein subunit

Non-irradiated clones Contig Identified Protein

NI 1-5 Malpy02019 CCAAT-box DNA binding proteinNI 1-11 Malpy01430 235 kDa rhoptry proteinNI 1-12 Malpy00905 Putative bir1NI 2-14 Malpy02672 Ribosomal protein S4NI 2-10 Malpy00013 Uncharacterized protein familyNI 2-12 Malpy00271 Erythrocyte membrane proteinNI 2-2 Malpy00014 Putative uncharacterized protein familyNI 2-19 Malpy00954 Putative yir4 proteinNI 1-1 Malpy02241 Hypothetical proteinDNI 2-3 Malpy01423 Heat shock protein 60DNI 2-7 Malpy00271 Uncharacterized protein familyNI 1-5 Malpy02019 Hypothetical protein

Screening with un-subtracted cDNA probes

Un-subtracted tester probe Un-subtracted driver probe

Screening with subtracted cDNA probes

Forward subtracted probe Reverse subtracted probe

++ +++

+

++

++++

- --- --

--

- ---

-

--

-

+

SD IN

SDIN

--

1

141 1

1

14

14 1415

15

15

15 26

2626

26

27

27

27

27

40

40

40

40

41

41

41

41

5252

52 52

5353

535366

6666

66

67

67

67

67

78

78

78

78

79

7979

79 92

92

92

92

93

93

93

93

104

104

104

104

Gamma-irradiated P.yoelii EEF subtracted cDNA library dot blots

Dot blots screened with un-subtracted cDNA probes

Dot blots screened with subtracted cDNA probes

Un-subtracted tester probe Un-subtracted driver probe

Forward subtracted probe Reverse subtracted probe

Non-irradiated P. yoelii EEF subtracted cDNA library dot blots

1

1 1

1

14 14

14 14

15 15

15 15

26 26

2626

27 27

2727

40 40

4040

41 41

4141

52 52

52 52

53 53

5353

66 66

66 66

67 67

67 67

78 78

7878

79 79

79 79

92 92

9292

9393

9393

104 104

104104

Erythrocyte membrane protein

Control

p235kDa rhoptry protein

Tubulin tyrosine ligase

yir

NI I

Liver Stage Parasites Blood Stage Parasites

Hep-17

500bp

200bp

Marker 1 2 3 4 5 6

7 LC

28SrRNA

MSP-1

Marker

500bp

CONCLUSION

-SSH technique enabled us to generate two cDNA libraries from the IEEF and the NIEEF.

-Semi-quantitative RT-PCR analysis of selected clones showed the effect of radiation on sporozoites DNA when exposed to 12,000 rads of radiation from a 137Cesium source to be up regulation, down regulation or abrogation of some liver stage genes.

-The expression of the EMP gene in the NIEEF confirms a progressive transformation that will transition the sporozoite into the erythrocytic stage while abrogation of the EMP gene in the IEEF confirms the development arrest of sporozoites at the trophozoite stages.

Acknowledgment

Dr. KellyDr. Sedegha

SSH Primers

The following oligonucleotides are used at a concentration of 10 µM. Whenever possible, oligonucleotides should be HPLC- or gel-purified. 1. cDNA synthesis primers: 5′-TTTTGTACAAGCT(T)30-3′ 2. Adapters: (Ad1) –5′-CTAATACGACTCACTATAGGGCTCGAGCGGCCGCCCGGG CAGGT-3′ 3′-GGCCCGTCCA-5′ (Ad2) – 5′-TGTAGCGTGAAGACGACAGAAAGGGCGTGGTGCGGAGG GCGGT-3′ 3′-GCCTCCCGCCA-5′ (Ad2R) – 5′-CTAATACGACTCACTATAGGGCAGCGTGGTCGCGGCCGA GGT-3′ 3′-GCCGGCTCCA-5′ 3. PCR primers: (P1) – 5′-CTAATACGACTCACTATAGGGC-3′ (P2) – 5′-TGTAGCGTGAAGACGACAGAA-3′ Nested primer 1 (NP1) – 5′-TCGAGCGGCCGCCCGGGCAGGT-3′ Nested primer 2 (NP2) – 5′-AGGGCGTGGTGCGGAGGGCGGT-3′ Nested primer 2R (NP2R) – 5′-AGCGTGGTCGCGGCCGAGG