Cloning of Malaria Genes using Perkisus marinus

Supported by HSF Grant R21

Plasmodium falciparum

• Protozoan parasite – etiological agent responsible for Malaria in humans

• Transmitted by female Anopheles mosquito• Most dangerous because of high complication and

mortality rates– Claims the lives of more children worldwide than

any other infectious disease– One million people die each year from malaria

Life cycle of apicomplexan parasites and structure of Apical complex

a host (the anopheles mosquito) is infected via an active invasion by the parasites which divide to produce sporozoites . Sporozoites enter a human’s bloodstream and quickly invade liver cells. The liver-stage parasites differentiate and undergo asexual replication resulting in tens of thousands of merozoites. Eventually, the cells burst, releasing merozoites which infect erythrocytes.

Apical complex structure: 1-polar ring, 2-conoid, 3-micronemes, 4-rhoptries, 5-nucleus, 6-nucleolus, 7-mitochondria, 8-posterior ring, 9-alveoli, 10-golgi apparatus, 11-micropore.

Apicoplast: single plastid surrounded by3 0r 4 membranes (lipid and heme biosynthesis), necessary for survival

Use of Homologous / Heterologous Systems for Vaccine Development

Prokaryotic systems• E. Coli expression system most commonly used for production

of recombinant proteins for pharmaceutical applicationsAdvantages: grow rapidly to high densities, inexpensive, easy to

transform, can produce high quantities of soluble recombination product

Disadvantages: absence of post-transcriptional modifications, amino acid substitutions or modifications, heterogeneous products, accumulation of recombinant proteins in inclusion bodies

• Overall success rate for obtaining soluble immunogenic recombinant products remains low– From 303 plasmodium genes, only 7% of the recombinant proteins

produced antibody titers– Of 1000 open reading frames resolved from plasmodium <7% were

expressed as soluble proteins– Plasmodium has an AT rich genome

Eukaryotic Systems• Main advantages – closer phylogenic relationship with parasites

– post translational modifications such as glycosylation, acylation, ability to form disulfide linkages, proteolytic processing, subcellular compartmentalization and secretion mechanisms to avoid accumulation and lower toxicity

• Yeast - Pichia pastoris and Sacchromyces Cerevisiae– Advantages: suitable for large scale fermentors, high protein yield– Disadvantages: N- and O- linked glycosylation patterns quantitatively and qualitatively

different from native parasite protein

• Mammalian cell lines – labor intensive and expensive, used for identification and characterization of plasmodium surface ligands, binding assays, and testing of vaccines

• Baculovirus-mediated expression systems- insect cells and larvea– Advantages:mg quantities of recombinant protein with some post-transcriptional

modification, can be upgraded to bioreactor scale– Disadvantages: time consuming generation of recombinant baculovirus, inablility to

synthesize complex glycans, lethality to host cells

Cell Free Expression systems

• Wheat germ cell • Advantages: no prior optimization for P. falciparum AT biased codon usage• Suitable for proteins from genes with codon bias with no need for post-

transcriptional modifications- Used to produce P. flaciparum DHFR-TS which had been resistant to

multiple attempts of expression in other expression systems- Analysis of 124 P. falciparum genes resulted in the production of 93

recombinant proteins

Homologous and Heterologous parasite-based expression systems

• Major advantage – faithfully display all desirable characteristics such as immunogenicity and biological activity, over expression or scaled-up culture size.

• Protozoan parasites as surrogate systems for expression of proteins of other parasites has been used successfully– Neospora caninum can express genes of Toxoplasmosis gondii– Toxoplasma-ts-4 mutants can express Leishmania antigen and elicit an immunee

response in BALC/c mice

• ‘Next door neighbor’ – optimal expression system – Phylogeny and systematic powerful tools to select species which may

exhibit the highest potential to produce recombinant proteins

Perkisus marinus• Protozoan parasite of the eastern oyster Crassostrea virginica .• Responsible for much of the oyster mortality of natural and farmed oyster

populations along the Atlantic and Gulf coasts of the United States since the 1950’s.

• The disease it causes is called Dermo and is characterized by proteolytic degradation of oyster tissues.

• P. marinus is a protozoan parasite within the dinoflagellate line, still close to the divergence point from the apicomplexans (P. falciparum) and shares multiple morphological features with Apicomplexa

• Advantages: easy to grow in culture, high cell densities ( 108 cells per ml),Non-pathnogenic to humans and inexpensive (1 L ~$31)

pPmMoe• Vector based on a highly expressed P. marinus gene tagged

with GFP • Optimized transfection conditions to introduce exogenous

DNA using electroporation• Constructed by PCR amplification and cloning into pCR4-Topo,

and introduction of PCR amplified GFP into later part of PmMoe gene.

• Named gene PmMoe from the latin moeniaum meaning walls or fortification of the city. pmMoe is targeted to the prominent cell walll of Perkinsus trophozoites.

pPmMoe-GFP

Additional modification:Introduction of MCS andHis-tag pPmMoeGFP-99

Genes cloned into pPmMoe-GFP-99 to date

Plasmodium Genes• Sera 4S and 5S: serine-repeat antigens - function unknown thought to

interact with the rhopotry proteins and facilitate invasion into host cells. Antibodies against recombinant Sera proteins have provided immunity to Aeotus monkeys(*)

• Ama1: apical membrane antigen 1 - essential membrane protein stored in the micronemes of the apicomlexa complex and transported to the parasite surface prior to and during host cell invasion (**).

HIV gene• gp120 : glycoprotein exposed on the surface of the HIV envelope. gp120 is

essential for virus entry into cells as it plays a vital role in seeking out specific cell surface receptors for entry.

*J Inselburg, D J Bzik, W B Li, K M Green, J Kansopon, B K Hahm, I C Bathurst, P J Barr and R N Rossan, Protective immunity induced in Aotus monkeys by recombinant SERA proteins of Plasmodium falciparum. Infect Immun. 1991 April; 59(4): 1247-1250

**Bannister, L. H., J. M. Hopkins, A. R. Dluzewski, G. Margos, I. T. Williams,M. J. Blackman, C. H. Kocken, A. W. Thomas, and G. H. Mitchell. 2003.Plasmodium falciparum apical membrane antigen 1 (PfAMA-1) is translocatedwithin micronemes along subpellicular microtubules during merozoitedevelopment. J. Cell Sci. 116:3825–3834.

Overall Cloning Strategy

RE digest with AatII and KpnI

pSera4S

AatII

KpnI

PCR amplifyAatII

kpnISera 5S

pGEMT

Clone into pGEMT

Check colonies by PCR amplification (inclusion or orientation)Plasmid minipreps of clones Having Sera4sSequencing

pGEMT

Sera4S

Sera4S RE digest with AatII and KpnI

pPmMoe-GFP-His-99

pGEMTSera4S

Ligate and transform JM109

PCR Amplification of Sera 4S using primers having AatII and KpnI ends

Sera

4S

Sera

5S

1kB

Sera 4S ~850 bpSera 5S ~800 bp

Confirmation of Sera 4S in pGEMT PCR amplification with primers having AatII and KpnI ends

1 kB

Chose clones 7, 11, and 12 for RE digestion and Sequencing

7 11 12

Restriction enzyme digestion AatII/KpnI of clones pGEMT-Sera4S-7, 11 and 12

1 kB Clone 7

Uncut cut Uncut cut Uncut cutClone 11 Clone 12

Sera 4S

Restriction enzyme digestion of pMHG-99 with AatII and KpnI

1kB

pMH

G-9

9 U

C

pMGH-99 Cut

pPmMoe GFP His-99 Sera 4S Sequencing

Directional cloning of Sera-5S

pGEMT- Sera5S clones

Sera 5S

Chose clones 4, 5, 6, 10, and 11 for digestion and sequencing

4 5 6 10 11

Restriction enzyme digestion of pGEMT-Sera 5S

clones with AatII/KpnI1

kB 5S-4

UC

5S-4

Cut

5S-5

UC

5S-5

Cut

5S-6

UC

5S-6

Cut

5S-1

0 U

C

5S-1

0 Cu

t

5S-1

1 U

C

5S-1

1 Cu

t

Sera 5S

Sera 5S run of AAAAAAA in primer binding site of pMGH99-5S clones

Moe Protein Recovered after Transfection of P. Marinus with pPmMoe-GFP-His

M L FT W1 W2 E1 E2 E3 FH1

75 kD

25 kD

61 kD

~30 kD

Qiagen Ni-TA column purification using a mixed population of (1.2 x 107)0.4% demonstrating fluorescence due to GFP

Summary and Future Directions• Sera 4S, a serine repeat protein, was successfully cloned into our

pPmMoe-GFP construct. • Sera 5S cloning was confounded by a run of AAAAs at the 3’ end of the

sera 5S gene• His-Tagged recombinant proteins can be successfully separated from

parasite protein lysates using Qiagen Ni-NTA columns • Ama1 and gp120 have been cloned (data not shown). These clones have

been sent for sequencing of joint regions .• Sera 4S, Ama1 and gp120 will be transfected into P. marinus to determine

recombinant protein expression using fluorescence microscopy and isolation of His-tagged recombinant proteins.