Download identification of vaccine and drug targets against

IDENTIFICATION OF VACCINE AND
DRUG TARGETS AGAINST MALARIA
HIRDESH KUMAR
KUSUMA SCHOOL OF BIOLOGICAL SCIENCES
INDIAN INSTITUTE OF TECHNOLOGY DELHI
MARCH 2016
©Indian Institute of Technology Delhi (IITD), New Delhi, 2016
IDENTIFICATION OF VACCINE AND
DRUG TARGETS AGAINST MALARIA
by
Hirdesh Kumar
Kusuma School of Biological Sciences
Submitted
In fulfilment of the requirements of the degree of
Doctor of Philosophy
to the
Indian Institute of Technology Delhi
March 2016
CERTIFICATE
This is to certify that the thesis entitled “Identification of vaccine and drug targets against
malaria”, being submitted by Mr. Hirdesh Kumar to the Indian Institute of Technology
Delhi for the award of the degree of “Doctor of Philosophy” is a record of the bonafide
research carried out by him, which has been prepared under our supervision and guidance in
conformity with rules and regulations of the Indian Institute of Technology Delhi. The results
therein have not been submitted in part or full to any other University or Institute for the award
of any Degree/Diploma.
Dr. James Gomes
Professor
Kusuma School of Biological
Sciences
Indian Institute of
Technology Delhi
New Delhi - 110016, India
Dr. B Jayaram
Professor
Department of Chemistry
and SCFBio
Indian Institute of
Technology Delhi
New Delhi - 110016, India
Date:
Place: New Delhi
i
Dr. Friedrich Frischknecht
Professor
Department of Infectious
Disease,
Heidelberg Clinic
Germany
Acknowledgements
Foremost, I would like to express my sincere gratitude to my supervisors. I am thankful to Prof
James Gomes for his continuous support throughout my PhD carrier, his motivation and regular
inputs into the problems. I am indebted to Prof. Frischknecht who helped me in developing the
scientific attitude and also for his invaluable suggestions throughout my PhD carrier. I am
indeed thankful to Prof. Jayaram for his motivation and time to time discussion. I sincerely
thank you all for being the sort of supervisors every student needs - astute, supportive,
enthusiastic and inspiring.
I would like to express my deep sense of gratitude to the following people for their direct or
indirect contribution in my thesis: Professors Aditya Mittal, Archana Chugh, Ashok Kumar
Patel, Bishwajit Kundu, Chinmoy Sankar Dey, Seyed E. Hasnain, Tapan Kumar Chaudhuri
and Vivekanandan Perumal for their teaching. My friends from Kusuma School of Biological
Sciences (KSBS), in particular Ashutosh and Vinay who taught me basic molecular biology
techniques and to Aditya and Suhas for their time to time discussions. Dr. C. R. Pillay (National
Institute of Malaria Research, New Delhi) who introduced me to the malaria biology, in-vitro
blood culture and microscopy of the Plasmodium parasite. Dr. Shikhar Gupta (Procter and
Gamble, Singapore) for encouraging me to apply for DAAD scholarship. Dr. Ranajit Shinde
(Institute of Microbial Technology, Chandigarh) and Dr. Rajendra Kumar (Umeå University,
Sweden) for their invaluable discussions on molecular dynamics simulations. Mahendra Awale
(University of Berne) to assist in automated virtual screening and Vivek Kumar (National
Institute of Pharmaceutical Education and Research, Mohali) for his all-time support and for
valuable discussions.
I would like to express my great regards to German Academic Exchange Service (DAAD) for
awarding me DAAD scholarship to work in Germany and giving opportunity to convert my
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idea into reality. My sincere thanks to Prof. Frischknecht who allowed me to work in his lab
and to test my hypothesis. I am thankful to Goethe institute for exciting German language
course. I would also like to thank to Miriam Griesheimer who nicely took care of official
documents that made my early days in Heidelberg much easier.
In Prof. Frischknecht’ laboratory, I am extremely thankful to Mirko Singer, Jessica Kehrer and
Gunnar Mair who introduced me to advanced molecular parasitology techniques and for the
in-depth discussions.
A special thanks to Miriam Ester, who is always nice to everyone even towards her mosquitoes.
My special thanks also to Dennis Klug and Ross Douglas for critically reviewing my work. My
sincere thanks to Kartik for all his support, stimulating discussions, for his company during
weekends and sleepless nights when we were working together, and of-course for zussamen
essen. I am also thankful to all other lab-mates including Catherine Moreau, Mendi Muthinja,
Benjamin Spreng, Katharina Quadt and Konrad Beyer for providing the wonderful working
environment. In Mueller’s lab, I am thankful to Ann-Kristin Mueller, Kirsten Heiss and Julia
Sattler for their time to time discussions and fruitful suggestions. Franziska to try adenoassociated viral production of LISP2. I am also thankful to animal-facility persons who took
care of my experimental animals.
I am also thankful to Jessica Kehrer, Gunnar Mair, Nikhil Taxak, Julia Sattler for reading my
thesis and making necessary corrections.
Last but not least, I am thankful to my parents who gave me strength to work. My brother to
provide me mental peace to think better and work harder. And to my lovely wife for her eternal
love, sound patience, all time support and encouragement, and for having faith in me.
Hirdesh Kumar
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Abstract
Malaria parasites have become resistant to promising artemisinin-combination therapy (ACT)
and leading vaccine candidate RTS,S has also failed to provide long-term protection in humans.
The Malaria Eradication Program needs a new generation of antimalarial drugs and effective
vaccines against the disease. An ideal antimalarial drug should arrest the parasite in all
development stages in humans and thus inhibit the target protein in each of these stages.
Similarly if vaccines are developed, these needs to be specific for a certain stage. For example,
parasites that lack genes that encode proteins with essential functions during asexual replication
or red blood cell invasion, cannot be made or evaluated for their potential as liver stage specific
vaccine. Genetically attenuated parasites (GAPs) that lack genes essential for the liver stage of
the malaria parasite, and therefore cause developmental arrest, have been developed as live
vaccines in rodent malaria models and recently been tested in humans. Combining the available
proteomics data from rodent- and human-parasites, I performed a systematic ortholog-based
analysis and identified potential vaccine candidates and drug targets. Next, using reverse
genetics approach, I studied the role of five such candidates in Plasmodium berghei ANKA: 2
vaccine candidates and 3 drug-targets. The gene of interests were individually deleted in the
parasites and the clonal lines were characterized throughout the life cycle (in C57BL/6, NMRI
and BALB/c mice; Anopheles stephensi mosquitoes and liver hepatocellular cells).
To test first vaccine candidate, I generated P. berghei parasite lines that lacked the entire coding
sequence for the late liver stage protein LISP2. The deletion of lisp2 leads to a nearly complete
arrest in late liver stage development. The N-terminus of the protein was sufficient to support
the complete liver stage development of the parasites. C57BL/6 mice that cleared lisp2(-)
parasites, remained protection against further wild-type challenge.
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Then, the second vaccine candidate was tested and ARP encoding gene in the parasites was
deleted. Thus developed mutant parasites have a growth-delay during blood stage development
and mice infected with such parasites eventually cleared the infection. Although able to form
ookinetes, arp(-) parasites did not form oocysts in mosquito midguts and were not transmitted
through mosquitoes. Mice given arp(-) parasites were immune to the subsequent challenge
with blood stages of P. berghei strain ANKA and P. yoelii YM and to the P. berghei strain
ANKA sporozoites.
For testing drug-targets, I hypothesized that depletion of house-cleaning enzymes involved in
the conversion of toxic metabolites could potentially lead to the arrest of parasite growth
because accumulation of these metabolites might kill the parasites. The non-canonical
nucleotides like dUTP, dITP and dXTP, which are generated during metabolism of canonical
nucleotides (trinucleotides of A, T, G and C) incorporate into the nascent DNA leading to DNA
break and further to cell death. Through chem-bioinformatics analyses, I predicted three housecleaning enzymes in Plasmodium: deoxyuridine 5’-triphosphate pyrophosphatase (dUTPase);
inosine 5’-triphosphate pyrophosphatase (ITPase) and NUDIX-domain containing protein
(NuDiP). These enzymes detoxify non-canonical nucleotides. Using reverse genetics approach,
I tested 3 candidates and found that dUTPase was essential for P. berghei blood stage while
NuDiP and ITPase were dispensable.
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Table of content
CERTIFICATE ......................................................................................................................... i
Acknowledgements ..................................................................................................................ii
Abstract .................................................................................................................................... iv
List of figures ............................................................................................................................ x
List of tables............................................................................................................................xii
Abbreviation ......................................................................................................................... xiii
Chapter 1: Introduction .......................................................................................................... 1
1.1 Global impact of the disease .............................................................................................. 2
1.2 Complexity of the disease .................................................................................................. 2
1.3 Life cycle of the parasite .................................................................................................... 3
1.4 Clinical features of malaria ................................................................................................ 5
1.5 Malaria complications ........................................................................................................ 6
1.5.1 Cerebral malaria .............................................................................................................. 6
1.5.2 Severe anaemia ................................................................................................................ 7
1.6 Diagnosis of malaria ........................................................................................................... 7
1.6.1 Microscopy ...................................................................................................................... 8
1.6.2 Rapid Diagnostic Test (RDT).......................................................................................... 8
1.7 Current antimalarial therapy ............................................................................................... 9
1.7.1 Artemisinin derivatives ................................................................................................... 9
1.7.2 Artemisinin combination therapy (ACT) ...................................................................... 10
1.8 Antimalarial drug resistance ............................................................................................. 11
1.8.1 Artemisinin resistance ................................................................................................... 11
1.8.2 Alternatives to artemisinin resistance............................................................................ 11
1.9 Vaccine against malaria.................................................................................................... 12
1.9.1 Pre-erythrocytic vaccine ................................................................................................ 12
1.10 P. berghei: A model rodent parasite ............................................................................... 15
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1.11 Scope of the research ...................................................................................................... 16
1.12 Definition of problems and objectives ........................................................................... 18
Chapter 2: In silico identification of vaccine and drug targets against malaria .............. 20
2.1 Introduction ...................................................................................................................... 21
2.2 Results and Discussion ..................................................................................................... 23
2.2.1 Search for GAPLS candidates ........................................................................................ 24
2.2.2 Search for multi-stage drug targets ............................................................................... 33
2.3 Conclusions ...................................................................................................................... 47
Chapter 3: Deletion of lisp2 arrests P. berghei ANKA liver stages in C57BL/6 mice and
protects against further infection ......................................................................................... 48
3.1 Introduction ...................................................................................................................... 50
3.2 Results and Discussion ..................................................................................................... 53
3.2.1 LISP2 is a Plasmodium specific protein with conserved, 6-Cys domain ...................... 54
3.2.2 Incomplete deletion of lisp2(-) did not result in a complete arrest ............................... 54
3.2.3 Complete disruption of lisp2 results in an almost complete arrest in the liver ............. 57
3.2.4 493-amino-acid long N-LISP2 is capable to complete liver stage development .......... 58
3.2.5 Immunization with lisp2(-) sporozoites confers dose dependent protection ................. 61
3.2.6 Additive attenuation in malaria parasites lacking two liver specific genes................... 63
3.3 Summary .......................................................................................................................... 64
Chapter 4: Plasmodium berghei arp(-) parasites are virulence attenuated blood stages and
induce protective immunity against experimental malaria ............................................... 66
4.1 Introduction ...................................................................................................................... 67
4.2 Results and Discussion ..................................................................................................... 69
4.2.1 ARP is a conserved Plasmodium protein with a “dsDNA_bind” domain .................... 69
4.2.2 PbARP is expressed in all stages .................................................................................. 70
4.2.3 ARP(-) parasites form attenuated blood stages in infected mice ................................... 70
4.2.4 ARP(-) parasites arrest during mosquito-stage development ........................................ 72
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4.2.5 Recovery from ARP(-) infection results in long-lasting malaria immunity .................. 73
4.2.6 Deciphering the role of ARP in the parasite.................................................................. 74
4.2.7 MYST is essential for P. berghei ANKA blood stages ................................................. 76
4.2.8 Can we predict GAPBS candidates? ............................................................................... 77
4.3 Summary .......................................................................................................................... 78
Chapter 5: Identification of essential house-cleaning enzymes in Plasmodium berghei .. 79
5.1 Introduction ...................................................................................................................... 81
5.2 Results and Discussion ..................................................................................................... 84
5.2.1 Identification of detoxifying enzymes using chem-bioinformatics analyses ................ 84
5.2.2 Three detoxifying genes are expressed during blood and mosquito stages................... 89
5.2.3 Failure to delete dUTPase in P. berghei ....................................................................... 91
5.2.4 Parasites lacking ITPase complete their life-cycle........................................................ 92
5.2.5 Nudix-domain containing protein (NuDiP) has dispensable role ................................. 97
5.2.6 PbdUTPase consists an additional insertional motif ................................................... 101
5.3 Summary ........................................................................................................................ 103
Chapter 6: Materials and methods ..................................................................................... 104
6.1 Identification of vaccine targets ..................................................................................... 104
6.1.1 Ortholog and paralog search in different Plasmodium species ................................... 104
6.1.2 Phyletic distribution .................................................................................................... 104
6.1.3 Percentage identity matrices ........................................................................................ 105
6.2 Identification of multi-stage drug-targets ....................................................................... 106
6.2.1 Unique, multi-stage expressed P. falciparum proteins................................................ 106
6.2.2 Plasmodium protein lacking similar human protein.................................................... 106
6.2.3 Search for similar crystal structure .............................................................................. 107
6.2.4 Search for essential genes............................................................................................ 107
6.3 Homology modeling and crystal structure analysis ....................................................... 107
6.4 Molecular docking of non-canonical nucleotides (chapter 5) ........................................ 108
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6.5 Expression analysis of GOIs .......................................................................................... 109
6.6 Preparation of knockout vectors ..................................................................................... 111
6.6.1 Transfection vectors .................................................................................................... 111
6.6.2 Cloning ........................................................................................................................ 111
6.7 Generation of mutant P. berghei ANKA parasites lacking GOI .................................... 116
6.7.1 Ethics statement ........................................................................................................... 116
6.7.2 Animals and parasites .................................................................................................. 116
6.7.3 Transfection ................................................................................................................. 116
6.8 Characterization of mutant parasites across the life cycle ............................................. 118
6.8.1 Limiting dilution.......................................................................................................... 118
6.8.2 Phenotypic characterization of GOI(-) blood stages ................................................... 119
6.8.3 Mosquito-stage infection of GOI(-) parasites ............................................................. 119
6.8.4 Infectivity of the mutant sporozoites ........................................................................... 121
6.9 Recycling of selection marker (Negative selection) ....................................................... 122
6.10 Immunization and challenges ....................................................................................... 123
6.10.1 arp(-) GAPBS ............................................................................................................. 123
6.10.2 lisp2(-) GAS .............................................................................................................. 123
Summary and conclusions ................................................................................................... 124
Conclusions .......................................................................................................................... 127
References ............................................................................................................................. 129
Biodata .................................................................................................................................. 141
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List of figures
Figure 1.1: Life cycle of Plasmodium falciparum ..................................................................... 4
Figure 2.1: Search space for identification of drug-targets and GAPLS. ................................. 23
Figure 2.2: Identification of liver stage candidate genes in human parasites .......................... 25
Figure 2.3: Percentage identity matrices of 20 shortlisted vaccine candidates........................ 27
Figure 2.4: Identification of multi-stage drug-targets in P. falciparum. .................................. 35
Figure 3.1: Percentage identity matrices of LISP2 in different Plasmodium species. ............. 54
Figure 3.2: LISP2: 6-Cys domain in Plasmodium genus......................................................... 55
Figure 3.3: Different attempts to delete lisp2 in P. berghei ANKA parasites. ........................ 55
Figure 3.4: lisp2 deletion strategy in Plasmodium berghei ..................................................... 56
Figure 3.5: Liver stage development of lisp2(-) sporozoites ................................................... 57
Figure 3.6: Mosquito stage development of lisp2(-) parasites. ................................................ 58
Figure 3.7: lisp2(-) parasites arrest during late liver stages ..................................................... 59
Figure 3.8 Recycling of selection marker ................................................................................ 59
Figure 3.9: Complementaiton of lisp2(-) parasites with N-LISP2 encoding sequence.. ......... 60
Figure 3.10: 493 amino acids of LISP2 rescue the liver stage arrest of lisp2(-) parasites....... 61
Figure 3.11: C57BL/6 mice immunized with different GAS. ................................................. 62
Figure 4.1: Gene and protein architecture of PbARP .............................................................. 69
Figure 4.2: Analysis of PbARP expression in P. berghei life cycle. ....................................... 71
Figure 4.3: Targeted gene disruption of ARP in P. berghei..................................................... 71
Figure 4.4: ARP (-) parasites showed delayed growth during blood stage ............................. 72
Figure 4.5: ARP depleted parasites form less ookinetes and form no oocyst .......................... 72
Figure 4.6: ARP(-) parasites generate protective immunity..................................................... 73
Figure 4.7: Schematic representation of ARP signaling network. ........................................... 75
Figure 5.1: Docking of non-canonical nucleotides in dUTPase and ITPase ........................... 86
x
Figure 5.2: A, Role of dUTPase, ITPase and NuDiP as detoxifying enzymes. ....................... 88
Figure 5.3: Comparison of active site region of ITPase and dUTPase .................................... 89
Figure 5.4: Expression analysis of 3 detoxifying genes.. ........................................................ 90
Figure 5.5: The preparation of parasites expressing GFP-tagged genes .................................. 90
Figure 5.6: Expression profile and subcellular localization of three proteins ......................... 92
Figure 5.8: Targeted gene disruption of ITPase in P. berghei................................................. 93
Figure 5.9: ITPase (-) parasites showed normal growth during blood stage ........................... 93
Figure 5.10: ITPase (-) parasites showed form normal mosquito stages ................................. 94
Figure 5.11: ITPase(-) parasites are delayed in vivo liver stage development. ....................... 95
Figure 5.12: Gliding motility of ITPase (-) (KO) and wild-type (WT) parasites .................... 96
Figure 5.13: Liver stage development of (ITPase(-)) parasites. .............................................. 96
Figure 5.14: Chem-Bioinformatics analysis of NuDiP. ........................................................... 98
Figure 5.15: Targeted gene disruption of NuDiP in P. berghei ............................................... 99
Figure 5.16: Characterization of NuDiP(-) parasites ............................................................. 100
Figure 5.17: Sequence alignment of the different dUTPases. ............................................... 102
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List of tables
Table 1.1: Distinction of different Plasmodium species based on microscopy ......................... 8
Table 2.1: Final shortlisted 20 liver stage specific GAP candidates in P. falciparum ............ 26
Table 2.2: 43 shortlisted multi-stage drug-targets against malaria .......................................... 36
Table 3.1: 6-Cys family members in Plasmodium................................................................... 52
Table 5.1: 24 “hydrolase” in P. berghei .................................................................................. 84
Table 5.2: Residues involved in H-bond interactions in docking studies. ............................... 86
Table 6.1: Homology modeling of shortlisted proteins ......................................................... 108
Table 6.2: Transfection vectors used to tag genes with GFP encoding sequence. ................ 109
Table 6.3: Details of primers used for different cloning reactions. ....................................... 114
Table 6.4: Details of vectors used in different knockout strategies ....................................... 116
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Abbreviation
5-FC
ᵒC
ACT
ARP
bp
BS
BSA
C
C57Bl/6
cDNA
CQ
CSP
hDHFR
DDT
DHFS
DMEM
DNA
dNTPs
ds
dUTPase
ECM
E.coli
eGFP
FBT
FCS
FDA
g
GA
GAP
GAPBS
GAPLS
GAS
gDNA
GFP
GOI
h
HAT
i.p.
i.v.
IFA
ITP
ITPase
iRBCs
kb
kDa
K/X
L
5-fluorocytosine
degree celcius
artemisinin combination therapy
apoptosis related protein
base pairs
blood stage
bovine serum albumin
celsius
C57 black 6, inbred mouse strain
complementary DNA
chloroquine
circumsporozoite protein
human dihydrofolate reductase
dichlorodiphenyltrichloroethane
dihydrofolate synthase
doulbecco’s Modified Eagles Medium
deoxyribonucleic acid
deoxynucleotides
double-stranded
deoxyuridine-triphosphatase
experimental cerebral malaria
Escherichia coli
enhanced green fluorescent protein
fresh blood transfer
fetal calf serum
food and drug administration
unit for centrifugation step
glutaraldehyde
genetically attenuated parasites
blood stage arresting GAP
liver stage arresting GAP
genetically attenuated sporozoites
genomic DNA
green fluorescent protein
gene of interest
hour
histone acetyltransferase
intraperitoneal
intravenous
immunofluoroscence assay
inosine triphosphate
inosine triphosphate (ITP) pyrophosphohydrolase
infected red blood cells
kilo base
kilo daltons
ketamine/xylazin
liter
xiii
LAVs
lisp2
MG
min
MYST
live attenuated vaccines
liver stage specific protein 2
midgut
minutes
protein under family that include MOZ, Ybf1/Sas3, Sas2 and Tip60 as
other members
NA
numerical aperture
NMRI
Naval Medical Research Institute, refers to an albino outbred mouse strain
nt
nucleotide
N-LISP2
N-terminus of LISP2
ORF
open reading frame
PBS
phosphate buffered saline
PCR
polymerase chain reaction
PDCD5
programmed cell death 5
Pb
Plasmodium berghei
Pf
Plasmodium falciparum
Py
Plasmodium yoelii
PFA
paraformaldehyde
p.i.
post infection
PM
plasma membrane
PVM
parasitophorous vacuole membrane
RDT
rapid diagnostic test
RPMI
Roswell Park Memorial Institute medium
RBC
red blood cell
RT
room temperature
RTS,S/AS01 central repeat region (R) of P. falciparum circumsporozoite protein (CSP)
flanked at both ends with T-cell epitopes (T) of CSP using the hepatitis B
surface antigen (S) as a carrier matrix. The additional S stands for its
expression in Saccharomyces cerevisiae (S) containing liposomal-based
Adjuvant System (AS01)
sec
seconds
sm
selection marker
ss
single-stranded
SD
standard deviation
SG
salivary gland
U
units
UTR
untranslated region
WHO
world health organization
WT
wildtype
yFCU
a bifunctional protein that combines yeast cytosine deaminase and uridyl
5FC
5’-fluorocytosine
xiv
Thesis organization
This thesis is organized in seven chapters. In the first chapter, the literature survey about
malaria and current antimalarial therapy have been described. This is followed by a brief
introduction about the definition of problem, motivation and specific objectives of the study.
Second chapter focuses on in silico identification of potential vaccine- and drug-targets against
the malaria parasites. In next three chapters, I tested two of previously identified vaccinecandidates and three predicted drug-targets. Chapter three involves generation of knockout P.
berghei parasites lacking lisp2, a late liver stage specific gene, and evaluation of their vaccine
potential. The fourth chapter constitutes the experiments to generate arp(-) parasites and testing
of their vaccine potential. In chapter five, a chem-bioinformatics based approach to shortlist
three house-cleaning drug-targets, is described. The essentiality of each of these candidates has
been evaluated. In the final chapter, material and methods used in the execution of the work
required for this research study are discussed. The biodata consisting of published articles,
conference proceeding, and abstracts are included at the end of the thesis.
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