Search for Potential Vaccine Candidates in silico from the Cryptosporidium
hominis and Cryptosporidium parvum genomes
E. A. Colby, under the mentorship of Dr. Ping Xu
Bioinformatics and Bioengineering Summer Institute, Virginia Commonwealth University, United States
August 11, 2004
ABSTRACT
A genomic analysis of Cryptosporidium hominis was carried out. The 4107 C. hominis open
reading frames (ORFs) were screened using sequence similarity searches, cellular location
algorithms, domain similarity searches, as well as major histocompatibility complex (MHC)
binding predictions. Each ORF was compared to its homologue in C. parvum in terms of percent
identity. 1048 ORFs were determined possibly surface located and to be vaccine candidates; 97
are discussed in this article.
INTRODUCTION
C. hominis and C. parvum are apicomplexan parasites of importance in public health today;
they have caused epidemics in the United States and abroad. C. parvum infects both bovine and
human hosts; C. hominis infects mainly humans. While C. hominis and C. parvum are
approximately 97% identical, the parasites are known to be close to Plasmodium falciparum, the
parasite of malaria. Infection causes severe diarrhea that can last for several weeks and may be
fatal in the very young, elderly or immunosuppressed. Transfer of the parasite from host to host
occurs through the fecal-oral route in water. This includes drinking water and recreational/pool
water. Resistance to chlorine water treatment of the two parasites renders the prospect of a
vaccine critical.
Currently, there is no vaccine that guarantees protective immunity. The two Cryptosporidium
genomes have recently been finished [1, 25]. Many computational prediction programs are
available for studying protein molecular function, cellular location, and biological processing [13,
14, 10, 22, 3, 4, 18]. The availability of these parasitic genomes and of computational prediction
algorithms for cellular location and secondary structure makes development of a vaccine timelier
and more tangible than ever before. This study focused on surface-exposed proteins as vaccine
candidates in order to facilitate maximum probability of antibody binding in vivo.
METHODS
(i) Amino acid sequences of the 4107 ORFs. The ORF sequences were obtained from the C. hominis genome
annotation [25] using GLIMMER [7, 21] software. ORFs were named “Chro.[chromosome#][ORF#]”.
(ii) Analysis of ORFs. SignalP V2.0.b2 [13, 14] was used to predict signal peptides and signal anchors.
Transmembrane helices were predicted by TM-HMM [10, 22]. BLASTP [3] searching against non-redundant
database in GenBank was used to elucidate gene function. Functional domains were predicted using PFAM [4];
domain matches with expected values of greater than 10-5 were not considered. SYFPEITHI [18] was used to search
for MHC binding motifs. Data was parsed using Perl scripts.
RESULTS
Of the 4107 ORFs screened, 235 were predicted to have signal anchors, and 431 were
predicted to have signal peptides, and 382 were predicted to have transmembrane helices. These
1048 ORFs are considered eligible vaccine candidates.
From these, only the 97 ORFs with functional domain matches confirming cellular location or
relating to virulence are discussed here. All contain several possible MHC binding motifs (not
shown). Currently, there are no prediction algorithms available that make the distinction
between organelle membrane-bound proteins and plasma membrane-bound proteins, but this
may be inferred, to some extent, from functional domain matches. See Table 1.
TABLE 1
Ch gene
Length (bp)
Location
Chro.70118
Chro.30245
Chro.20039
Chro.80171
Chro.20456
Chro.10133
Chro.20432
Chro.20285
Chro.10338
Chro.20173
Chro.70533
Chro.30465
Chro.30069
Chro.40463
Chro.30064
Chro.40323
Chro.30401
Chro.50164
Chro.40312
Chro.50220
Chro.40351
Chro.60622
Chro.60244
Chro.20404
Chro.70203
Chro.30397
Chro.20323
Chro.40094
Chro.80388
Chro.10032
Chro.10391
Chro.70575
Chro.20427
Chro.10390
Chro.70354
Chro.40272
Chro.40405
Chro.40052
Chro.60033
Chro.30468
Chro.30153
Chro.30220
Chro.20090
Chro.50147
Chro.70061
Chro.70043
1419
5340
1347
1251
4449
1362
2463
381
1374
1842
1803
4290
1167
642
1602
1515
234
1059
1671
1644
1614
2340
4860
312
4668
2820
1173
1833
2661
1278
1521
1884
1347
2055
1911
1797
3897
555
2556
1008
1392
1707
3741
840
3000
4353
M
M
M
M
M
M
M
M
M
M
M
M
M
M
M
M
M
M
M
M
S/M
S/M
S/M
S/M
S/M
S/M
S/M
S/M
S/M
S/M
S/M
S/M
S/M
S/M
S/M
S/M
S/M
S/M
S/M
S/M
S/M
S/M
S/M
S/M
S/M
S/M
Function
Protein secretion
Voltage-gated potassium channel
Nucleoside transporter
Similar, human GDP-fucose transporter 1
Calcium transporter
Phosphatidyl serine synthase
Patatin-like phospholipase
Calcium transporter
Zinc transporter
Calcium binding EGF domain
Amino acid transporter
Calcium transporter
Similar, human GDP-fucose transporter 1
Major Facilitator Superfamily (MFS)
Amino acid transporter
Sugar (and other) transporter; MFS
CorA-like Mg2+ transporter
ZIP Zinc transporter
Amino acid transporter
Amino acid transporter
Oocyst wall protein
Mucin-like glycoprotein
Oocyst wall protein
EF hand
LCCL domain
Sodium/calcium exchanger
Thrombospondin type 1 domain
Kazal-type serine protease inhibitor domain
Oocyst wall protein
Peptidase C13 family
Thrombospondin type 1 domain
Oocyst wall protein
EF hand
Thrombospondin type 1 domain
LMBR1-like membrane protein
Ankyrin repeats; Sushi domain (SCR repeat)
Kazal-type serine protease inhibitor domain
MFS
Oocyst wall protein
Peptidase M16 inactive domain
Unknown function, but known to be transmembrane
Calcium binding EGF domain
LCCL domain
SPFH domain / Band 7 family
Calcium transporter
LCCL; F5/8 type C domain
%ID Cp
99.79
97.58
98.44
97.84
98.58
97.14
98.05
92.47
98.25
98.21
97.84
96.16
34.1
99.53
98.69
95.63
100
97.45
97.85
97.45
99.26
88.64
98.58
93.18
98.52
97.66
97.19
28.34
96.84
96.95
92.11
98.57
98.37
95.92
96.38
97.83
52.24
99.46
94.61
97.02
98.28
97.72
99.52
98.93
97.2
98.35
Chro.60194
1035
S/M
Apyrase
Chro.80295
2916
S/M
Sushi domain
Chro.20014
4605
S/M
Similar, ABC transporter
Chro.30475
3825
S/M
Peptidase M16 inactive domain
Chro.60103
1968
S/M
Thrombospondin type 1 domain
Chro.80136
1521
TM
Mucin-like glycoprotein
Chro.70475
4995
TM
SCP-like extracellular protein
Chro.20017
4755
TM
Similar, ABC transporter
Chro.40157
4407
TM
Similar, ABC transporter
Chro.20159
1722
TM
Major Facilitator Superfamily (MFS)
Chro.70384
1905
TM
MFS
Chro.80449
1827
TM
MFS
Chro.30058
4944
TM
Similar, ABC transporter
Chro.80431
1371
TM
Amino acid transporter
Chro.30489
1617
TM
Amino acid transporter
Chro.40505
1680
TM
Amino acid transporter
Chro.70499
4173
TM
Similar, ABC transporter
Chro.80017
1410
TM
Amino acid transporter
Chro.70473
1563
TM
MFS
Chro.80188
2373
TM
MFS
Chro.30352
1248
TM
MFS
Chro.20091
1812
TM
MFS
Chro.40314
1284
TM
CDP-alcohol phosphatidyltransferase
Chro.70464
3387
TM
Calcium transporter
Chro.30090
4758
TM
Similar, ABC transporter
Chro.10380
822
TM
Phosphatidylinositol N-acetylglucosaminyltransferase
Chro.80246
4473
TM
Calcium transporter
Chro.60540
3849
TM
Similar, ABC transporter
Chro.40306
4584
TM
Calcium transporter
Chro.60617
2097
TM
Similar, ABC transporter
Chro.40053
1071
TM
CDP-alcohol phosphatidyltransferase
Chro.40158
3105
TM
Similar, ABC transporter
Chro.60627
1929
TM
Similar, ABC transporter
Chro.50196
2010
TM
N-acetylglucosaminyl transferase component (Gpi1)
Chro.30458
843
TM
Sugar (and other) transporter; MFS
Chro.60238
699
TM
BT1 family
Chro.60067
990
TM
ZIP Zinc transporter
Chro.70498
3327
TM
Similar, ABC transporter
Chro.10157
2310
TM
Similar, ABC transporter
Chro.50257
1125
TM
Translocation protein Sec62
Chro.30424
666
TM
Calcium transporter
Chro.10286
516
TM
Copper transporter
Chro.50149
396
TM
Defender Against Death (DAD)
Chro.10084
1443
TM
Similar, ABC transporter
Chro.20166
1776
TM
CorA-like Mg2+ transporter
Chro.70210
3687
TM
Oocyst wall protein
Chro.70309
1719
TM
Similar, ABC transporter
Chro.20067
1278
TM
Nucleotide-sugar transporter, possibly in golgi membrane
Chro.20284
1242
TM
Nucleotide-sugar transporter, possibly in golgi membrane
Chro.20283
225
TM
Nucleotide-sugar transporter, possibly in golgi membrane
TM = Transmembrane; contains one or more transmembrane helices as predicted by TM-HMM
S/M = Secretory/membrane; contains signal peptide as predicted by SignalP
M = Membrane; contains signal anchor as predicted by SignalP
Cp = C. Parvum
Ch = C. hominis
97.68
95.83
98.12
96.94
29.09
94.14
98.68
25.47
97.69
98.61
98.11
98.36
97.27
99.78
97.03
97.86
97.18
99.79
97.5
94.69
96.4
97.68
96.73
99.47
99.05
99.27
98.46
98.05
98.63
98.86
96.36
97.39
98.29
97.61
99.29
97.85
96.31
99.1
98.05
99.73
95.4
97.09
98.48
25.1
99.66
24.34
97.91
99.06
98.07
94.83
DISCUSSION
There are primarily three kinds of proteins in Table 1: transporters, surface and adhesion
proteins, and proteins with domains related to virulence.
Transporters were selected as candidates not only due to their transmembrane location, but
also due to their potential for blocking essential parasitic cell processes, such as the import of
nucleosides and amino acids from the host. It was revealed that these single-celled parasites lack
the ability to synthesize such essential molecules on their own. It is conjectured that antibody
binding of parasitic transporters will inhibit transporter function, thus causing the parasites to
starve and die. Referring to Table 1, members of the Major Facilitator Superfamily (MFS) clan
are transporters. EF hands have a subclass of buffering/transport proteins. Members of the BT1
family are also thought to be transporters.
Oocyst wall proteins, the Band 7 family protein, the LMBR1-like membrane protein, and the
SCP-like extracellular protein are confirmed surface proteins. Other surface proteins include the
mucin-like glycoproteins; mucin is a cellular adhesion molecule that, in humans, is located on
the surface of leukocytes and attaches to the selectin on another cell. Sushi domains, ankyrin
repeats, N-acetylglucosaminyl transferase (Gpi1) components, and some calcium binding EGF
(Epidermal Growth Factor) domains are also involved in cellular adhesion. Proteins containing
any of these domains are most likely on the parasitic cell surface and may also facilitate antibody
binding due to their adhesion properties.
Proteins with thrombospondin repeats are likely to be related to virulence; circumsporozoite
surface protein 2 and TRAP proteins of Plasmodium (among other parasitic organisms) contain
one or more instance of this repeat. It has been involved in cell-cell interaction, inhibition of
angiogenesis and apoptosis. The thrombospondin repeat shows signs of origination by exon
shuffling, and proteins containing this repeat may not be the best vaccine candidates because
they are highly variable. However, this information is useful for annotation purposes.
Proteins that had domain matches such as “Phosphatidyl serine synthase,” “Patatin-like
phospholipase,” “CDP-alcohol phosphatidyltransferase,” and “Phosphatidylinositol Nacetylglucosaminyltransferase” may also be related to virulence; the mechanism for infection
involves rearrangement of the host epithelial cell’s lipid bilayer to accommodate parasite
attachment. Phosphatidyl serine synthase is a membrane bound protein which catalyses the
replacement of the head group of a phospholipid (phosphotidylcholine or
phosphotidylethanolamine) by L-serine. Patatin is a storage protein but it also has the enzymatic
activity of lipid acyl hydrolase, catalyzing the cleavage of fatty acids from membrane lipids.
CDP-alcohol phosphatidyltransferase is involved in phospholipid biosynthesis.
Phosphatidylinositol N-acetylglucosaminyltransferase is involved in cell anchoring via the
transfer of N-acetylglucosamine (GlcNAc) from UDP-N-acetylglucosamine to
phosphatidylinositol (PI). For these reasons, and because they have some chance of being on the
parasitic cell surface, it is suggested that these proteins be studied to determine if they have a role
in anchoring the parasite to the host epithelial cell.
Proteins with LCCL domains are definitely related to virulence. A previous study identified
these proteins as secretory proteins that are secreted during infection stages [23]. Although
vaccination by these proteins has not been proven effective, documentation of all the proteins in
the genome that contain both signal peptides and LCCL domains may be useful for annotation
purposes or for future studies.
Possibly relating to virulence are proteins containing the Kazal-type serine protease inhibitor
domain or the peptidase M16 inactive domain; both families include digestive enzyme inhibitors
and degraders. Apyrase is known to be involved in the inhibition of host platelet aggregation by
nucleotide hydrolysis at the extracellular level. The peptidase C13 family contains a globin
degrading enzyme. Defender Against Death (DAD) is a plasma membrane protein that prevents
apoptosis.
To identify vaccine candidates for both C. parvum and C. hominis, we only include the
candidate ORFs of C. hominis having greater than 90% identity with homologues in C. parvum
be subject to further consideration for use in a vaccine.
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