© 2005 Nature Publishing Group
A protein interaction network of the malaria
parasite Plasmodium falciparum
Douglas J. LaCount1,2*, Marissa Vignali2*, Rakesh Chettier3, Amit Phansalkar3, Russell Bell3, Jay R. Hesselberth2,
Lori W. Schoenfeld1,2, Irene Ota3, Sudhir Sahasrabudhe3, Cornelia Kurschner3, Stanley Fields1,2
& Robert E. Hughes3†
Plasmodium falciparum causes the most severe form of malaria
and kills up to 2.7 million people annually1. Despite the global
importance of P. falciparum, the vast majority of its proteins
have not been characterized experimentally. Here we identify
P. falciparum protein–protein interactions using a high-
the difficulties in expressing P. falciparum proteins in Saccharo-
myces cerevisiae. From more than 32,000 yeast two-hybrid screens
with P. falciparum protein fragments, we identified 2,846 unique
interactions, most of which include at least one previously
uncharacterized protein. Informatic analyses of network connec-
tivity, coexpression of the genes encoding interacting fragments,
and enrichment of specific protein domains or Gene Ontology
annotations2were used to identify groups of interacting proteins,
including one implicated in chromatin modification, transcrip-
tion, messenger RNA stability and ubiquitination, and another
implicated in the invasion of host cells. These data constitute the
first extensive description of the protein interaction network for
this important human pathogen.
The 80% AT content of the P. falciparum genome3hinders protein
expression in heterologous systems4and limits both conventional
biochemical approaches and comprehensive analyses of this organ-
ism’s proteins. We overcame this problem by applying a yeast two-
hybrid approach that makes use of protein fusions carrying three
elements: the Gal4 DNA-binding or activation domain, a random
fragment of a P. falciparum protein, and an enzyme that allows the
growth of auxotrophic yeast deleted for the cognate gene (Sup-
plementary Fig. 1). This procedure was designed to select only those
yeast transformants that contained plasmids encoding in-frame and
expressed fragments of P. falciparum proteins. In addition, this
procedure enabled us to generate libraries of DNA-binding domain
fusions (‘baits’) from which randomly chosen transformants could
be individually screened in parallel against a library of activation
domain fusions (‘preys’). Our libraries were derived from RNA
isolated from mixed intra-erythrocytic-stage parasites—the stage
responsible for pathogenesis in humans—and thus lack genes
expressed exclusively in the liver, gametocyte or mosquito stages.
Sequence analysis of the inserts from 4,456 DNA-binding domain
fusions identified more than 2,000 non-overlapping gene fragments
representing 1,295 different P. falciparum genes expressed through-
out the intraerythrocytic cycle (Supplementary Fig. 2a), indicating
that our libraries are complex. Because relatively small fragments of
P. falciparum genes (about 450 base pairs on average) were cloned,
narrowly defined protein–protein interaction domains could be
Coverage of the proteome was obtained by performing more than
32,000 yeast two-hybrid screens, of which 11% yielded positives in
which the identities of both interacting protein fragments were
determined (Supplementary Table 1). Because the complete set of
putative interactions contains both true and false positives, we first
sought to eliminate the most obvious class of false positives, namely
those protein fragments with many partners, which seem to be
‘promiscuous’ in the two-hybrid assay. Indeed, although the vast
majority of fragments identified relatively few partners, some inter-
acted with many partners (up to a maximum of 207). To identify
promiscuous protein fragments, we sequentially applied k-means
clustering analysis (with k ¼ 2) to prey and bait fragments to define
two populations based on the number of interacting partners. This
approach identified 13 promiscuous prey fragments with more than
31 partners, and 28 promiscuous bait fragments with more than 25
partners (Supplementary Table 2), resulting in the removal of 2,155
interactions involving these fragments from the data set. Because the
remaining interactions are listed as pairs of interacting proteins
rather than fragments, the total number of partners for a given
protein can exceed the thresholds used to remove promiscuous
fragments if the protein contains multiple non-promiscuous frag-
ments. Although this analysis removes a significant number of
non-specific interactions, other classes of false positive, including
promiscuous fragments that resulted in fewer interactions than our
threshold values and two-hybrid pairs resulting from mutations in
the plasmids or reporter strain, remain in the data set. False positives
have been noted in other high-throughput two-hybrid data sets (for
example those for proteins of Drosophila5,6), and can make up a
substantial portion of the reported interactions.
data set (Supplementary Table 3, also available from the PlasmoDB
(http://www.plasmodb.org/) and BIND (http://bind.ca/) databases)
highly interconnected, scale-free network7containing 1,267 proteins
linked by 2,823 interactions. An additional 41 proteins are present in
small groups of one or two interactions. All categories of proteins
seem to have been sampled approximately in proportion to their
set includes 23 interactions that were previously observed either in
Plasmodium or between orthologous proteins (Supplementary
Table 4). In all, 82% of the interactions include at least one protein
annotated as ‘hypothetical,’ and 33% of the interactions include two
hypothetical proteins. The difficulties in expressing P. falciparum
proteins in heterologous systems precluded experimental confir-
mation of the interactions by another method. To circumvent this
problem, we used several independent bioinformatic analyses to
uncover biologically interesting regions of the network.
1Howard Hughes Medical Institute,2Departments of Genome Sciences and Medicine, University of Washington, Box 357730, Seattle, Washington 98195, USA.3Prolexys
Pharmaceuticals, Inc., 2150 West Dauntless Avenue, Salt Lake City, Utah 84111, USA. †Present address: Buck Institute, Novato, California 94945, USA.
*These authors contributed equally to this work.
Vol 438|3 November 2005|doi:10.1038/nature04104
© 2005 Nature Publishing Group
Because high degrees of local network interconnectivity can
identify sets of functionally related proteins8, we surveyed the net-
work for groups of proteins with a greater number of connections
thanwouldbe expected bychance. We parsed the network into 1,308
primary subnetworks containing a protein, its direct binding part-
ners and all interactions between them,and calculated a connectivity
coefficient (defined as the number of interactions divided by the
number of proteins). A comparison of the distribution of connec-
tivity coefficients present among the experimentally observed sub-
networks with those derived from randomized subnetworks of equal
size showed an enrichment for highly connected subnetworks in
the real data (Fig. 1a); 96 subnetworks showed a higher degree of
interconnectivity than expected by chance (P # 0.05) when com-
pared with the mean of the connectivity coefficients from 100
randomized subnetworks of the same size (Supplementary Table 5).
Several of these 96 subnetworks shared a common set of interactions
interconnectivity. This region overlaps the complex with the highest
identifies densely connected areas of protein interaction networks
identified a group of interacting proteins likely to integrate chroma-
tin modification, transcriptional regulation, mRNA stability and
ubiquitination (Fig. 1b). Whereas 11% of the interactions in the
whole data set were observed in two or more independent experi-
ments, more than 40% of the interactions in this group were
observed in multiple independent experiments.
This set of interacting proteins is centred on PF08_0034, the
P. falciparum orthologue of the yeast histone acetyltransferase
Gcn5. The interactions established by PfGcn5 are mediated by an
indicating that this group might represent a Plasmodium-specific
pathway that regulates gene expression. Other potential chromatin-
modifying proteins in this group include PFF1440w, a protein
containing a PHD domain, bromodomain and SET domain that
potentially recognizes acetylated nucleosomes and acts as a histone
methyltransferase11,12, and PFF1470c, an orthologue13of yeast DNA
Pol2 (Pol1), which is involved in DNA replication and chromatin
silencing at telomeres14. Two putative transcription factors are
also present in this group: PF11_0241, which contains a Myb
DNA-binding domain (and directly interacts with both PF08_0034
and a BTB/POZ domain, which is found in some transcription
factors involved in the recruitment of histone deacetylase com-
plexes15. These interactions indicate that chromatin-modifying com-
plexes might be targeted to specific regionsin the genome to regulate
transcription and are of particular significance given the apparently
unique features of gene expression of the parasite16,17and the dearth
of recognizable transcription factors encoded by its genome18,19. The
presence of three ubiquitin metabolism proteins (the RING finger
and forkhead-associated (FHA)-domain protein PFL0275w, the
HECT-domain protein PFF1365c and the UCH-domain protein
PFI0225w) indicates that ubiquitination might be involved in regu-
lating the stability or activity of proteins in this group; Gcn5-
containing HAT complexes can deubiquitinate histones20. This
group also contains MAL8P1.104, the P. falciparum orthologue of
Caf1, the major mRNA deadenylase in yeast and a member of
the Ccr4–Not complex. Indeed, this group seems analogous to
Ccr4–Not, which also integrates transcription regulation, chromatin
modification, ubiquitination and RNA stability21. Other interactions
implicated in Plasmodium nucleic acid metabolism18,19are shown in
Supplementary Fig. 4.
Interacting proteins from S. cerevisiae tend to have similar mRNA
abundance profiles22; positively correlated mRNA expression is
therefore generally taken as an indication that a putative interaction
is more likely to be real. However, this correlation is not as evident in
the large-scale C. elegans protein interaction data23, and two genes
need not share similar expression profiles for their proteins to be
present in the cell at the same time. For example, one gene may be
expressed constitutively whereas the other is induced under
certain conditions, or their proteins may have different half-lives.
Indeed, the time of maximal accumulation for a substantial
portion of P. falciparum proteins is shifted relative to the time of
P. falciparum protein interaction data and mRNA abundance, we
compared our core data set to data from two genome-scale gene
expression studies that addressedthe timing of mRNAaccumulation
during the P. falciparum life cycle16,17. We calculated Pearson corre-
lation coefficients (PCCs) for each protein pair and averaged these
values for proteins with more than one partner. We identified 82
proteins with average PCCs significantly higher than expected
compared with mean PCCs from 100 randomizations in which the
remained constant (P # 0.05; Supplementary Table 6). Several of
Figure 1 | Connectivity analysis. a, Distribution of connectivity coefficients
from experimental (solid lines) and randomized (dashed line) subnetworks
network. The graph shows interactions (lines) between proteins (circles)
involved in transcription or chromatin metabolism (blue) and ubiquitin
metabolism (orange); proteins with no additional supporting evidence
linking them to these processes are shown in grey. Thin lines indicate
interactions observed in a single yeast two-hybrid experiment; thick lines
show interactions found in reciprocal orientation or in two or more
PlasmoDB; when available, common names, putative functions and
domains are shown in parentheses. Only proteins with two or more
interactions with other group members are shown; additional partners for
these proteins are listed in Supplementary Table 3.
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these 82 proteins are expressed during schizogony, the time at which
new merozoites are being formed. In addition, when we compared
the interaction data set with the gene clusters defined in ref. 17 based
on mRNA abundance profiles, we identified cluster 15 as having
a higher than expected number of interactions among proteins
within the cluster (19 interactions observed, 10 predicted from a
randomized network, P , 0.05; Supplementary Table 7).
Cluster 15 contains proteins implicated in the invasion of host
cells, including merozoite surface protein 1 (MSP1, PFI1475w), an
essential protein that coats the surface of merozoites and is thought
to be required for the invasion of red blood cells. This potential
vaccine candidate has several conserved blocks of sequence, some of
which establish interactions with uncharacterized, coexpressed pro-
teins that might also have a function in the invasion of host cells
proteins, we screened our data set for pairs of interacting proteins
with expression patterns similar to that of MSP1 (that is, expression
peaking at the time when new merozoites are formed and repressed
in the early phase of the 48-h infection cycle). We identified 103
interactions among 89proteins,75ofwhicharelinkedtogether inan
extended region of the network (Supplementary Fig. 6). The core
of this set of proteins (Fig. 2) recapitulates a previously shown
interaction between MSP1 and MSP9 (PFL1385c)25and links 19
uncharacterized proteins to 16 proteins that are involved in the
invasion of host cells or are localized to the merozoite surface. Of the
19 uncharacterized proteins, 6 have been detected in merozoites by
mass spectrometry26and 4 have predicted signal peptides27. Consist-
entwith PFD0230c(a putative typeIdipeptidyl aminopeptidasethat
interacts with MSP3) having a function in the invasion of host cells
is the observation by M. Klemba and D. Goldberg (personal
communication) that it seems to localize to the apical region of
developing merozoites. Other interactions link merozoite surface
proteins (MSP1 and MSP9) to proteins localized to rhoptries
(RhopH1, RhopH2 and RhopH3), indicating the potential for
transient interactions that occur during the invasion of host cells
after the contents of the rhoptries have been released.
biological processes, enrichment of particular domains in subnet-
works can implicate proteins relevant to a process. Similarly, enrich-
(http://www.geneontology.org) can also implicate proteins from a
subnetwork in biological processes. We therefore searched the
P. falciparum interaction network for primary subnetworks
Figure 2 | Interactions between uncharacterized P. falciparum proteins and
proteins involved in the invasion of host cells. Gene names and thin and
thick lines are as in Fig. 1. Squares, proteins with a predicted signal peptide;
triangles, at least one predicted transmembrane domain; circles, no
predicted transmembrane domains; green, peptides from that protein were
detectedby massspectrometry inmerozoites26;grey,proteinnotdetectedin
merozoites. Only proteins with mRNA expression profiles similar to that of
MSP1 are shown; additional partners for these proteins are listed in
Supplementary Table 3.
Figure 3 | Subnetworks with shared protein domains and GO
annotations. Gene names and thin and thick lines are as in Fig. 1; when
in parentheses. Nodes coloured red share the feature indicated at the top of
the panel; nodes coloured grey do not. Only partners of the central protein
that contain the indicated feature are shown; additional partners for these
proteins are listed in Supplementary Table 3.
NATURE|Vol 438|3 November 2005
© 2005 Nature Publishing Group
of RNA recognition motifs (RRM; Fig. 3a), indicating that the
proteins in these subnetworks might be involved in RNA processing
or splicing. For example, PF07_0082, a hypothetical protein, inter-
acts with three proteins that have RRM domains and a fourth that
might be an orthologue of the Drosophila splicing factor suppressor
splicing factor Cwc2, interacts with four putative RNA-binding
proteins and an orthologue of the yeast splicing protein Prp40.
orthologues of yeast splicing factors Prp4 and Prp18, and human
alternative splicing factor 2; the putative P. falciparum Prp4 protein
in turn binds to the P. falciparum orthologue of yeast Prp9
(PFI1215w, not shown). PFI1715w also interacts with three proteins
bearing SNF2 helicase domains (Fig. 3b). Given that proteins con-
taining SNF2 domains are involved in chromatin remodelling,
PFI1715w mightprovide alink between gene expression andsplicing
in P. falciparum. In some cases, domain and GO annotation enrich-
ment indicate unexpected alternative functions for P. falciparum
proteins. We found an orthologue of the yeast nucleosome assembly
protein Nap1 (PFL0185c) in interactions with nine ribosomal
proteins (Fig. 3c) and a protein involved in ribosome biogenesis
(PF11_0259, Rrs1; not shown), possibly indicating a role for
PFL0185c in ribosome assembly or translation. Similarly, a putative
subunit of the N-terminal acetyltransferase complex (PFL2120w)
interacts with the cytoplasmic tails of four PfEMP-1 (P. falciparum
erythrocyte membrane protein-1) proteins (Fig. 3d) and two other
proteins that are known or predicted to be exported to the host cell
acetylation (or binding to PFL2120w) might be involved in protein
trafficking to this compartment. Other examples of domain enrich-
ment implicate uncharacterized P. falciparum proteins in protein
folding (Fig. 3e) and a possible protein kinase signalling cascade
Last, we compared our set of interactions with the more than 150
parasite-derivedproteins knownorpredicted to beexported intothe
host cell cytoplasm28–30. These proteins extensively modify infected
red blood cells, establishing a new secretory system in the cytoplasm
and generating knob-like structureson the cell surface. We identified
15 interactions among 19 exported proteins, which might provide
insight into the structure of parasite-mediated modifications of the
host cell (Suppementary Fig. 7).
The data set of putative protein interactions described here
greatly exceeds the number of previously known interactions for
P. falciparum and provides the basis for focused experiments on a
variety of biological processes. As this network reflects pathways and
processes of the parasite, this information should be relevant both to
our understanding of the basic biology of the organism and to the
discovery of new drug and vaccine targets. Although the difficulties
in working with this organism currently preclude the types of
experimental validation that are available in model organisms, we
expect that the accumulation and integration of large-scale gene and
protein expression studies and protein interaction data sets
will continue to provide informatics-based approaches towards
understanding this human parasite.
Bait and prey construction. Complementary DNA was generated from
poly(A)þRNA isolated from mixed-staged (strain 3D7)-infected erythrocytes
(a gift from K. Ganesan and P. Rathod) and inserted between the Gal4
transcriptional activation domain and the Schizosaccharomyces pombe URA4
coding region of pOAD.102 (prey plasmid) or the Gal4 DNA-binding domain
and the S. cerevisiae MET2 coding region of pOBD.111 (bait plasmid). Yeast
transformed with bait or prey plasmids were plated on medium lacking uracil
(prey) or methionine (bait) to select for transformants expressing the markers
fused to the cDNA inserts. Additional information about the plasmids, yeast
strains and library construction can be found in Supplementary Methods.
Yeast two-hybrid process. Individual bait colonies were picked at random and
clonally expanded in liquid medium in 96-well plates. Aliquots from the prey
libraries were added to each well; mating occurred overnight. Matings were
plated on medium that selected for the mating event, the expression of the
reporter genes ADE2 and HIS3. The cDNA inserts from yeast that grew on this
selection medium were amplified by polymerase chain reaction and then
sequenced. The identities of inserts were determined by querying the sequences
against the annotated P. falciparum genes in PlasmoDB version 4.0 and the
genome sequences from PlasmoDB version 3.3 that were excluded from version
4.0. Additional details are provided in Supplementary Methods.
Removal of false-positive bait and prey fragments. Activation and DNA-
binding domain inserts were treated as protein fragments and independently
grouped into two populations by k-means clustering on the basis of their
number of partners. Interactions involving fragments from groups with the
greater number of partners were deemed promiscuous and removed from the
final data set. Additional information is provided in Supplementary Methods.
Computational analysis. The identification of local regions of the protein
interaction network with enhanced connectivity, comparisons of protein
interaction data with P. falciparum microarray data sets from refs 16 and
17, and the discovery of proteins whose partners were enriched for protein
domain or GO annotations were performed as described in Supplementary
Received 14 April; accepted 1 August 2005.
1.Breman, J. G. The ears of the hippopotamus: manifestations, determinants, and
estimates of the malaria burden. Am. J. Trop. Med. Hyg. 64, 1– -11 (2001).
Ashburner, M. et al. Gene ontology: tool for the unification of biology. The
Gene Ontology Consortium. Nature Genet. 25, 25– -29 (2000).
Gardner, M. J. et al. Genome sequence of the human malaria parasite
Plasmodium falciparum. Nature 419, 498– -511 (2002).
Sibley, C. H. et al. Yeast as a model system to study drugs effective against
apicomplexan proteins. Methods 13, 190– -207 (1997).
Formstecher, E. et al. Protein interaction mapping: a Drosophila case study.
Genome Res. 15, 376– -384 (2005).
Giot, L. et al. A protein interaction map of Drosophila melanogaster. Science 302,
1727– -1736 (2003).
Barabasi, A. L. & Oltvai, Z. N. Network biology: understanding the cell’s
functional organization. Nature Rev. Genet. 5, 101– -113 (2004).
Rives, A. W. & Galitski, T. Modular organization of cellular networks. Proc. Natl
Acad. Sci. USA 100, 1128– -1133 (2003).
Bader, G. D. & Hogue, C. W. An automated method for finding molecular
complexes in large protein interaction networks. BMC Bioinformatics 4, 2
10. Fan, Q., An, L. & Cui, L. Plasmodium falciparum histone acetyltransferase, a
yeast GCN5 homologue involved in chromatin remodeling. Eukaryot. Cell 3,
264– -276 (2004).
11. Rea, S. et al. Regulation of chromatin structure by site-specific histone H3
methyltransferases. Nature 406, 593– -599 (2000).
12. Ragvin, A. et al. Nucleosome binding by the bromodomain and PHD finger of
the transcriptional cofactor p300. J. Mol. Biol. 337, 773– -788 (2004).
13. Li, L., Stoeckert, C. J. Jr & Roos, D. S. OrthoMCL: identification of ortholog
groups for eukaryotic genomes. Genome Res. 13, 2178– -2189 (2003).
14. Iida, T. & Araki, H. Noncompetitive counteractions of DNA polymerase epsilon
and ISW2/yCHRAC for epigenetic inheritance of telomere position effect in
Saccharomyces cerevisiae. Mol. Cell. Biol. 24, 217– -227 (2004).
15. Huynh, K. D. & Bardwell, V. J. The BCL-6 POZ domain and other POZ domains
interact with the co-repressors N-CoR and SMRT. Oncogene 17, 2473– -2484
16. Bozdech, Z. et al. The transcriptome of the intraerythrocytic developmental
cycle of Plasmodium falciparum. PLoS Biol. 1, E5 (2003).
17. Le Roch, K. G. et al. Discovery of gene function by expression profiling of the
malaria parasite life cycle. Science 301, 1503– -1508 (2003).
18. Coulson, R. M., Hall, N. & Ouzounis, C. A. Comparative genomics of
transcriptional control in the human malaria parasite Plasmodium falciparum.
Genome Res. 14, 1548– -1554 (2004).
19. Aravind, L., Iyer, L. M., Wellems, T. E. & Miller, L. H. Plasmodium biology:
genomic gleanings. Cell 115, 771– -785 (2003).
20. Daniel, J. A. et al. Deubiquitination of histone H2B by a yeast
acetyltransferase complex regulates transcription. J. Biol. Chem. 279,
1867– -1871 (2004).
21. Collart, M. A. Global control of gene expression in yeast by the Ccr4-Not
complex. Gene 313, 1– -16 (2003).
22. Ge, H., Walhout, A. J. & Vidal, M. Integrating ‘omic’ information: a bridge
between genomics and systems biology. Trends Genet. 19, 551– -560 (2003).
23. Li, S. et al. A map of the interactome network of the metazoan C. elegans.
Science 303, 540– -543 (2004).
24. Le Roch, K. G. et al. Global analysis of transcript and protein levels across the
Plasmodium falciparum life cycle. Genome Res. 14, 2308– -2318 (2004).
NATURE|Vol 438|3 November 2005
© 2005 Nature Publishing Group Download full-text
25. Li, X. et al. A co-ligand complex anchors Plasmodium falciparum merozoites to
the erythrocyte invasion receptor band 3. J. Biol. Chem. 279, 5765– -5771 (2004).
26. Florens, L. et al. A proteomic view of the Plasmodium falciparum life cycle.
Nature 419, 520– -526 (2002).
27. Kissinger, J. C. et al. The Plasmodium genome database. Nature 419, 490– -492
28. Cooke, B. M., Lingelbach, K., Bannister, L. H. & Tilley, L. Protein trafficking in
Plasmodium falciparum-infected red blood cells. Trends Parasitol. 20, 581– -589
29. Hiller, N. L. et al. A host-targeting signal in virulence proteins reveals a
secretome in malarial infection. Science 306, 1934– -1937 (2004).
30. Marti, M., Good, R. T., Rug, M., Knuepfer, E. & Cowman, A. F. Targeting malaria
virulence and remodeling proteins to the host erythrocyte. Science 306,
1930– -1933 (2004).
Supplementary Information is linked to the online version of the paper at
Acknowledgements We thank P. Duffy, J. Feagin and C. H. Sibley for reading the
manuscript critically, A. Gauntlett for technical assistance, and W. Hol for
helpful discussions. This work was supported by a grant from the NIH. J.R.H.
was supported by an NIH Kirschstein NRSA post-doctoral fellowship. S.F. is an
Investigator of the Howard Hughes Medical Institute.
Author Information Reprints and permissions information is available at
npg.nature.com/reprintsand permissions.The authors declare no competing
financial interests. Correspondence and requests for materials should be
addressed to S.F. (email@example.com) or R.E.H.
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