RE S EAR CH | R E P O R T S
A forward genetic screen identifies erythrocyte CD55 as essential for Plasmodium falciparum invasion Elizabeth S. Egan,1,2 Rays H. Y. Jiang,1,3 Mischka A. Moechtar,1 Natasha S. Barteneva,4 Michael P. Weekes,5 Luis V. Nobre,5 Steven P. Gygi,6 Joao A. Paulo,6 Charles Frantzreb,1 Yoshihiko Tani,7 Junko Takahashi,7 Seishi Watanabe,8 Jonathan Goldberg,1 Aditya S. Paul,1 Carlo Brugnara,9 David E. Root,10 Roger C. Wiegand,10* John G. Doench,10 Manoj T. Duraisingh1,10† Efforts to identify host determinants for malaria have been hindered by the absence of a nucleus in erythrocytes, which precludes genetic manipulation in the cell in which the parasite replicates. We used cultured red blood cells derived from hematopoietic stem cells to carry out a forward genetic screen for Plasmodium falciparum host determinants. We found that CD55 is an essential host factor for P. falciparum invasion. CD55-null erythrocytes were refractory to invasion by all isolates of P. falciparum because parasites failed to attach properly to the erythrocyte surface. Thus, CD55 is an attractive target for the development of malaria therapeutics. Hematopoietic stem cell–based forward genetic screens may be valuable for the identification of additional host determinants of malaria pathogenesis.
S
Frequency 0.2 0.4 0.6
32.3%
0.05%
15%
4% 0.35%
1.95% 0.05%
0.6
Top underrepresented genes
0.4
C8orf55 HNRNPK BCL2L1 TUBA1C PL-5283 GPBP1L1 ALAS2 CCT7 SLC12A4 UROD ATP6AP2 PSMD3 SLC25A37 KPNB1 SULT1A4 PDZK1IP1 ISG15 FECH
Day 6: Lentivirus infection
0.2 0 0.2
Department of Immunology and Infectious Diseases, Harvard T. H. Chan School of Public Health, Boston, MA, USA. 2Division of Infectious Diseases, Boston Children’s Hospital, Boston, MA, USA. 3Department of Global Health and Center for Drug Discovery and Innovation, University of South Florida, Tampa, FL, USA. 4Department of Pediatrics, Harvard Medical School and Program in Cellular and Molecular Medicine, Boston Children’s Hospital, Boston, MA, USA. 5Cambridge Institute for Medical Research, University of Cambridge, Cambridge, UK. 6Department of Cell Biology, Harvard Medical School, Boston, MA, USA. 7Japanese Red Cross Kinki Block Blood Center, Osaka, Japan. 8Japanese Red Cross Kyushu Block Blood Center, Fukuoka, Japan. 9 Department of Laboratory Medicine, Boston Children’s Hospital and Harvard Medical School, Boston, MA, USA. 10 The Broad Institute of Harvard and Massachussetts Insititute of Technology, Cambridge, MA, USAA. *Present address: Preceres, Cambridge, MA, USA. †Corresponding author. E-mail:
[email protected]
Fig. 1. Pooled shRNA screen for genes that regulate terminal erythroid differentiation. (A) Schematic of pooled shRNA screen. CD34+ HSCs were induced toward erythroid development, transduced with shRNA library on Day 6, and selected with puromycin. On day 19, shRNA proviruses in orthochromatic erythroblasts were quantified by means of Illumina (San Diego, CA) sequencing. (B) Change in relative abundance of 5530 shRNAs in erythrocyte proteome library after 19 days of differentiation. (C) RNAi gene enrichment ranking (RIGER) analysis of 116 candidate genes based on magnitude of erythropoiesis phenotype. NES, normalized enrichment score. (D) Predicted localization of library components, with percent of genes that influence erythropoiesis in red.
Increased
16.2%
1
SLC14A1 ST13 PSMA4 ADSL BPGM YOD1 RAB21 EIF2AK1
Terminal differentiation
30.1%
-3 −2 −1 0 1 2 Log2 normalized shRNA counts (mean day 19-day 0)
Days 7-11: Select for transduced cells
0.4
Top overrepresented genes 0.6 0%
Blood Groups
EndoMembrane Cytoskeleton Cytosol
Quantify shRNAs by deep sequencing
SCIENCE sciencemag.org
Nucleus
20%
40%
60%
80%
100%
Erythropoiesis effect
Day 19: Harvest DNA from remaining cells
Membrane
Analysis
demiologic approaches (3, 4). Genome-wide association studies have searched for host determinants of malaria, but functional validation in the erythrocyte remains challenging owing to the absence of a nucleus (5–7). Recent advances in the ex vivo production of erythrocytes now enable generation of genetically altered cells that support P. falciparum
Decreased
0.0
Day 0: Initiate CD34+ cell culture
Adjusted average NES
Proliferation and lentivirus infection
evere malaria is caused by Plasmodium falciparum and is one of the leading causes of mortality among children globally (1). During infection, parasites invade and replicate within human erythrocytes (2). Host erythrocyte polymorphisms that confer resistance to severe malaria have been identified with epi-
infection (8–11). Here, we used ex vivo–cultured red blood cells (cRBCs) in a forward genetic screen in order to identify host determinants of malaria infection. The human erythrocyte is a terminally differentiated minimal cell that lacks organelles and a nucleus and has a small proteome (12). To identify erythrocyte proteins that influence P. falciparum infection, we designed a screening strategy involving RNA interference (RNAi)–based knockdown of gene expression in hematopoietic progenitor cells, induction of ex vivo erythropoiesis, and last, infection of terminally differentiated erythroblasts with P. falciparum. Because gene knockdowns that affect erythroid development could have potent yet nonspecific effects on parasites in this approach, we first screened the erythrocyte proteome so as to identify genes that influence
Mitochondrion
8 MAY 2015 • VOL 348 ISSUE 6235
7 11
Downloaded from www.sciencemag.org on August 24, 2015
MALARIA
R ES E A RC H | R E PO R TS
Fig. 2. Pooled shRNA screen to identify host determinants of P. falciparum infection. (A) Schematic of blood group shRNA screen. HPCs were transduced with pooled lentivirus library expressing 308 shRNAs targeting 42 blood group genes. Knockdown cRBCs infected with P. falciparum were sorted, and shRNAs were quantified by means of deep sequencing. (B) RIGER analysis ranking results for three independent experiments. Genes were ranked according to NES scores (green heat map). Arrows indicate top hits; asterisks indicate additional candidates.
1
Experiment:
*
6 4 4 2
0
0
D C sh
-1
-1
pT
44 D C
-1
-1
55 D C
D
44 sh
Em
pT C sh
55
2 2
-1
4
sh
CD44 KD CD55 KD
3 *
6
0
Control Unstained
2 * *
6
Em
shCD55-1
Parasitemia (%)
shCD44-1
*
8
10 µm
sh
Control
sh
Normalized to mode
*
D
Day 19
C
Day 14
sh
Day 9
pT
α-CD55
44
α-CD44
D
Quantify remaining shRNAs
Gene Rank > 20
C
FACS GFP cells
Mock infection
Gene Rank1
Em
24 hr
--------------------------------------------------
Parasite infection (24 hr)
P. falciparum infection
3
sh
Days 7-17: Selection and terminal differentiation
--------------------------------
Screen 2
sh
Day 6: Lentivirus infection
A4GALT ABCB6 * ABCG2 ABO ACHE AQP1 * AQP3 * ART4 B3GALNT1 * BCAM * BSG C1GALT1 C1GALT1C1 CD151 CD44 CD55 CD99 * CR1 DARC ERMAP FUT1 FUT2 FUT3 FUT6 FUT7 GCNT2 GYP A GYPB GYPC GYPE ICAM4 KEL Luciferase RHAG RHCE RHD SEC1 SEMA7A SLC14A1 SLC4A1 ST8SIA6 XG XK
1
shRNAs underrepresented in the differentiated erythroblasts would target genes important for erythropoiesis. The erythropoiesis screen yielded sufficient cells for full coverage of the library (>1500 cells per shRNA) (fig. S1B) (14), and deep sequencing of shRNA proviruses isolated from the terminally differentiated erythroid cells revealed a normal distribution (fig. S1C). Of shRNAs, 4.4% were depleted more than 50% in day-19 cRBCs relative to the original library pool, whereas 2% were enriched (Fig. 1B), indicating that these shRNAs can influence erythropoiesis. We ranked genes according to the depletion or enrichment of multiple shRNAs (15) and identified 116 candidates that grouped into categories relevant to terminal erythroid differentiation, including heme metabolism, protein turnover, and apoptosis (Fig. 1C, figs. S1D and S2, and data sets S2 and S3). We validated four top hits (fig. S3 and data set S4). This functional analysis of erythropoiesis provides a framework with which to study host determinants of malaria infection. To identify factors that influence host susceptibility to P. falciparum infection, we chose to focus on a small subset of the erythrocyte proteome: 42 genes encoding human blood groups. All known P. falciparum receptors fall within this group, and the shRNAs targeting these genes did not appear to affect erythroid development (Fig. 1D). Also, focusing on a small gene set increased the sensitivity to a level required for the inherently complex parasite screen (16). We transduced hematopoietic progenitor cells (HPCs) with a pooled lentivirus shRNA library targeting the blood group genes (Fig. 2A). At the late orthochromatic erythroblast stage, we infected the knockdown cells with a green fluorescent protein (GFP)–expressing line of P. falciparum strain 3D7. We isolated the parasitized cells and quantified the relative abundance of each shRNA in the population by means of deep sequencing (fig. S4). In parallel, we quantified the abundance of each shRNA in a control population of knockdown cRBCs not exposed to parasites. Hairpins
-1
Day 0: Initiate CD34+ cell culture
differentiate to the orthochromatic erythroblast stage (when parasite infection can occur) (13). After 19 days, we quantified each shRNA in the surviving orthochromatic erythroblasts relative to the original library, with the prediction that
55
Proliferation, transduction, and diferentiation
terminal differentiation of erythrocytes (Fig. 1A, fig. S1A, and data set S1). CD34+ hematopoietic stem cells (HSCs) were transduced with our pooled erythrocyte proteome short hairpin RNA (shRNA) library and induced to proliferate and
Fig. 3. Validation of CD44 and CD55 as host factors required for P. falciparum invasion. (A) CD44 and CD55 levels on day 19/20 cRBCs expressing CD44, CD55, or control shRNAs (EmpT). Detection was performed by means of antibody staining and flow cytometry. (B) Morphology of differentiating cRBCs depleted for CD44 and CD55, detected by means of May-Grünwald and Giemsa staining. (C) P. falciparum strain 3D7 invasion assays in control, CD44 knockdown, and CD55 knockdown cRBCs.Three independent biological replicates from two distinct bone marrow donors are shown. Mean T SD, n = 2 or 3 assays. *P < 0.05, onetailed t test.
7 12
8 MAY 2015 • VOL 348 ISSUE 6235
sciencemag.org SCIENCE
RE S EAR CH | R E P O R T S
Laboratory-adapted strains
p>0.05 p
