Sickle-trait hemoglobin reduces adhesion to both CD36 and EPCR by Plasmodium falciparum-infected erythrocytes
Jens E V Petersen, Joseph W Saelens, Elizabeth Freedman, Louise Turner, Thomas Lavstsen, Rick M Fairhurst, Mahamadou Diakité, Steve M Taylor, Jens E V Petersen, Joseph W Saelens, Elizabeth Freedman, Louise Turner, Thomas Lavstsen, Rick M Fairhurst, Mahamadou Diakité, Steve M Taylor
Abstract
Sickle-trait hemoglobin protects against severe Plasmodium falciparum malaria. Severe malaria is governed in part by the expression of the Plasmodium falciparum Erythrocyte Membrane Protein 1 (PfEMP1) that are encoded by var genes, specifically those variants that bind Endothelial Protein C Receptor (EPCR). In this study, we investigate the effect of sickle-trait on parasite var gene expression and function in vitro and in field-collected parasites. We mapped var gene reads generated from RNA sequencing in parasite cultures in normal and sickle-cell trait blood throughout the asexual lifecycle. We investigated sickle-trait effect on PfEMP1 interactions with host receptors CD36 and EPCR using static adhesion assays and flow cytometry. Var expression in vivo was compared by assembling var domains sequenced from total RNA in parasites infecting Malian children with HbAA and HbAS. Sickle-trait did not alter the abundance or type of var gene transcripts in vitro, nor the abundance of overall transcripts or of var functional domains in vivo. In adhesion assays using recombinant host receptors, sickle-trait reduced adhesion by 73-86% to CD36 and 83% to EPCR. Similarly, sickle-trait reduced the surface expression of EPCR-binding PfEMP1. In conclusion, Sickle-cell trait does not directly affect var gene transcription but does reduce the surface expression and function of PfEMP1. This provides a direct mechanism for protection against severe malaria conferred by sickle-trait hemoglobin. Trial Registration: ClinicalTrials.gov Identifier: NCT02645604.
Conflict of interest statement
The authors have declared that no competing interests exist.
Figures
References
- Taylor SM, Parobek CM, Fairhurst RM. Haemoglobinopathies and the clinical epidemiology of malaria: a systematic review and meta-analysis. Lancet Infect Dis. 2012;12: 457–468. doi: 10.1016/S1473-3099(12)70055-5
- Archer NM, Petersen N, Clark MA, Buckee CO, Childs LM, Duraisingh MT. Resistance to Plasmodium falciparum in sickle cell trait erythrocytes is driven by oxygen-dependent growth inhibition. Proc Natl Acad Sci. 2018;115: 7350–7355. doi: 10.1073/pnas.1804388115
- Friedman MJ. Oxidant damage mediates variant red cell resistance to malaria. Nature. 1979. doi: 10.1038/280245a0
- Pasvol G, Weatherall DJ, Wilson RJM. Cellular mechanism for the protective effect of haemoglobin S against P. falciparum malaria [22]. Nature. 1978. doi: 10.1038/274701a0
- Cyrklaff M, Srismith S, Nyboer B, Burda K, Hoffmann A, Lasitschka F, et al.. Oxidative insult can induce malaria-protective trait of sickle and fetal erythrocytes. Nat Commun. 2016. doi: 10.1038/ncomms13401
- Waldecker M, Dasanna AK, Lansche C, Linke M, Srismith S, Cyrklaff M, et al.. Differential time-dependent volumetric and surface area changes and delayed induction of new permeation pathways in P. falciparum-infected hemoglobinopathic erythrocytes. Cell Microbiol. 2017. doi: 10.1111/cmi.12650
- Cholera R, Brittain NJ, Gillrie MR, Lopera-Mesa TM, Diakite SAS, Arie T, et al.. Impaired cytoadherence of Plasmodium falciparum-infected erythrocytes containing sickle hemoglobin. Proc Natl Acad Sci. 2008;105: 991–996. doi: 10.1073/pnas.0711401105
- Cyrklaff M, Sanchez CP, Kilian N, Bisseye C, Simpore J, Frischknecht F, et al.. Hemoglobins S and C interfere with actin remodeling in Plasmodium falciparum-infected erythrocytes. Science (80-). 2011;334: 1283–1286. doi: 10.1126/science.1213775
- Opi DH, Ochola LB, Tendwa M, Siddondo BR, Ocholla H, Fanjo H, et al.. Mechanistic studies of the negative epistatic malaria-protective interaction between sickle cell trait and α+thalassemia. EBioMedicine. 2014. doi: 10.1016/j.ebiom.2014.10.006
- Lansche C, Dasanna AK, Quadt K, Fröhlich B, Missirlis D, Tétard M, et al.. The sickle cell trait affects contact dynamics and endothelial cell activation in Plasmodium falciparum-infected erythrocytes. Commun Biol. 2018. doi: 10.1038/s42003-018-0223-3
- Rask TS, Hansen DA, Theander TG, Gorm Pedersen A, Lavstsen T. Plasmodium falciparum erythrocyte membrane protein 1 diversity in seven genomes—divide and conquer. PLoS Comput Biol. 2010;6: e1000933. doi: 10.1371/journal.pcbi.1000933
- Lavstsen T, Salanti A, Jensen ATR, Arnot DE, Theander TG. Sub-grouping of Plasmodium falciparum 3D7 var genes based on sequence analysis of coding and non-coding regions. Malar J. 2003;2: 27. doi: 10.1186/1475-2875-2-27
- Roberts DJ, Craig AG, Berendt AR, Pinches R, Nash G, Marsh K, et al.. Rapid switching to multiple antigenic and adhesive phenotypes in malaria. Nature. 1992. doi: 10.1038/357689a0
- Salanti A, Staalsoe T, Lavstsen T, Jensen ATR, Sowa MPK, Arnot DE, et al.. Selective upregulation of a single distinctly structured var gene in chondroitin sulphate A-adhering Plasmodium falciparum involved in pregnancy-associated malaria. Mol Microbiol. 2003;49: 179–191. doi: 10.1046/j.1365-2958.2003.03570.x
- Jespersen JS, Wang CW, Mkumbaye SI, Minja DT, Petersen B, Turner L, et al.. Plasmodium falciparum var genes expressed in children with severe malaria encode CIDRα1 domains. EMBO Mol Med. 2016;8: 839–850. doi: 10.15252/emmm.201606188
- Bernabeu M, Danziger SA, Avril M, Vaz M, Babar PH, Brazier AJ, et al.. Severe adult malaria is associated with specific PfEMP1 adhesion types and high parasite biomass. Proc Natl Acad Sci U S A. 2016. [cited 25 May 2016]. doi: 10.1073/pnas.1524294113
- Turner L, Lavstsen T, Berger SS, Wang CW, Petersen JE V, Avril M, et al.. Severe malaria is associated with parasite binding to endothelial protein C receptor. Nature. 2013;498: 502–5. doi: 10.1038/nature12216
- Lavstsen T, Turner L, Saguti F, Magistrado P, Rask TS, Jespersen JS, et al.. Plasmodium falciparum erythrocyte membrane protein 1 domain cassettes 8 and 13 are associated with severe malaria in children. Proc Natl Acad Sci. 2012;109: E1791–E1800. doi: 10.1073/pnas.1120455109
- Sanchez CP, Karathanasis C, Sanchez R, Cyrklaff M, Jäger J, Buchholz B, et al.. Single-molecule imaging and quantification of the immune-variant adhesin VAR2CSA on knobs of Plasmodium falciparum-infected erythrocytes. Commun Biol. 2019. doi: 10.1038/s42003-019-0429-z
- Dahlbäck M, Lavstsen T, Salanti A, Hviid L, Arnot DE, Theander TG, et al.. Changes in var gene mRNA levels during erythrocytic development in two phenotypically distinct Plasmodium falciparum parasites. Malar J. 2007;6: 78. doi: 10.1186/1475-2875-6-78
- Gölnitz U, Albrecht L, Wunderlich G. Var transcription profiling of Plasmodium falciparum 3D7: assignment of cytoadherent phenotypes to dominant transcripts. Malar J. 2008;7: 14. doi: 10.1186/1475-2875-7-14
- Hsieh F-L, Turner L, Bolla JR, Robinson C V., Lavstsen T, Higgins MK. The structural basis for CD36 binding by the malaria parasite. Nat Commun. 2016;7: 12837. doi: 10.1038/ncomms12837
- Hempel C, Wang CW, Kurtzhals JAL, Staalsø T. Binding of Plasmodium falciparum to CD36 can be shielded by the glycocalyx. Malar J. 2017;16: 193. doi: 10.1186/s12936-017-1844-6
- Duffy F, Bernabeu M, Babar PH, Kessler A, Wang CW, Vaz M, et al.. Meta-analysis of plasmodium falciparum var signatures contributing to severe Malaria in African children and Indian adults. MBio. 2019. doi: 10.1128/mBio.00217-19
- Robinson BA, Welch TL, Smith JD. Widespread functional specialization of Plasmodium falciparum erythrocyte membrane protein 1 family members to bind CD36 analysed across a parasite genome. Mol Microbiol. 2003. doi: 10.1046/j.1365-2958.2003.03378.x
- Bouwens EAM, Stavenuiter F, Mosnier LO. Mechanisms of anticoagulant and cytoprotective actions of the protein C pathway. Journal of Thrombosis and Haemostasis. 2013. pp. 242–253. doi: 10.1111/jth.12247
- Claessens A, Adams Y, Ghumra A, Lindergard G, Buchan CC, Andisi C, et al.. A subset of group A-like var genes encodes the malaria parasite ligands for binding to human brain endothelial cells. Proc Natl Acad Sci U S A. 2012. doi: 10.1073/pnas.1120461109
- Avril M, Tripathi AKK, Brazier AJJ, Andisi C, Janes JHH, Soma VLL, et al.. A restricted subset of var genes mediates adherence of Plasmodium falciparum-infected erythrocytes to brain endothelial cells. Proc Natl Acad Sci. 2012;109: E1782–E1790. doi: 10.1073/pnas.1120534109
- Gillrie MR, Avril M, Brazier AJ, Davis SP, Stins MF, Smith JD, et al.. Diverse functional outcomes of Plasmodium falciparum ligation of EPCR: Potential implications for malarial pathogenesis. Cell Microbiol. 2015;17: 1883–1899. doi: 10.1111/cmi.12479
- Petersen JE V, Bouwens EAM, Tamayo I, Turner L, Wang CW, Stins M, et al.. Protein C system defects inflicted by the malaria parasite protein PfEMP1 can be overcome by a soluble EPCR variant. Thromb Haemost. 2015;114: 1038–1048. doi: 10.1160/TH15-01-0018
- Lau CKY, Turner L, Jespersen JS, Lowe ED, Petersen B, Wang CW, et al.. Structural conservation despite huge sequence diversity allows EPCR binding by the pfemp1 family implicated in severe childhood malaria. Cell Host Microbe. 2015;17: 118–129. doi: 10.1016/j.chom.2014.11.007
- Dorovini-Zis K, Schmidt K, Huynh H, Fu W, Whitten RO, Milner D, et al.. The neuropathology of fatal cerebral malaria in Malawian children. Am J Pathol. 2011;178: 2146–2158. doi: 10.1016/j.ajpath.2011.01.016
- Milner DA, Whitten RO, Kamiza S, Carr R, Liomba G, Dzamalala C, et al.. The systemic pathology of cerebral malaria in African children. Front Cell Infect Microbiol. 2014;4: 104. doi: 10.3389/fcimb.2014.00104
- Kessler A, Dankwa S, Bernabeu M, Harawa V, Danziger SA, Duffy F, et al.. Linking EPCR-Binding PfEMP1 to Brain Swelling in Pediatric Cerebral Malaria. Cell Host Microbe. 2017. doi: 10.1016/j.chom.2017.09.009
- Silverstein RL, Febbraio M. CD36, a scavenger receptor involved in immunity, metabolism, angiogenesis, and behavior. Science Signaling. 2009. doi: 10.1126/scisignal.272re3
- Subramani R, Quadt K, Jeppesen AE, Hempel C, Vang Petersen JE, Hassenkam T, et al.. Plasmodium Falciparum-infected erythrocyte knob density is linked to the pfemp1 variant expressed. MBio. 2015. doi: 10.1128/mBio.01456-15
- Langreth SG, Reese RT, Motyl MR, Trager W. Plasmodium falciparum: Loss of knobs on the infected erythrocyte surface after long-term cultivation. Exp Parasitol. 1979. doi: 10.1016/0014-4894(79)90101-2
- Quadt KA, Barfod L, Andersen D, Bruun J, Gyan B, Hassenkam T, et al.. The Density of Knobs on Plasmodium falciparum-Infected Erythrocytes Depends on Developmental Age and Varies among Isolates. PLoS One. 2012;7. doi: 10.1371/journal.pone.0045658
- Kilian N, Dittmer M, Cyrklaff M, Ouermi D, Bisseye C, Simpore J, et al.. Haemoglobin s and c affect the motion of maurer’s clefts in plasmodium falciparum-infected erythrocytes. Cell Microbiol. 2013. doi: 10.1111/cmi.12102
- Mkumbaye SI, Wang CW, Lyimo E, Jespersen JS, Manjurano A, Mosha J, et al.. The severity of Plasmodium falciparum infection is associated with transcript levels of var genes encoding endothelial protein C receptor-binding P. falciparum erythrocyte membrane protein 1. Infect Immun. 2017. doi: 10.1128/IAI.00841-16
- Tonkin-Hill GQ, Trianty L, Noviyanti R, Nguyen HHT, Sebayang BF, Lampah DA, et al.. The Plasmodium falciparum transcriptome in severe malaria reveals altered expression of genes involved in important processes including surface antigen–encoding var genes. PLoS Biol. 2018. doi: 10.1371/journal.pbio.2004328
- Cranmer SL, Magowan C, Liang J, Coppel RL, Cooke BM. An alternative to serum for cultivation of Plasmodium falciparum in vitro. Trans R Soc Trop Med Hyg. 1997;91: 363–5. Available: doi: 10.1016/s0035-9203(97)90110-3
- Lopera-Mesa TM, Doumbia S, Konaté D, Anderson JM, Doumbouya M, Keita AS, et al.. Effect of red blood cell variants on childhood malaria in Mali: A prospective cohort study. Lancet Haematol. 2015;2: e140–e149. doi: 10.1016/S2352-3026(15)00043-5
- Venkatesan M, Amaratunga C, Campino S, Auburn S, Koch O, Lim P, et al.. Using CF11 cellulose columns to inexpensively and effectively remove human DNA from Plasmodium falciparum-infected whole blood samples. Malar J. 2012. doi: 10.1186/1475-2875-11-41
- Wang CW, Lavstsen T, Bengtsson DC, Magistrado PA, Berger SS, Marquard AM, et al.. Evidence for in vitro and in vivo expression of the conserved VAR3 (type 3) plasmodium falciparum erythrocyte membrane protein 1. Malar J. 2012;11: 129. doi: 10.1186/1475-2875-11-129
- Bolger AM, Lohse M, Usadel B. Trimmomatic: A flexible trimmer for Illumina sequence data. Bioinformatics. 2014. doi: 10.1093/bioinformatics/btu170
- Patro R, Duggal G, Love MI, Irizarry RA, Kingsford C. Salmon provides fast and bias-aware quantification of transcript expression. Nat Methods. 2017. doi: 10.1038/nmeth.4197
- Soneson C, Love MI, Robinson MD. Differential analyses for RNA-seq: transcript-level estimates improve gene-level inferences. F1000Research. 2015. doi: 10.12688/f1000research.7563.2
- Li H, Handsaker B, Wysoker A, Fennell T, Ruan J, Homer N, et al.. The Sequence Alignment/Map format and SAMtools. Bioinformatics. 2009. doi: 10.1093/bioinformatics/btp352
- Pehrson C, Heno KK, Adams Y, Resende M, Mathiesen L, Soegaard M, et al.. Comparison of functional assays used in the clinical development of a placental malaria vaccine. Vaccine. 2017. doi: 10.1016/j.vaccine.2016.12.028
- Schindelin J, Arganda-Carreras I, Frise E, Kaynig V, Longair M, Pietzsch T, et al.. Fiji: an open-source platform for biological-image analysis. Nat Methods. 2012;9: 676–682. doi: 10.1038/nmeth.2019
- Ritz C, Baty F, Streibig JC, Gerhard D. Dose-response analysis using R. PLoS One. 2015. doi: 10.1371/journal.pone.0146021
- Ellis B, Haaland P, Hahne F, Meur N Le, Gopalakrishnan N, Spidlen J, et al.. FlowCore: Basic structures for flow cytometry data. R Packag version 201. 2020.
- Sarkar D, Le meur N, Gentleman R. Using flowViz to visualize flow cytometry data. Bioinformatics. 2008. doi: 10.1093/bioinformatics/btn021
- Hahne F, Gopalakrishnan N, Khodabakhshi A, Wong C, Lee K. flowStats: Statistical methods for the analysis of flow cytometry data. R Packag version 420. 2020.
- Phu V, Jiang W, Gottardo R, Finak G. GgCyto: Next generation open-source visualization software for cytometry. Bioinformatics. 2018. doi: 10.1093/bioinformatics/bty441
Source: PubMed