Haptoglobin is a plasma protein of mammals that plays a crucial role in vascular homeostasis by binding free haemoglobin released from ruptured red blood cells. Trypanosoma brucei can exploit this by internalising haptoglobin-haemoglobin complex to acquire host haem. Here, we investigated the impact of haptoglobin deficiency (Hp-/-) on T. brucei brucei infection and the parasite´s capacity to internalise haemoglobin in a Hp-/- mouse model. The infected Hp-/- mice exhibited normal disease progression, with minimal weight loss and no apparent organ pathology, similarly to control mice. While the proteomic profile of mouse sera significantly changed in response to T. b. brucei, no differences in the infection response markers of blood plasma between Hp-/- and control Black mice were observed. Similarly, very few quantitative differences were observed between the proteomes of parasites harvested from Hp-/- and Black mice, including both endogenous proteins and internalised host proteins. While haptoglobin was indeed absent from parasites isolated from Hp-/-mice, haemoglobin peptides were unexpectedly detected in parasites from both Hp-/- and Black mice. Combined, the data support the dispensability of haptoglobin for haemoglobin internalisation by T. b. brucei during infection in mice. Since the trypanosomes knock-outs for their haptoglobin-haemoglobin receptor (HpHbR) internalised significantly less haemoglobin from Hp-/- mice compared to those isolated from Black mice, it suggests that T. b. brucei employs also an HpHbR-independent haptoglobin-mediated mode for haemoglobin internalisation. Our study reveals a so-far hidden flexibility of haemoglobin acquisition by T. b. brucei and offers novel insights into alternative haemoglobin uptake pathways.

Centre Algatech Institute of Microbiology Czech Academy of Sciences Třeboň Czechia

Faculty of Science University of South Bohemia České Budějovice Czechia

Institute of Parasitology Biology Centre Czech Academy of Sciences České Budějovice Czechia

Institute of Physiology Czech Academy of Sciences Prague Czechia

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Lukeš J, Kachale A, Votýpka J, Butenko A, Field MC. African trypanosome strategies for conquering new hosts and territories: the end of monophyly? Trends Parasitol. (2022) 38:724–36. doi: 10.1016/j.pt.2022.05.011 PubMed DOI

Kořený L, Oborník M, Lukeš J. Make it, take it, or leave it: heme metabolism of parasites. PloS Pathog. (2013) 9:e1003088. doi: 10.1371/journal.ppat.1003088 PubMed DOI PMC

Cenci U, Moog D, Curtis BA, Tanifuji G, Eme L, Lukeš J, et al. . Heme pathway evolution in kinetoplastid protists. BMC Evolutionary Biol. (2016) 16:109. doi: 10.1186/s12862-016-0664-6 PubMed DOI PMC

Tripodi KEJ, Menendez Bravo SM, Cricco JA. Role of heme and heme-proteins in trypanosomatid essential metabolic pathways. Enzyme Research 2011. (2011) p:1–12. doi: 10.4061/2011/873230 PubMed DOI PMC

Widener J, Nielsen MJ, Shiflett A, Moestrup S, Hajduk S. Haemoglobin is a co-factor of human trypanosome lytic factor. PloS Pathog. (2007) 3:e129. doi: 10.1371/journal.ppat.0030129 PubMed DOI PMC

Higgins MK, Lane-Serff H, MacGregor P, Carrington M. A receptor’s tale: an eon in the life of a trypanosome receptor. PloS Pathog. (2017) 13:e1006055. doi: 10.1371/journal.ppat.1006055 PubMed DOI PMC

Higgins MK, Tkachenko O, Brown A, Reed J, Raper J, Carrington M. Structure of the trypanosome haptoglobin–haemoglobin receptor and implications for nutrient uptake and innate immunity. Proc Natl Acad Sci. (2013) 110:1905–10. doi: 10.1073/pnas.1214943110 PubMed DOI PMC

Vanhollebeke B, De Muylder G, Nielsen MJ, Pays A, Tebabi P, Dieu M. A haptoglobin-haemoglobin receptor conveys innate immunity to trypanosoma brucei in humans. Science. (2008) 320:677–81. doi: 10.1126/science.1156296 PubMed DOI

Lane-Serff H, MacGregor P, Peacock L, Macleod OJS, Kay C, Gibson W, et al. . Evolutionary diversification of the trypanosome haptoglobin-haemoglobin receptor from an ancestral haemoglobin receptor. eLife. (2016) 5:e13044. doi: 10.7554/eLife.13044 PubMed DOI PMC

Kristiansen M, Graversen JH, Jacobsen C, Sonne O, Hoffman H, Law SKA, et al. . Identification of the haemoglobin scavenger receptor. Nature. (2001) 409:198–201. doi: 10.1038/35051594 PubMed DOI

Etzerodt A, Kjolby M, Nielsen MJ, Maniecki MB, Svendsen P, Moestrup SK, et al. . Plasma clearance of haemoglobin and haptoglobin in mice and effect of CD163 gene targeting disruption. Antioxidants Redox Signaling. (2013) 18:2254–63. doi: 10.1089/ars.2012.4605 PubMed DOI

Changmai P, Horáková E, Long S, Černotíková-Stříbrná E, McDonald LM, Bontempi EJ, et al. . Both human ferredoxins equally efficiently rescue ferredoxin deficiency in Trypanosoma brucei . Mol Microbiol. (2013) 89:135–51. doi: 10.1111/mmi.12264 PubMed DOI

Puustinen A, Wikström M. The heme groups of cytochrome o from Escherichia coli. Proc Natl Acad Sci. (1991) 88:6122–6. doi: 10.1073/pnas.88.14.6122 PubMed DOI PMC

Horáková E, Lecordier L, Cunha P, Sobotka R, Changmai P, Langedijk CJM, et al. . Heme-deficient metabolism and impaired cellular differentiation as an evolutionary trade-off for human infectivity in Trypanosoma brucei gambiense. Nat Commun. (2022) 13:7075. doi: 10.1038/s41467-022-34501-4 PubMed DOI PMC

Watanabe J, Chou KJ, Liao JC, Miao Y, Meng H-H, Ge H, et al. . Differential association of haemoglobin with proinflammatory high density lipoproteins in atherogenic/hyperlipidemic mice. J Biol Chem. (2007) 282:23698–707. doi: 10.1074/jbc.M702163200 PubMed DOI

Stødkilde K, Torvund-Jensen M, Moestrup SK, Andersen CBF. Structural basis for trypanosomal haem acquisition and susceptibility to the host innate immune system. Nat Commun. (2014) 5:5487. doi: 10.1038/ncomms6487 PubMed DOI

Pamir N, Pan C, Plubell DL, Hutchins PM, Tang C, Wimberger J, et al. . Genetic control of the mouse HDL proteome defines HDL traits, function, and heterogeneity. J Lipid Res. (2019) 60:594–608. doi: 10.1194/jlr.M090555 PubMed DOI PMC

Watanabe J, Grijalva V, Hama S, Barbour K, Berger FG, Navab M, et al. . Haemoglobin and its scavenger protein haptoglobin associate with apoA-1-containing particles and influence the inflammatory properties and function of high density lipoprotein. J Biol Chem. (2009) 284:18292–301. doi: 10.1074/jbc.M109.017202 PubMed DOI PMC

Du R, Winarsih I, Ho B, Ding JL. Lipid-free apolipoprotein A-I exerts an antioxidative role against cell-free haemoglobin. Am J Clin Exp Immunol. (2012) 1:33–48. PubMed PMC

Vanhollebeke B, Uzureau P, Monteyne D, Pérez-Morga D, Pays E. Cellular and Molecular Remodeling of the Endocytic Pathway during Differentiation of Trypanosoma brucei Bloodstream Forms. Eukaryotic Cell. (2010) 9:1272–82. doi: 10.1128/EC.00076-10 PubMed DOI PMC

Vanhollebeke B, Pays E. The trypanolytic factor of human serum: many ways to enter the parasite, a single way to kill. Mol Microbiol. (2010) 76:806–14. doi: 10.1111/j.1365-2958.2010.07156.x PubMed DOI

Pays E, Vanhollebeke B, Uzureau P, Lecordier L, Pérez-Morga D. The molecular arms race between African trypanosomes and humans. Nat Rev Microbiol. (2014) 12:575–84. doi: 10.1038/nrmicro3298 PubMed DOI

Barker C, Barbour KW, Berger FG, Hajduk SL. Activity of human trypanosome lytic factor in mice. Mol Biochem Parasitol. (2001) 117:129–36. doi: 10.1016/S0166-6851(01)00339-5 PubMed DOI

Ramos S, Jeney V, Figueiredo A, Paixão T, Sambo MR, Quinhentos V, et al. . Targeting circulating labile heme as a defense strategy against malaria. Life Sci Alliance. (2024) 7:e202302276. doi: 10.26508/lsa.202302276 PubMed DOI PMC

Horáková E, Changmai P, Vancová M, Sobotka R, Abbeele JVD, Vanhollebeke B, et al. . The Trypanosoma brucei TbHrg protein is a heme transporter involved in the regulation of stage-specific morphological transitions. J Biol Chem. (2017) 292:6998–7010. doi: 10.1074/jbc.M116.762997 PubMed DOI PMC

Perner J, Hatalova T, Cabello-Donayre M, Urbanova V, Sojka D, Frantova H, et al. . Haem-responsive gene transporter enables mobilization of host haem in ticks. Open Biol. (2021) 11:210048. doi: 10.1098/rsob.210048 PubMed DOI PMC

Cabello-Donayre M, Orrego LM, Herráez E, García-Hernández R, Pérez-Victoria JM. New insights on heme uptake in leishmania spp. Int J Mol Sci. (2022) 23:10501. doi: 10.3390/ijms231810501 PubMed DOI PMC

Swenson SA, Moore CM, Marcero JR, Medlock AE, Reddi AR, Khalimonchuk O, et al. . From synthesis to utilization: the ins and outs of mitochondrial heme. Cells. (2020) 9:579. doi: 10.3390/cells9030579 PubMed DOI PMC

Acestor N, Zíková A, Dalley RA, Anupama A, Panigrahi AK, Stuart KD, et al. . Trypanosoma brucei mitochondrial respiratome: composition and organization in procyclic form. Mol Cell Proteomics. (2011) 10:M110.006908. doi: 10.1074/mcp.M110.006908 PubMed DOI PMC

Peacock AC, Zíková A, Dalley RA, Anupama A, Panigrahi AK, Stuart KD, et al. . Haptoglobin levels in serum of various strains of mice. Science. (1967) 158:1703–4. doi: 10.1126/science.158.3809.1703 PubMed DOI

Madsen M, Gelderman AH, Ragland RH, HA, HoffmaN. Molecular characterization of the haptoglobin·Haemoglobin receptor CD163. J Biol Chem. (2004) 279:51561–7. doi: 10.1074/jbc.M409629200 PubMed DOI

Moestrup S, Møller H. CD163: a regulated haemoglobin scavenger receptor with a role in the anti-inflammatory response. Ann Med. (2009) 36:347–54. doi: 10.1080/07853890410033171 PubMed DOI

Calomeno NA, Moreira RS, Fernandes LA, Batista F, Marques J, Wagner G, et al. . Serum proteomic signature of Trypanosoma evansi –infected mice for identification of potential biomarkers. Veterinary Parasitol. (2021) 290:109342. doi: 10.1016/j.vetpar.2021.109342 PubMed DOI

Huntoon KM, Rybchyn MS, Easterbrook-Smith SB, Henriques C, Wilson MR. The acute phase protein haptoglobin regulates host immunity. J Leukocyte Biol. (2008) 84:170–81. doi: 10.1189/jlb.0208100 PubMed DOI PMC

Huntoon KM, Russell L, Tracy E, Barbour KW, Li Q, Shrikant PA, et al. . A unique form of haptoglobin produced by murine hematopoietic cells supports B-cell survival, differentiation and immune response. Mol Immunol. (2013) 55:345–54. doi: 10.1016/j.molimm.2013.03.008 PubMed DOI PMC

Lipschitz DA, Allegre A, Cook JD. The clinical significance of ferritinuria. Blood. (1980) 55:260–4. doi: 10.1182/blood.V55.2.260.260 PubMed DOI

Balla J, Vercellotti GM, Jeney V, Yachie A, Varga Z, Jacob HS, et al. . Heme, heme oxygenase, and ferritin: how the vascular endothelium survives (and dies) in an iron-rich environment. Antioxidants Redox Signaling. (2007) 9:2119–38. doi: 10.1089/ars.2007.1787 PubMed DOI

Liu F-T, Stowell SR. The role of galectins in immunity and infection. Nat Rev Immunol. (2023) 23:479–94. doi: 10.1038/s41577-022-00829-7 PubMed DOI PMC

Rodriguez A, García GA, Bua J, Cerliani JP, Postan M, Tasso LM, et al. . Galectin-1 prevents infection and damage induced by trypanosoma cruzi on cardiac cells. PloS Negl Trop Dis. (2015) 9:e0004148. doi: 10.1371/journal.pntd.0004148 PubMed DOI PMC

Sack GH. Serum Amyloid A (SAA) Proteins. Springer: Subcellular Biochemistry, Vol. 94. (2020). pp. 421–36. PubMed

Noborn F, Ancsin JB, Ubhayasekera W, Kisilevsky R, Li J-P. Heparan sulfate dissociates serum amyloid A (SAA) from acute-phase high-density lipoprotein, promoting SAA aggregation. J Biol Chem. (2012) 287:25669–77. doi: 10.1074/jbc.M112.363895 PubMed DOI PMC

Johnston HE, Yadav K, Kirkpatrick JM, Biggs GS, Oxley D, Kramer HB, et al. . Solvent precipitation SP3 (SP4) enhances recovery for proteomics sample preparation without magnetic beads. Analytical Chem. (2022) 94:10320–8. doi: 10.1021/acs.analchem.1c04200 PubMed DOI PMC