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Haptoglobin is dispensable for haemoglobin uptake by Trypanosoma brucei

ABSTRACT: Trypanosoma brucei requires haemoglobin for survival in the mammalian bloodstream. Haptoglobin (Hp), a host protein known to bind haemoglobin, was determined as a key component for haemoglobin acquisition by the parasite. Here, we investigated the impact of haptoglobin deficiency on T. brucei brucei infection and the parasite´s capacity to internalise haemoglobin in the 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 have been observed between Hp-/- and control Black mice. Similarly, very few quantitative differences have been observed between the proteomes of parasites harvested from Hp-/- and Black mice, entailing 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. This was verified by western blotting, EM immunolabeling of parasites cryosections, and analytical quantification of haem cofactors in the parasites extracts. Combined, the data support the Hp dispensability for haemoglobin internalisation by T. b. brucei during infection in mice. To further explore this phenomenon, we have generated T. b. brucei haptoglobin-haemoglobin receptor (HpHbR) knock-outs and used them to infect Hp-/- and control mice. The HpHbR-knock-out trypanosomes internalised significantly less haemoglobin from Hp-/- mice compared to those isolated from Black mice strongly suggesting that T. b. brucei employs an HpHbR-independent Hp-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.

INSTRUMENT(S): Orbitrap Exploris 480

ORGANISM(S): Trypanosoma Brucei Brucei Treu927 Mus Musculus (mouse)

TISSUE(S): Blood Plasma

DISEASE(S): Trypanosomiasis

SUBMITTER: Marek Vrbacky

LAB HEAD: Jan Perner

PROVIDER: PXD052778 | Pride | 2024-07-16

REPOSITORIES: Pride

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Project description:Zinc finger and SCAN domain containing 10 (Zscan10) was identified as a novel transcription factor in osteoclast differentiation by our previous study. However, the biological functions of Zscan10 have not been fully understood except its role in maintenance of genome stability and pluripotency in embryonic stem cells. Therefore, the purpose of this study is clarification of Zscan10 function in somatic cells, especially during osteoclast differentiation. First, Zscan10 KO RAW264 (KO) cells were established using CRISPR/Cas9 system and single cell sorting. Then, Control (Ctrl) and KO cells were differentiated into osteoclast by RANKL stimulation. As a result, TRAP activity and expression levels of differentiation marker genes, such as Nfatc1, were significantly increased and the expression of inhibitory factors, such as Irf8, was decreased in KO cells as compared to Ctrl cells. These results suggested that Zscan10 might regulate transcription of the genes which negatively control osteoclastogenesis. To understand the transcriptomes controlled by Zscan10, RNA-seq was performed and the stringent analyses identified significantly down-regulated Haptoglobin (Hp) in KO cells. Additionally, Zscan10 binding sequence was located near the genomic region of Hp gene locus. ChIP against Zscan10 followed by qPCR for the region revealed that Zscan10 binds to the region located near Hp gene locus and transcript start site, suggesting that Zscan10 may regulate transcription of Hp. Next, to examine the effect of Hp under Zscan10 mediated osteoclastogenesis, KO cells were treated with recombinant Hp (rHp). As a result, Hp treatment could suppress the elevated TRAP activity of KO cells without affecting cell viability. Furthermore, Hp KO mice exhibit decreased bone mass and increased osteoclast number, and Hp has been reported to be involved in suppression of osteoclastogenesis. Also, In hemolytic disease, Hp had been decreased and also bone density. These facts suggested that Zscan10 negatively regulates osteoclast differentiation through transcription of Hp.

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Haptoglobin is dispensable for haemoglobin uptake by Trypanosoma brucei

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