Pierrick Uzureau

Association of Trypanolytic ApoL1 Variants with Kidney Disease in African-Americans

Out of Africa Kidney disease is more common in African Americans than in Americans of European descent, and genetics is likely to be a major contributing factor. Genovese et al. (p. 841, published online 15 July) now show that African Americans who carry specific sequence variants in a gene on chromosome 22 encoding apolipoprotein L-1 (APOL1) have an increased risk of developing hypertension-attributed end-stage kidney disease or focal segmental glomerulosclerosis. These variants are absent from European chromosomes. Among the functions ascribed to APOL1 is the ability to lyse and kill trypanosomes. Intriguingly, APOL1 derived from the risk alleles, but not the “wild-type” allele, killed Trypanosoma brucei rhodesiense, which causes African sleeping sickness. Genetic variants associated with kidney disease in African Americans may confer protection against trypanosomes. African Americans have higher rates of kidney disease than European Americans. Here, we show that, in African Americans, focal segmental glomerulosclerosis (FSGS) and hypertension-attributed end-stage kidney disease (H-ESKD) are associated with two independent sequence variants in the APOL1 gene on chromosome 22 {FSGS odds ratio = 10.5 [95% confidence interval (CI) 6.0 to 18.4]; H-ESKD odds ratio = 7.3 (95% CI 5.6 to 9.5)}. The two APOL1 variants are common in African chromosomes but absent from European chromosomes, and both reside within haplotypes that harbor signatures of positive selection. ApoL1 (apolipoprotein L-1) is a serum factor that lyses trypanosomes. In vitro assays revealed that only the kidney disease–associated ApoL1 variants lysed Trypanosoma brucei rhodesiense. We speculate that evolution of a critical survival factor in Africa may have contributed to the high rates of renal disease in African Americans.

Mechanism of Trypanosoma brucei gambiense resistance to human serum

The African parasite Trypanosoma brucei gambiense accounts for 97% of human sleeping sickness cases. T. b. gambiense resists the specific human innate immunity acting against several other tsetse-fly-transmitted trypanosome species such as T. b. brucei, the causative agent of nagana disease in cattle. Human immunity to some African trypanosomes is due to two serum complexes designated trypanolytic factors (TLF-1 and -2), which both contain haptoglobin-related protein (HPR) and apolipoprotein LI (APOL1). Whereas HPR association with haemoglobin (Hb) allows TLF-1 binding and uptake via the trypanosome receptor TbHpHbR (ref. 5), TLF-2 enters trypanosomes independently of TbHpHbR (refs 4, 5). APOL1 kills trypanosomes after insertion into endosomal/lysosomal membranes. Here we report that T. b. gambiense resists TLFs via a hydrophobic β-sheet of the T. b. gambiense-specific glycoprotein (TgsGP), which prevents APOL1 toxicity and induces stiffening of membranes upon interaction with lipids. Two additional features contribute to resistance to TLFs: reduction of sensitivity to APOL1 requiring cysteine protease activity, and TbHpHbR inactivation due to a L210S substitution. According to such a multifactorial defence mechanism, transgenic expression of T. b. brucei TbHpHbR in T. b. gambiense did not cause parasite lysis in normal human serum. However, these transgenic parasites were killed in hypohaptoglobinaemic serum, after high TLF-1 uptake in the absence of haptoglobin (Hp) that competes for Hb and receptor binding. TbHpHbR inactivation preventing high APOL1 loading in hypohaptoglobinaemic serum may have evolved because of the overlapping endemic area of T. b. gambiense infection and malaria, the main cause of haemolysis-induced hypohaptoglobinaemia in western and central Africa.

Adenylate Cyclases of Trypanosoma brucei Inhibit the Innate Immune Response of the Host

Tricky Tryps African trypanosomes, responsible for human sleeping sickness, are known for their powerful strategies of immune evasion, in particular antigenic variation. Adding another facet to this adaptive potential, Salmon et al. (p. 463, published online 14 June; see the cover) now show that early after infection, these parasites subvert the first line of innate host defense by inhibiting tumor necrosis factor-α synthesis in myeloid cells. This occurs through the stress-induced synthesis and release of cyclic adenosine monophosphate by phagocytosed parasites. The findings provide a long-sought function for the abundant and diverse adenylate cyclases in salivarian trypanosomes. Furthermore, this altruistic host colonization strategy, in which a proportion of parasites are sacrificed so that others can thrive, also highlights the selective advantage of population behavior in infection. Parasites release cyclic adenosine monophosphate when swallowed up by myeloid cells, thereby turning off a host defense pathway. The parasite Trypanosoma brucei possesses a large family of transmembrane receptor–like adenylate cyclases. Activation of these enzymes requires the dimerization of the catalytic domain and typically occurs under stress. Using a dominant-negative strategy, we found that reducing adenylate cyclase activity by about 50% allowed trypanosome growth but reduced the parasite’s ability to control the early innate immune defense of the host. Specifically, activation of trypanosome adenylate cyclase resulting from parasite phagocytosis by liver myeloid cells inhibited the synthesis of the trypanosome-controlling cytokine tumor necrosis factor–α through activation of protein kinase A in these cells. Thus, adenylate cyclase activity of lyzed trypanosomes favors early host colonization by live parasites. The role of adenylate cyclases at the host-parasite interface could explain the expansion and polymorphism of this gene family.

The molecular arms race between African trypanosomes and humans

Humans can survive bloodstream infection by African trypanosomes, owing to the activity of serum complexes that have efficient trypanosome-killing ability. The two trypanosome subspecies that are responsible for human sleeping sickness — Trypanosoma brucei rhodesiense and Trypanosoma brucei gambiense — can evade this defence mechanism by expressing distinct resistance proteins. In turn, sequence variation in the gene that encodes the trypanosome-killing component in human serum has enabled populations in western Africa to restore resistance to T. b. rhodesiense, at the expense of the high probability of developing kidney sclerosis. These findings highlight the importance of resistance to trypanosomes in human evolution.

Cytokinesis of Trypanosoma brucei bloodstream forms depends on expression of adenylyl cyclases of the ESAG4 or ESAG4-like subfamily.

Antigenic variation of the parasite Trypanosoma brucei operates by monoallelic expression of a variant surface glycoprotein (VSG) from a collection of multiple telomeric expression sites (ESs). Each of these ESs harbours a long polycistronic transcription unit containing several expression site‐associated genes (ESAGs). ESAG4 copies encode bloodstream stage‐specific adenylyl cyclases (AC) and belong to a larger gene family of around 80 members, the majority of which, termed genes related to ESAG4 (GRESAG4s), are not encoded in ESs and are expressed constitutively in the life cycle. Here we report that ablation of ESAG4 from the active ES did not affect parasite growth, neither in culture nor upon rodent infection, and did not significantly change total AC activity. In contrast, inducible RNAi‐mediated knock‐down of an AC subfamily that includes ESAG4 and two ESAG4‐like GRESAG4 (ESAG4L) genes, decreased total AC activity and induced a lethal phenotype linked to impaired cytokinesis. In the Δesag4 line compensatory upregulation of apparently functionally redundant ESAG4L genes was observed, suggesting that the ESAG4/ESAG4L‐subfamily ACs are involved in the control of cell division. How deregulated adenylyl cyclases or cAMP might impair cytokinesis is discussed.

Characterization of a TFIIH homologue from Trypanosoma brucei.

Trypanosomes are protozoans showing unique transcription characteristics. We describe in Trypanosoma brucei a complex homologous to TFIIH, a multisubunit transcription factor involved in the control of transcription by RNA Pol I and RNA Pol II, but also in DNA repair and cell cycle control. Bioinformatics analyses allowed the detection of five genes encoding four putative core TFIIH subunits (TbXPD, TbXPB, Tbp44, Tbp52), including a novel XPB variant, TbXPBz. In all cases sequences known to be important for TFIIH functions were conserved. We performed a molecular analysis of this core complex, focusing on the two subunits endowed with a known enzymatic (helicase) activity, XPD and XPB. The involvement of these T. brucei proteins in a bona fide TFIIH core complex was supported by (i) colocalization by immunofluorescence in the nucleus, (ii) direct physical interaction of TbXPD and its interacting regulatory subunit Tbp44 as determined by double‐hybrid assay and tandem affinity purification of the core TFIIH, (iii) involvement of the core proteins in a high molecular weight complex and (iv) occurrence of transcription, cell cycle and DNA repair phenotypes upon either RNAi knock‐down or overexpression of essential subunits.

A Trypanosoma brucei Kinesin Heavy Chain Promotes Parasite Growth by Triggering Host Arginase Activity

Background In order to promote infection, the blood-borne parasite Trypanosoma brucei releases factors that upregulate arginase expression and activity in myeloid cells. Methodology/Principal findings By screening a cDNA library of T. brucei with an antibody neutralizing the arginase-inducing activity of parasite released factors, we identified a Kinesin Heavy Chain isoform, termed TbKHC1, as responsible for this effect. Following interaction with mouse myeloid cells, natural or recombinant TbKHC1 triggered SIGN-R1 receptor-dependent induction of IL-10 production, resulting in arginase-1 activation concomitant with reduction of nitric oxide (NO) synthase activity. This TbKHC1 activity was IL-4Rα-independent and did not mirror M2 activation of myeloid cells. As compared to wild-type T. brucei, infection by TbKHC1 KO parasites was characterized by strongly reduced parasitaemia and prolonged host survival time. By treating infected mice with ornithine or with NO synthase inhibitor, we observed that during the first wave of parasitaemia the parasite growth-promoting effect of TbKHC1-mediated arginase activation resulted more from increased polyamine production than from reduction of NO synthesis. In late stage infection, TbKHC1-mediated reduction of NO synthesis appeared to contribute to liver damage linked to shortening of host survival time. Conclusion A kinesin heavy chain released by T. brucei induces IL-10 and arginase-1 through SIGN-R1 signaling in myeloid cells, which promotes early trypanosome growth and favors parasite settlement in the host. Moreover, in the late stage of infection, the inhibition of NO synthesis by TbKHC1 contributes to liver pathogenicity.

Identification and characterization of two trypanosome TFIIS proteins exhibiting particular domain architectures and differential nuclear localizations.

Nuclear transcription of Trypanosoma brucei displays unusual features. Most protein‐coding genes are organized in large directional gene clusters, which are transcribed polycistronically by RNA polymerase II (pol II) with subsequent processing to generate mature mRNA. Here, we describe the identification and characterization of two trypanosome homologues of transcription elongation factor TFIIS (TbTFIIS1 and TbTFIIS2‐1). TFIIS has been shown to aid transcription elongation by relieving arrested pol II. Our phylogenetic analysis demonstrated the existence of four independent TFIIS expansions across eukaryotes. While TbTFIIS1 contains only the canonical domains II and III, the N‐terminus of TbTFIIS2‐1 contains a PWWP domain and a domain I. TbTFIIS1 and TbTFIIS2‐1 are expressed in procyclic and bloodstream form cells and localize to the nucleus in similar, but distinct, punctate patterns throughout the cell cycle. Neither TFIIS protein was enriched in the major pol II sites of spliced‐leader RNA transcription. Single RNA interference (RNAi)‐mediated knock‐down and knockout showed that neither protein is essential. Double knock‐down, however, impaired growth. Repetitive failure to generate a double knockout of TbTFIIS1 and TbTFIIS2‐1 strongly suggests synthetical lethality and thus an essential function shared by the two proteins in trypanosome growth.

Resistance to normal human serum reveals Trypanosoma lewisi as an underestimated human pathogen

Human-infectious trypanosomes such as Trypanosoma cruzi, T. brucei rhodesiense, and T. b. gambiense can be discriminated from those only infecting animals by their resistance to normal human serum (NHS). These parasites are naturally resistant to trypanolysis induced by the human-specific pore-forming serum protein apolipoprotein L1 (ApoL-1). T. lewisi, a worldwide distributed parasite, has been considered as rat-specific and non-pathogenic to the natural hosts. Here we provide evidence that 19 tested T. lewisi isolates from Thailand and China share resistance to NHS. Further investigation on one selected isolate CPO02 showed that it could resist at least 90% NHS or 30 μg/ml recombinant human ApoL-1 (rhApoL-1) in vitro, in contrast to T. b. brucei which could not survive in 0.0001% NHS and 0.1 μg/ml rhApoL-1. In vivo tests in rats also demonstrated that this parasite is fully resistant to lysis by NHS. Together with recent reports of atypical human infection by T. lewisi, these data allow the conclusion that T. lewisi is potentially an underestimated and thus a neglected human pathogen.

Quantitative analysis of platelets aggregates in 3D by digital holographic microscopy

Platelet spreading and retraction play a pivotal role in the platelet plugging and the thrombus formation. In routine laboratory, platelet function tests include exhaustive information about the role of the different receptors present at the platelet surface without information on the 3D structure of platelet aggregates. In this work, we develop, a method in Digital Holographic Microscopy (DHM) to characterize the platelet and aggregate 3D shapes using the quantitative phase contrast imaging. This novel method is suited to the study of platelets physiology in clinical practice as well as the development of new drugs.