Didier Salmon

The genes and transcripts of an antigen gene expression site from T. brucei

The AnTat 1.3A antigen gene expression site of T. brucei was cloned from genomic libraries of the 200 kb expressor chromosome. In addition to the antigen gene, it contains seven putative coding regions (ESAGs, for expression site-associated genes), as well as a RIME retroposon. The polypeptide encoded by ESAG 4 shows homology to yeast adenylate cyclase, and possesses structural features of a transmembrane protein. The expression site is transcribed by a pol l-like polymerase in the parasite bloodstream form only, but sequences similar to ESAGs 5, 4, and 2 are also transcribed constitutively elsewhere, by a polymerase sensitive to alpha-amanitin. Ultraviolet irradiation, which seems to block RNA processing, allows the tentative mapping of a transcription promoter about 45 kb upstream of the antigen gene.

A novel heterodimeric transferrin receptor encoded by a pair of VSG expression site-associated genes in T. brucei

In T. brucei, a transferrin-binding protein has been found to share sequence homology with pESAG-7 and -6, the products of two related genes present in the VSG gene polycistronic transcription unit. When expressed in Xenopus oocytes, they appear as N-glycosylated proteins secreted in the medium (pESAG-7) and GPI anchored to the membrane (pESAG-6). These proteins are able to homo- or heterodimerize, probably through association in the same orientation. Only heterodimers can bind Tf, possibly two molecules per dimer. A comparison of Tf binding to pESAG-7/6-expressing oocytes and trypanosomes suggests that pESAG-7/6 is the Tf receptor of the parasite. In trypanosomes, the majority of pESAG-7/6 is released from the membrane and associates, together with Tf, with a glycosylated matrix present in the lumen of the flagellar pocket. Both pESAG-7/6 and Tf are internalized via coated pits and vesicles. These observations suggest a novel mode of Tf binding and uptake in trypanosomes.

Characterization of the ligand‐binding site of the transferrin receptor in Trypanosoma brucei demonstrates a structural relationship with the N‐terminal domain of the variant surface glycoprotein

The Trypanosoma brucei transferrin (Tf) receptor is a heterodimer encoded by ESAG7 and ESAG6, two genes contained in the different polycistronic transcription units of the variant surface glycoprotein (VSG) gene. The sequence of ESAG7/6 differs slightly between different units, so that receptors with different affinities for Tf are expressed alternatively following transcriptional switching of VSG expression sites during antigenic variation of the parasite. Based on the sequence homology between pESAG7/6 and the N‐terminal domain of VSGs, it can be predicted that the four blocks containing the major sequence differences between pESAG7 and pESAG6 form surface‐exposed loops and generate the ligand‐binding site. The exchange of a few amino acids in this region between pESAG6s encoded by different VSG units greatly increased the affinity for bovine Tf. Similar changes in other regions were ineffective, while mutations predicted to alter the VSG‐like structure abolished the binding. Chimeric proteins containing the N‐terminal dimerization domain of VSG and the C‐terminal half of either pESAG7 or pESAG6, which contains the ligand‐binding domain, can form heterodimers that bind Tf. Taken together, these data provided evidence that the T.brucei Tf receptor is structurally related to the N‐terminal domain of the VSG and that the ligand‐binding site corresponds to the exposed surface loops of the protein.

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.

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.

Monitoring of the Parasite Load in the Digestive Tract of Rhodnius prolixus by Combined qPCR Analysis and Imaging Techniques Provides New Insights into the Trypanosome Life Cycle

Background Here we report the monitoring of the digestive tract colonization of Rhodnius prolixus by Trypanosoma cruzi using an accurate determination of the parasite load by qPCR coupled with fluorescence and bioluminescence imaging (BLI). These complementary methods revealed critical steps necessary for the parasite population to colonize the insect gut and establish vector infection. Methodology/Principal Findings qPCR analysis of the parasite load in the insect gut showed several limitations due mainly to the presence of digestive-derived products that are thought to degrade DNA and inhibit further the PCR reaction. We developed a real-time PCR strategy targeting the T. cruzi repetitive satellite DNA sequence using as internal standard for normalization, an exogenous heterologous DNA spiked into insect samples extract, to precisely quantify the parasite load in each segment of the insect gut (anterior midgut, AM, posterior midgut, PM, and hindgut, H). Using combined fluorescence microscopy and BLI imaging as well as qPCR analysis, we showed that during their journey through the insect digestive tract, most of the parasites are lysed in the AM during the first 24 hours independently of the gut microbiota. During this short period, live parasites move through the PM to establish the onset of infection. At days 3–4 post-infection (p.i.), the parasite population begins to colonize the H to reach a climax at day 7 p.i., which is maintained during the next two weeks. Remarkably, the fluctuation of the parasite number in H remains relatively stable over the two weeks after refeeding, while the populations residing in the AM and PM increases slightly and probably constitutes the reservoirs of dividing epimastigotes. Conclusions/Significance These data show that a tuned dynamic control of the population operates in the insect gut to maintain an equilibrium between non-dividing infective trypomastigote forms and dividing epimastigote forms of the parasite, which is crucial for vector competence.

Simultaneous depletion of Atm and Mdl rebalances cytosolic Fe-S cluster assembly but not heme import into the mitochondrion of Trypanosoma brucei.

ABC transporter mitochondrial 1 (Atm1) and multidrug resistance‐like 1 (Mdl) are mitochondrial ABC transporters. Although Atm1 was recently suggested to transport different forms of glutathione from the mitochondrion, which are used for iron‐sulfur (Fe‐S) cluster maturation in the cytosol, the function of Mdl remains elusive. In Trypanosoma brucei, we identified one homolog of each of these genes, TbAtm and TbMdl, which were downregulated either separately or simultaneously using RNA interference. Individual depletion of TbAtm and TbMdl led to limited growth defects. In cells downregulated for TbAtm, the enzymatic activities of the Fe‐S cluster proteins aconitase and fumarase significantly decreased in the cytosol but not in the mitochondrion. Downregulation of TbMdl did not cause any change in activities of the Fe‐S proteins. Unexpectedly, the simultaneous downregulation of TbAtm and TbMdl did not result in any growth defect, nor were the Fe‐S cluster protein activities altered in either the cytosolic or mitochondrial compartments. Additionally, TbAtm and TbMdl were able to partially restore the growth of the Saccharomyces cerevisiae Δatm1 and Δmdl2 null mutants, respectively. Because T. brucei completely lost the heme b biosynthesis pathway, this cofactor has to be obtained from the host. Based on our results, TbMdl is a candidate for mitochondrial import of heme b, which was markedly decreased in both TbMdl and TbAtm + TbMdl knockdowns. Moreover, the levels of heme a were strongly decreased in the same knockdowns, suggesting that TbMdl plays a key role in heme a biosynthesis, thus affecting the overall heme homeostasis in T. brucei.

The GTPase TcRjl of the human pathogen Trypanosoma cruzi is involved in the cell growth and differentiation

The protozoan parasite Trypanosoma cruzi, the etiological agent of Chagas Disease, undergoes through a complex life cycle where rounds of cell division and differentiation occur initially in the gut of triatominae vectors and, after transmission, inside of infected cells in vertebrate hosts. Members of the Ras superfamily of GTPases are molecular switches which play pivotal regulatory functions in cell growth and differentiation. We have previously described a novel GTPase in T. cruzi, TcRjl, which belongs to the RJL family of Ras-related GTP binding proteins. Here we show that most of TcRjl protein is found bound to GTP nucleotides and may be locked in this stage. In addition, we show that TcRjl is located close to the kinetoplast, in a region corresponding possibly to flagellar pocket of the parasite and the expression of a dominant-negative TcRjl construct (TcRjlS37N) displays a significative growth phenotype in reduced serum medium. Remarkably, overexpression of TcRjl inhibits differentiation of epimastigotes to trypomastigote forms and promotes the accumulation of intermediate differentiation stages. Our data suggest that TcRjl might play a role in the control of the parasite growth and differentiation.

In vitro metacyclogenesis of Trypanosoma cruzi induced by starvation correlates with a transient adenylyl cyclase stimulation as well as with a constitutive upregulation of adenylyl cyclase expression

The Trypanosoma cruzi adenylyl cyclase (AC) multigene family encodes different isoforms (around 15) sharing a variable large N-terminal domain, which is extracellular and receptor-like, followed by a transmembrane helix and a conserved C-terminal catalytic domain. It was proposed that these key enzymes in the cAMP signalling pathway allow the parasite to sense its changing extracellular milieu in order to rapidly adapt to its new environment, which is generally achieved through a differentiation process. One of the critical differentiation events the parasitic protozoan T. cruzi undergoes during its life cycle, known as metacyclogenesis, occurs in the digestive tract of the insect and corresponds to the differentiation from noninfective epimastigotes to infective metacyclic trypomastigote forms. By in vitro monitoring the activity of AC during metacyclogenesis, we showed that both the activity of AC and the intracellular cAMP content follow a similar pattern of transient stimulation in a two-step process, with a first activation peak occurring during the first hours of nutritional stress and a second peak between 6 and 48 h, corresponding to the cellular adhesion. During this differentiation process, a general mechanism of upregulation of AC expression of both mRNA and protein is triggered and in particular for a major subclass of these enzymes that are present in various gene copies commonly associated to the THT gene clusters. Although the scattered genome distribution of these gene copies is rather unusual in trypanosomatids and seems to be a recent acquisition in the evolution of the T. cruzi clade, their encoded product redistributed on the flagellum of the parasite upon differentiation could be important to sense the extracellular milieu.

Trypanosoma brucei FKBP12 Differentially Controls Motility and Cytokinesis in Procyclic and Bloodstream Forms

ABSTRACT FKBP12 proteins are able to inhibit TOR kinases or calcineurin phosphatases upon binding of rapamycin or FK506 drugs, respectively. The Trypanosoma brucei FKBP12 homologue (TbFKBP12) was found to be a cytoskeleton-associated protein with specific localization in the flagellar pocket area of the bloodstream form. In the insect procyclic form, RNA interference-mediated knockdown of TbFKBP12 affected motility. In bloodstream cells, depletion of TbFKBP12 affected cytokinesis and cytoskeleton architecture. These last effects were associated with the presence of internal translucent cavities limited by an inside-out configuration of the normal cell surface, with a luminal variant surface glycoprotein coat lined up by microtubules. These cavities, which recreated the streamlined shape of the normal trypanosome cytoskeleton, might represent unsuccessful attempts for cell abscission. We propose that TbFKBP12 differentially affects stage-specific processes through association with the cytoskeleton.