Streptococcus pyogenes Cas9 ribonucleoprotein delivery for efficient, rapid and marker-free gene editing in Trypanosoma and Leishmania

Kinetoplastids are unicellular eukaryotic flagellated parasites found in a wide range of hosts within the animal and plant kingdoms. They are known to be responsible in humans for African sleeping sickness ( Trypanosoma brucei ), Chagas disease ( Trypanosoma cruzi ), and various forms of leishmaniasis ( Leishmania spp.), as well as several animal diseases with important economic impact (African trypanosomes, including T. congolense ). Understanding the biology of these parasites necessarily implies the ability to manipulate their genomes. In this study, we demonstrate that transfection of a ribonucleoprotein complex, composed of recombinant Streptococcus pyogenes Cas9 ( Sp Cas9) and an in vitro -synthesized guide RNA, results in rapid and efficient genetic modifications of trypanosomatids, in marker-free conditions. This approach was successfully developed to inactivate, delete and mutate candidate genes in various stages of the life cycle of T. brucei and T. congolense , and Leishmania promastigotes. The functionality of Sp Cas9 in these parasites now provides, to the research community working on these parasites, a rapid and efficient method of genome editing, without requiring plasmid construction and selection by antibiotics. Importantly, this approach is adaptable to any wild-type parasite, including field isolates

PLoS Biol

Microorganisms must make the right choice for nutrient consumption to adapt to their changing environment. As a consequence, bacteria and yeasts have developed regulatory mechanisms involving nutrient sensing and signaling, known as "catabolite repression," allowing redirection of cell metabolism to maximize the consumption of an energy-efficient carbon source. Here, we report a new mechanism named "metabolic contest" for regulating the use of carbon sources without nutrient sensing and signaling. Trypanosoma brucei is a unicellular eukaryote transmitted by tsetse flies and causing human African trypanosomiasis, or sleeping sickness. We showed that, in contrast to most microorganisms, the insect stages of this parasite developed a preference for glycerol over glucose, with glucose consumption beginning after the depletion of glycerol present in the medium. This "metabolic contest" depends on the combination of 3 conditions: (i) the sequestration of both metabolic pathways in the same subcellular compartment, here in the peroxisomal-related organelles named glycosomes; (ii) the competition for the same substrate, here ATP, with the first enzymatic step of the glycerol and glucose metabolic pathways both being ATP-dependent (glycerol kinase and hexokinase, respectively); and (iii) an unbalanced activity between the competing enzymes, here the glycerol kinase activity being approximately 80-fold higher than the hexokinase activity. As predicted by our model, an approximately 50-fold down-regulation of the GK expression abolished the preference for glycerol over glucose, with glucose and glycerol being metabolized concomitantly. In theory, a metabolic contest could be found in any organism provided that the 3 conditions listed above are met

Identification of a novel and ancestral machinery involved in mitochondrial membrane branching in Trypanosoma brucei

ABSTRACT African trypanosomes are eukaryotic parasites that exist in two main replicative forms; the procyclic form in the midgut of the insect vector, the tsetse fly Glossina spp. and the bloodstream form responsible for diseases in humans and cattle. Unlike most other eukaryotes, where mitochondria continuously fuse and divide, trypanosome mitochondria form a single and continuously interconnected network that only divides during cytokinesis. The machineries governing mitochondrial remodeling and interconnection, however, remain largely unknown. We characterize a dynamin-related protein (DRP) from T. brucei ( Tb DBF, previously called Tb MfnL) that depicts sequence similarities with Opa1 and Mfn, mammalian DRPs involved mitochondrial fusion. We showed that Tb DBF has closely related homologues in several organisms that are devoid of Mfn and Opa1, such as eukaryotes from different phyla, prokaryotes and archaea. Tb DBF is the first member of this new protein family to be functionally characterized. It localizes to the mitochondrial periphery and, upon overexpression, induces a strong increase in the interconnection and branching of mitochondrial filaments in a GTPase dependent manner. Its overexpression also promotes a major increase in cellular and mitochondrial volume and an increased consumption of the two major carbon sources used by the parasite (glucose and proline), as well as ethanolamine, a precursor of phosphatidyl-ethanolamine involved in membrane biogenesis and shaping. We propose that mitochondrial Tb DBF is a component of an ancestral membrane remodeling machinery that contributes to the formation of intermitochondrial connections

The susceptibility of Trypanosoma congolense and Trypanosoma brucei to isometamidium chloride and its synthetic impurities.

International audienceSince the 1950s, the chemotherapy of animal African trypanosomosis in cattle has essentially relied on only two compounds: isometamidium chloride (ISM), a phenanthridine, and diminazene aceturate, an aromatic diamidine. The commercial formulations of ISM, including Veridium(®) and Samorin(®), are a mixture of different compounds: ISM is the major component, mixed with the red isomer, blue isomer and disubstituted compound. To investigate the pharmacological effects of these individual compounds ISM, the blue and red isomers and the disubstituted compound were synthesised and purified by HPLC. The activity of each compound was analysed both in vitro, and in mice in vivo. For the in vitro analysis, a drug sensitivity assay was developed in 96-well tissue culture plates to determine the effective concentration which killed 50% of trypanosome population within 48 h of drug exposure (IC50). All compounds tested in vitro possessed trypanocidal activity, and purified ISM was the most active. Veridium(®) and Samorin(®) had similar IC50 values to purified ISM for both Trypanosoma congolense and Trypanosoma brucei brucei. The disubstituted compound had the highest IC50 values whereas intermediate IC50 values were obtained for the blue and red isomers. In vivo, single-dose tests were used to evaluate the trypanocidal and prophylactic activity against T. congolense. Interestingly, the prophylactic effect two months post treatment was as efficient with ISM, Veridium(®), Samorin(®) and the disubstituted compound at the highest dose of 1mg/kg whereas the red and blue isomers both showed much lower prophylactic activity. This study on T. congolense implies that it is necessary to limit the quantity of the blue and red isomers in the commercial mixture. Finally, the in vitro sensitivity assay may be useful for screening new trypanocides but also for the testing and detection of resistant trypanosome isolates

: Rebamipide and colonic barrier

International audienceOur aim was to study the effect of a mucosal protective agent, rebamipide, on the colonic barrier and the immune response in colitis-prone interleukin-10 deficient (IL-10-/-) C57BL/6 mice infected with Helicobacter hepaticus. After sacrifice, in all mice, control, or previously infected with H. hepaticus, or previously infected and treated with rebamipide enema, a histological examination of colonic samples was performed, intestinal permeability was studied in Ussing chamber, and mesenteric lymph node proliferation and cytokine secretion were measured. Mice treated with rebamipide, presented a reinforcement of the distal colonic epithelial barrier, an increase of mesenteric lymph node cells proliferation and of IFNΓ and IL-12 secretion. These results indicate that in IL-10-/- mice with mild colitis, rectally administered rebamipide reinforces the distal colonic barrier and has a slight Th1 immuno-stimulatory effect on mesenteric lymph node cells. These properties could be helpful in the management of some inflammatory bowel diseases

<i>Trypanosoma brucei gambiense</i> Infections in Mice Lead to Tropism to the Reproductive Organs, and Horizontal and Vertical Transmission

<div><p><i>Trypanosoma brucei gambiense</i>, transmitted by the tsetse fly, is the main causative agent of Human African trypanosomosis in West Africa and poses a significant health risk to 70 million people. Disease progression varies depending on host immunity, but usually begins with a haemo-lymphatic phase, followed by parasite invasion of the central nervous system.</p><p>In the current study, the tropism of <i>T</i>. <i>b</i>. <i>gambiense</i> 1135, causing a low level chronic ‘silent’ infection, was monitored in a murine model using bioluminescence imaging and PCR. A tropism to the reproductive organs, in addition to the central nervous system, after 12–18 months of infection was observed. Bioluminescent analysis of healthy females crossed with infected males showed that 50%, 62.5% and 37.5% of the female mice were subsequently positive for parasites in their ovaries, uteri and brain respectively. Although PCR confirmed the presence of parasites in the uterus of one of these mice, the blood of all mice was negative by PCR and LAMP. Subsequently, bioluminescent imaging of the offspring of infected female mice crossed with healthy males indicated parasites were present in the reproductive organs of both male (80%) and female (60%) offspring.</p><p>These findings imply that transmission of <i>T</i>. <i>b</i>. <i>gambiense</i> 1135 occurs horizontally, most probably via sexual contact, and vertically in a murine model, which raises the possibility of a similar transmission in humans. This has wide reaching implications. Firstly, the observations made in this study are likely to be valid for wild animals acting as a reservoir for <i>T</i>. <i>b</i>. <i>gambiense</i>. Also, the reproductive organs may act as a refuge for parasites during drug treatment in a similar manner to the central nervous system. This could leave patients at risk of a relapse, ultimately allowing them to act as a reservoir for subsequent transmission by tsetse and possibly, horizontally and vertically.</p></div

Investigation of the horizontal transmission of <i>T</i>. <i>b</i>. <i>gambiense</i> 1135 (Rluc) by crossing healthy female (n = 8) mice with infected male mice (n = 5).

<p>BLI signal from <i>ex vivo</i> organs of female mice (n = 8) crossed with <i>T</i>. <i>b</i>. <i>gambiense</i> 1135 infected males (n = 5) examined 7 months after crossing. A. BLI of <i>ex vivo</i> organs of a representative female mouse (mouse 229, see <a href="http://www.plosntds.org/article/info:doi/10.1371/journal.pntd.0004350#pntd.0004350.s008" target="_blank">S3 Table</a>). Ov, ovaries; Ut, uterus; B, brain; SC, spinal cord; Sp, spleen; Li, liver; Lu, lungs; K, kidneys; In, intestines; H, heart. The colour scale to the right of the image indicates the colour intensity in ph/sr/cm²/s. B. Percentage of positive female mice per organ. C. 1.8% agarose gel of PCR using pMUTec/TBingi nested primers on organs of female mice 229 and 228. Lane 1: spinal cord negative control; lane 2: spleen negative control; lane 3: ovary mouse 229; lane 4: uterus mouse 229 (band 201 bp); lane 5: spinal cord negative control; lane 6 spinal cord mouse 229; lane 7: spleen mouse 229; lane 8: ovary mouse 228; lane 9: uterus mouse 228; lane 10: spinal cord mouse 228; lane 11: genomic DNA <i>T</i>. <i>b</i>. <i>brucei</i> (nested PCR of the first PCR positive control); lane 12: genomic DNA <i>T</i>. <i>b</i>. <i>brucei</i> (nested PCR positive control); lane M: GeneRuler DNA ladder (ThermoScientific).</p

Analysis of vertical transmission by BLI of <i>ex vivo</i> organs of offspring (2–3 months old) of <i>T</i>. <i>b</i>. <i>gambiense</i> 1135 (Rluc) infected females (n = 10) crossed with healthy males (n = 5).

<p>BLI of <i>ex vivo</i> organs of a representative single (A) male (mouse 32486(1), see <a href="http://www.plosntds.org/article/info:doi/10.1371/journal.pntd.0004350#pntd.0004350.s009" target="_blank">S4 Table</a>) and (B) female (mouse 32925–1, see <a href="http://www.plosntds.org/article/info:doi/10.1371/journal.pntd.0004350#pntd.0004350.s009" target="_blank">S4 Table</a>) offspring. Ov, ovaries; Ut, uterus; T, testes; SV, seminal vesicles; B, brain; SC, spinal cord; Sp, spleen; Li, liver; Lu, lungs; K, kidneys; In, intestines; H, heart. The colour scale to the right of the image indicates the colour intensity in ph/sr/cm²/s. Percentage of (C) male and (D) female mice positive by BLI per organ.</p

Clinical signs observed in female mice (n = 16) following 12–18 months of infection with <i>T</i>. <i>b</i>. <i>gambiense</i> 1135 (Rluc).

<p>Mice were infected i.p. with 1–5 × 10⁶<i>T</i>. <i>b</i>. <i>gambiense</i> 1135 (Rluc) parasites and clinical signs monitored over 12–18 months.</p