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 Trypanosoma 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 (SpCas9) 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 SpCas9 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 but requires only cloning and PCR screening of the clones. Importantly, this approach is adaptable to any wild-type parasite.

Glycerol suppresses glucose consumption in trypanosomes through metabolic contest.

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.

Gluconeogenesis is essential for trypanosome development in the tsetse fly vector.

In the glucose-free environment that is the midgut of the tsetse fly vector, the procyclic form of Trypanosoma brucei primarily uses proline to feed its central carbon and energy metabolism. In these conditions, the parasite needs to produce glucose 6-phosphate (G6P) through gluconeogenesis from metabolism of non-glycolytic carbon source(s). We showed here that two phosphoenolpyruvate-producing enzymes, PEP carboxykinase (PEPCK) and pyruvate phosphate dikinase (PPDK) have a redundant function for the essential gluconeogenesis from proline. Indeed, incorporation of 13C-enriched proline into G6P was abolished in the PEPCK/PPDK null double mutant (Δppdk/Δpepck), but not in the single Δppdk and Δpepck mutant cell lines. The procyclic trypanosome also uses the glycerol conversion pathway to feed gluconeogenesis, since the death of the Δppdk/Δpepck double null mutant in glucose-free conditions is only observed after RNAi-mediated down-regulation of the expression of the glycerol kinase, the first enzyme of the glycerol conversion pathways. Deletion of the gene encoding fructose-1,6-bisphosphatase (Δfbpase), a key gluconeogenic enzyme irreversibly producing fructose 6-phosphate from fructose 1,6-bisphosphate, considerably reduced, but not abolished, incorporation of 13C-enriched proline into G6P. In addition, the Δfbpase cell line is viable in glucose-free conditions, suggesting that an alternative pathway can be used for G6P production in vitro. However, FBPase is essential in vivo, as shown by the incapacity of the Δfbpase null mutant to colonise the fly vector salivary glands, while the parental phenotype is restored in the Δfbpase rescued cell line re-expressing FBPase. The essential role of FBPase for the development of T. brucei in the tsetse was confirmed by taking advantage of an in vitro differentiation assay based on the RNA-binding protein 6 over-expression, in which the procyclic forms differentiate into epimastigote forms but not into mammalian-infective metacyclic parasites. In total, morphology, immunofluorescence and cytometry analyses showed that the differentiation of the epimastigote stages into the metacyclic forms is abolished in the Δfbpase mutant.

Trypanosoma brucei gambiense Infections in Mice Lead to Tropism to the Reproductive Organs, and Horizontal and Vertical Transmission.

Trypanosoma brucei gambiense, 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. In the current study, the tropism of T. b. gambiense 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. These findings imply that transmission of T. b. gambiense 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 T. b. gambiense. 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.

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

Since 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.

Hepatic lesions observed in hepatitis C virus transgenic mice infected by Helicobacter hepaticus.

BACKGROUND: The heterogeneity of hepatitis C virus (HCV) infection cannot always be explained by HCV genotypes or host genetic factors, raising the issue of possible cofactors. A new form of hepatitis leading to liver cancer was discovered in 1992 in mice, owing to an infection by Helicobacter hepaticus. Moreover, several studies showed an association between the presence of HCV and Helicobacter in the liver of patients with severe liver diseases suggesting a possible synergism between the two pathogens. In an HCV transgenic mouse model with a B6C3F1 background, the combination of H. hepaticus infection and the HCV transgene resulted in a significantly greater incidence and multiplicity of preneoplastic and neoplastic liver foci in males. OBJECTIVES: Because the mouse genetic background is a major determinant in the development of liver disease, our aim was to test the synergism between HCV and H. hepaticus infection using transgenic mice with a more sensitive genetic background to H. hepaticus infection. METHODS: For this purpose, four groups of mice were followed up to 14 months, the presence of H. hepaticus was monitored by PCR and hepatic lesions were looked for. RESULTS: We found that H. hepaticus, but not the HCV transgene, increased the number of hepatic lesions. The presence of carcinoma was more likely to occur on a background of hepatitis, and the overall lesions were more frequent in the presence of steatosis. The effect of the mouse genetic background was greater than the effect of the HCV transgene and was sufficient to promote lesions particularly via its sensitivity to H. hepaticus infection. CONCLUSIONS: Genetic susceptibility may be a more important factor than expected. Indeed, the synergism between HCV and H. hepaticus infection involved in liver disease may be highly host dependent.

Helicobacter infection induces podosome assembly in primary hepatocytes in vitro.

Helicobacter pylori (H. pylori) infection may contribute to many extragastric diseases including liver cirrhosis and hepatocellular carcinoma. However, the exact mechanism by which H. pylori induces the liver damage is largely unknown. We used cultured mouse primary hepatocytes as an in vitro model to investigate different aspects of liver physiology and pathology. In this study, we show that primary hepatocytes are able to assemble actin-based cytoskeletal structures called podosomes at the ventral plasma membrane. These structures are positive for podosome markers such as cortactin, vinculin and integrins and comprise proteolytic potential. Infection with the pathogen H. pylori further stimulates the formation of podosomes in primary hepatocytes. The use of pharmacological inhibitors reveals that this response is mediated, at least in part, by TGFß, a cytokine known to regulate podosome formation in endothelial cells. Similar results are obtained with the hepatoma cell line Huh7. Podosome formation is associated with increased hepatocyte degrading capacities but also with reduced cell motility. Therefore, podosome assembly translates into hepatocyte malfunction. Our study supports the hypothesis that hepatocytes can also assemble podosomes under pathological conditions in vivo.

Helicobacter pylori infection recruits bone marrow-derived cells that participate in gastric preneoplasia in mice.

BACKGROUND & AIMS: Studies in animal models have shown that bone marrow-derived cells (BMDC) could be involved in the formation of carcinomas of the upper gastrointestinal tract, including gastric carcinoma. Most gastric carcinomas in humans have been associated with chronic infection with Helicobacter pylori; we investigated the bacteria's potential to induce premalignant lesions in mice and studied the kinetics of BMDC settlement in the gastric epithelium. METHODS: C57BL/6J female chimeric mice with BMDCs from male donors that express green fluorescent protein were infected with human-derived and mouse-adapted strains of H pylori and followed. We assessed development of pathologic features and recruitment of BMDC to the gastric mucosa using immunohistochemistry and fluorescent in situ hybridization analyses of gastric tissue sections. RESULTS: Infection of mice with different strains of H pylori led to the development of chronic inflammation, hyperplasia, and mucinous metaplasia, and, later in life, of pseudointestinal metaplasia and dysplasia. After 1 year, gastric glands that contained green fluorescent protein-positive male cells were detected in 50%-90% of female chimeric mice infected with H pylori strains; the presence of these glands correlated with the development of pseudointestinal metaplasia. Twenty-two percent of H pylori-induced dysplastic lesions were composed of glands that contained epithelial BMDCs. CONCLUSIONS: H pylori infection leads to development of chronic inflammation, hyperplasia, metaplasia, and dysplasia, as well as the recruitment and accumulation of BMDC in the gastric epithelial mucosa. Nearly 25% of dysplastic lesions include cells that originate from the BM.

Effect of rebamipide on the colonic barrier in interleukin-10-deficient mice.

Our 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 IFNgamma 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.

Relationship between the severity of hepatitis C virus-related liver disease and the presence of Helicobacter species in the liver: a prospective study.

AIM: To determine the presence of Helicobacter species DNA in the liver of chronic hepatitis C (CHC) patients with and without cirrhosis as compared to controls, and to identify the bacterial species involved. METHODS: Seventy-nine consecutive patients (HBV and HIV negative) with a liver sample obtained after liver biopsy or hepatic resection were studied: 41 with CHC without cirrhosis, 12 with CHC and cirrhosis, and 26 controls (HCV negative). Polymerase chain reactions (PCRs) targeting Helicobacter 16S rDNA and species-specific were performed on DNA extracted from the liver. A gastric infection with H pylori was determined by serology and confirmed by 13C-urea breath test. RESULTS: Overall, Helicobacter 16S rDNA was found in 16 patients (20.2%). Although positive cases tended to be higher in CHC patients with cirrhosis (41.6%) than in those without cirrhosis (17.0%) or in controls (15.4%), the difference was not statistically significant (P = 0.08). H pylori-like DNA was identified in 12 cases and H. pullorum DNA in 2, while 2 cases remained unidentified. Gastric infection with H pylori was found in only 2 of these patients. CONCLUSION: Our results do not confirm the association of Helicobacter species DNA in the liver of CHC patients with advanced liver disease. The lack of correlation between positive H pylori serology and the presence of H pylori-like DNA in the liver may indicate the presence of a variant of this species.

Is Helicobacter pylori a true microaerophile?

BACKGROUND: There is no general consensus about the specific oxygen and carbon dioxide requirements of the human pathogen Helicobacter pylori. This bacterium is considered a microaerophile and consequently, it is grown under atmospheres at oxygen tensions 5-19% and carbon dioxide tensions 5-10%, both for clinical and basic and applied research purposes. The current study compared the growth of H. pylori in vitro, under various gas atmospheres, and determined some specific changes in the physiology of bacteria grown under different oxygen partial pressures. METHODS: Measurements of bacterial growth under various conditions were carried out employing classical solid and liquid culture techniques. Enzymatic activities were measured using spectrophotometric assays. RESULTS: H. pylori and all the other Helicobacter spp. tested had an absolute requirement for elevated carbon dioxide concentrations in the growth atmosphere. In contrast with other Helicobacter spp., H. pylori can tolerate elevated oxygen tensions when grown at high bacterial concentrations. Under 5% CO(2), the bacterium showed similar growth in liquid cultures under oxygen tensions from microaerobic (< 5%) to fully aerobic (21%) at cell densities higher than 5 x 10(5) cfu/ml for media supplemented with horse serum and 5 x 10(7) cfu/ml for media supplemented with beta-cyclodextrin. Evidence that changes occurred in the physiology of H. pylori was obtained by comparing the activities of ferredoxin:NADH (nicotinamide adenine dinucleotide) oxidoreductases of bacteria grown under microaerobic and aerobic atmospheres. CONCLUSIONS: H. pylori is a capnophile able to grow equally well in vitro under microaerobic or aerobic conditions at high bacterial concentrations, and behaved like oxygen-sensitive microaerophiles at low cell densities. Some characteristics of H. pylori cells grown in vitro under microaerobic conditions appeared to mimic better the physiology of organisms grown in their natural niche in the human stomach.

Construction of replicative and integrative plasmids for setting up the in vivo expression technology in Helicobacter pylori.

Some pathogenic factors of Helicobacter pylori, a bacterium involved in peptic ulcer and gastric cancer, have already been identified using either global or particular approach, but there are still some orphan genes and unidentified pathogenic factors. One of the methods used successfully for the identification of virulence genes of many pathogens is the in vivo expression technology. We describe here the construction and sequences of three different plasmids, one integrative and two replicatives, for the identification of virulence genes by using in vivo expression technology in H. pylori, and of potential use in other bacteria such as Campylobacter spp. Moreover, the use of the green fluorescent protein could allow to classify the genes according to the strength of their expression and to identify those which are repressed upon interaction with gastric mucosa.