Drug uptake and modulation of drug resistance in Leishmania by an aquaglyceroporin

Leishmaniasis is a protozoan parasitic disease that affects 12 million people worldwide. The first line choice for the treatment of this disease is antimonial drugs. In the endemic regions, resistance to this class of drugs is a major impediment to treatment. Microbes often become resistant to drugs by mutation or down-regulation of uptake systems, but the uptake system for the antimonial drugs in Leishmania is unknown. In other organisms, aquaglyceroporins have been shown to facilitate uptake of trivalent metalloids. In this study, we report the identification and characterization of aquaglyceroporins from Leishmania major (LmAQP1) and Leishmania tarentolae (LtAQP1), respectively. These Leishmania proteins have the conserved signature motifs of aquaglyceroporins. Transfection of LmAQP1 into three species of Leishmania, L. tarentolae, Leishmania infantum, and L. major, produced hypersensitivity to both As(III) and Sb(III) in all three strains. Increased production of LmAQP1 was detected by immunoblotting. Drug-resistant parasites with various mutations leading to resistance mechanisms became hypersensitive to both metalloids after expression of LmAQP1. Increased rates of uptake of As(III) or Sb(III) correlated with metalloid sensitivity of the wild type and drug-resistant transfectants. Transfection of LmAQP1 in a Pentostam-resistant field isolate also sensitized the parasite in the macrophage-associated amastigote form. One allele of LmAQP1 was disrupted in L. major, and the resulting cells became 10-fold more resistant to Sb(III). This is the first report of the uptake of a metalloid drug by an aquaglyceroporin in Leishmania, suggesting a strategy to reverse resistance in the field.     

Effect of anaerobic fungi on in vitro feed digestion by mixed rumen microflora of buffalo

Five strains of anaerobic fungi isolated from the faeces of wild (hog deer, Cervus porcinus; blackbuck, Antelope cervicapra; spotted deer, Axis axis; nilgai, Baselophus tragocamelus) and rumen liquor of domestic (sheep, Ovies aries) ruminants showing high fibrolytic enzyme producing ability were added to mixed rumen microflora of buffalo to study their effect on the digestibility of lignocellulosic feed (wheat straw and wheat bran in the ratio of 80:20), enzyme production and fermentation end products in in vitro conditions. Among the 5 isolates studied, FNG5 (isolated from nilgai) showed the highest stimulating effect on apparent digestibility (35.31 +/- 1.61% vs. 28.61 +/- 1.55%; P < 0.05), true digestibility (43.64 +/- 1.73% vs. 35.37 +/- 1.65%; P < 0.01), neutral detergent fiber digestibility (29.30 +/- 2.58% vs. 18.47 +/- 2.12; P < 0.01) of feed 24 h after inoculation compared to the control group. The production of carboxymethyl cellulase, xylanase, acetyl esterase and beta-glucosidase was significantly (P < 0.05) higher in the FNG5 inoculated incubation medium. There was no improvement in the digestibility and enzyme production on the addition of the other 4 isolates. Total volatile fatty acid levels as well as the concentration of acetate, propionate, isobutyrate and valerate were significantly higher in the FNG5 added group as compared to the control group. The fungal isolate FNG5 from nilgai, a wild ruminant, was found to be superior to the other isolates tested and appears to have a potential to be used as a feed additive for improving fiber degradation in domestic ruminants.     

Diversity of the killer cell Ig-like receptors of rhesus monkeys

Because the killer cell Ig-like receptors (KIRs) have only been characterized in humans and chimpanzees, we do not have a full understanding of their evolutionary history. Therefore, cDNAs encoding the KIR molecules of five rhesus monkeys were characterized, and were found to differ from the KIR molecules identified in humans and chimpanzees. Whereas only one KIR2DL4 molecule is detected in humans and chimpanzees, two distinct KIR2DL4 homologues were identified in the monkeys. Although the two human KIR3DL molecules are limited in their polymorphism, the KIR3DL homologues in the monkeys were highly polymorphic. Up to five KIR3DL homologues were identified in each monkey that was studied, and eleven distinct KIR3DL molecules were detected in the five rhesus monkeys. Two novel families of KIR molecules were identified in the rhesus monkeys, KIR3DH and KIR1D. The KIR3DH molecules have three Ig domains, transmembrane domains homologous to KIR2DL4 molecules that contain an arginine, and short cytoplasmic domains. With these features, the KIR3DH molecules resemble the activating forms of the human KIR molecules. The KIR1D molecule encodes only one complete Ig domain before a frame-shift in the second Ig domain occurs, leading to early termination of the molecule. Multiple splice variants of KIR1D exist that encode at least one Ig domain, as well as transmembrane and cytoplasmic domains. The extensive diversity of the rhesus monkey KIR3DL homologues and the novel KIR3DH and KIR1D molecules suggests that the KIR family of molecules has evolved rapidly during the evolution of primates.     

A novel recombinant Leishmania donovani p45, a partial coding region of methionine aminopeptidase, generates protective immunity by inducing a Th1 stimulatory response against experimental visceral leishmaniasis

The development of a vaccine against visceral leishmaniasis (VL) conferring long-lasting immunity remains a challenge. Identification and proteomic characterization of parasite proteins led to the detection of p45, a member of the methionine aminopeptidase family. To our knowledge the present study is the first known report that describes the molecular and immunological characterization of p45. Recombinant Leishmania donovani p45 (rLdp45) induced cellular responses in cured hamsters and generated Th1-type cytokines from peripheral blood mononuclear cells of cured/endemic VL patients. Immunization with rLdp45 exerted considerable prophylactic efficacy (～85%) supported by an increase in mRNA expression of iNOS, IFN-γ, TNF-α and IL-12 and decrease in TGF-β and IL-4, indicating its potential as a vaccine candidate against VL.     

Cholesterol-rich microdomains as docking platforms for respiratory syncytial virus in normal human bronchial epithelial cells

Respiratory syncytial virus (RSV) is one of the major causes of respiratory infections in children, and it is the main pathogen causing bronchiolitis in infants. The binding and entry mechanism by which RSV infects respiratory epithelial cells has not yet been determined. In this study, the earliest stages of RSV infection in normal human bronchial epithelial cells were probed by tracking virions with fluorescent lipophilic dyes in their membranes. Virions colocalized with cholesterol-containing plasma membrane microdomains, identified by their ability to bind cholera toxin subunit B. Consistent with an important role for cholesterol in RSV infection, cholesterol depletion profoundly inhibited RSV infection, while cholesterol repletion reversed this inhibition. Merger of the outer leaflets of the viral envelope and the cell membrane appeared to be triggered at these sites. Using small-molecule inhibitors, RSV infection was found to be sensitive to Pak1 inhibition, suggesting the requirement of a subsequent step of cytoskeletal reorganization that could involve plasma membrane rearrangements or endocytosis. It appears that RSV entry depends on its ability to dock to cholesterol-rich microdomains (lipid rafts) in the plasma membrane where hemifusion events begin, assisted by a Pak1-dependent process.     

Expression, purification and characterization of fourth FAS1 domain of TGFβIp-associated corneal dystrophic mutants

Corneal dystrophies (CDs) are a group of inherited bilateral disorders affecting the corneal tissue of the eye. Most of these CDs in the stromal layer of the cornea have been associated with mutations found on the TGFBI gene that codes for a 683-amino acid transforming growth factor induced protein (TGFβIp). These mutations have been found to induce atypical aggregation and progressive accumulation of protein aggregates in the cornea that leads to loss of corneal transparency and hence blindness. At present, 65 distinct pathogenic mutations have been identified in TGFBI that are associated with different clinical phenotypes. More than 80% of these missense mutations occur in the 4th FAS (fasciclin-like) 1 domain. Current treatment includes surgical intervention, which often involves high recurrence rates. Hence, it is imperative to examine the properties of the TGFβIp and the pathogenic mutant proteins to understand the pathology of the disease mechanism and to develop potent therapeutics. Here, we report the recombinant expression, purification, characterization and the effect of four clinically significant pathogenic TGFβIp mutants - R555W, H572R, A620D, and H626R on the biophysical properties of the wild type (WT) 4th FAS1 domain of TGFβIp. While a higher proportion of the R555W, H572R and H626R mutants of the 4th FAS1 domains remained stable, the A620D mutant largely existed as inclusion bodies in native state and aggregates under physiological conditions. These mutants present a unique platform to examine protein aggregation-prone diseases wherein single amino-acid mutations present distinct pathogenic phenotypes. Though pathogenically and phenotypically diverse, these mutants do not exhibit variations in secondary structure and stability, except for the A620D mutant, when examined by CD and UV spectroscopy.     

Redox potential-driven repeated batch ethanol fermentation under very-high-gravity conditions

When Saccharomyces cerevisiae was cultivated under ～200 g glucose/l condition, the time point at which glucose was completely utilized coincided with the moment at which the slope of a redox potential profile changed from negative or zero to positive. Based on this feature, a redox potential-driven glucose-feeding fermentation operation was developed, and resulted in a self-cycling period of 14.25 ± 0.4 h. The corresponding ethanol concentration was maintained at 88.4 ± 1.0 g/l with complete glucose conversion, and the cell viabilities increased from 80% in the transition period to 97.2 ± 1.1%, implying the occurrence of yeast acclimatization. In contrast, a pre-determined 36-h manually adjusted period was chosen to oscillate yeast cells under ～250 g glucose/l conditions, which resulted in 106.76 ± 0.7 g ethanol/l and 15.19 ± 1.3 g glucose/l remaining at the end of each cycle. Compared to the equivalent batch and continuous ethanol fermentation processes, the annual ethanol productivity of the reported fermentation operation is 2.4% and 13.2% greater, respectively in ～200 g feeding glucose/l conditions.     

Leishmania donovani Triose Phosphate Isomerase: A Potential Vaccine Target against Visceral Leishmaniasis

Visceral leishmaniasis (VL) is one of the most important parasitic diseases with approximately 350 million people at risk. Due to the non availability of an ideal drug, development of a safe, effective, and affordable vaccine could be a solution for control and prevention of this disease. In this study, a potential Th1 stimulatory protein- Triose phosphate isomerase (TPI), a glycolytic enzyme, identified through proteomics from a fraction of Leishmania donovani soluble antigen ranging from 89.9–97.1 kDa, was assessed for its potential as a suitable vaccine candidate. The protein- L. donovani TPI (LdTPI) was cloned, expressed and purified which exhibited the homology of 99% with L. infantum TPI. The rLdTPI was further evaluated for its immunogenicity by lymphoproliferative response (LTT), nitric oxide (NO) production and estimation of cytokines in cured Leishmania patients/hamster. It elicited strong LTT response in cured patients as well as NO production in cured hamsters and stimulated remarkable Th1-type cellular responses including IFN-ã and IL-12 with extremely lower level of IL-10 in Leishmania-infected cured/exposed patients PBMCs in vitro. Vaccination with LdTPI-DNA construct protected naive golden hamsters from virulent L. donovani challenge unambiguously (∼90%). The vaccinated hamsters demonstrated a surge in IFN-ã, TNF-á and IL-12 levels but extreme down-regulation of IL-10 and IL-4 along with profound delayed type hypersensitivity and increased levels of Leishmania-specific IgG2 antibody. Thus, the results are suggestive of the protein having the potential of a strong candidate vaccine.