Expression of biopterin transporter (BT1) protein in Leishmania

The present work focuses on the growth phase regulated expression of biopterin transporter gene (BT1) from the LD1 locus on chromosome 35 of Leishmania donovani. Antiserum against recombinant BT1 detected a polypeptide of 45 kDa of equal intensity at lag, log and stationary phases of promastigote growth, both in L. donovani strain LSB-7.1 (MHOM/BL/67/ITMAP263), and strain LSB-146.1 (HOM/IR/95/X81), a natural isolate from Isfehan, Iran that caused cutaneous leishmaniasis. However, in both these strains an additional polypeptide of higher molecular mass (50 kDa) was also observed during lag phase only. In addition, polypeptides of 40, 20, 18 and 16 kDa were seen only during the lag and log phases of both strains. Analysis of L. donovani single, double and triple (null) BT1 knockout mutants confirmed that the 45-kDa polypeptide was the BT1 gene product, as it was absent in the null mutant. These results indicate that 45-kDa BT1 protein in Leishmania is consistently and constitutively expressed in all the growth stages of the parasite

Role of biopterin transporter (BT1) gene on growth and infectivity of Leishmania

Leishmania are known to be auxotrophic for pteridines that are known to play a critical role in the parasites survival. In the present work the role of biopterin transporter in the growth of the parasite and infectivity in to macrophages has been worked out. The role of biopterin transporter in the susceptibility of Leishmania to antimonial compounds has also been demonstrated. This role has been verified by using attenuated strains of Leishmania with single, double, and triple (null) biopterin (BT1) mutants made by targeted gene replacement with specific antibiotic markers. Growth analysis of these mutants revealed that wild type, single and double knock out cell lines maintained high growth rates in the medium supplemented with biopterin and folate, whereas the triple knock out or null BT1 mutants were unable to grow in the absence of supplemental biopterin. Using wild type and null BT1 mutants, we examined the role of BT1 gene in infectivity and parasite survival. The cell lines with amplified BT1 gene showed increased infectivity and survival in the macrophages where as the cell lines with disrupted BT1 gene showed reduced infectivity and survival in the macrophages. We also examined the interaction between pteridine and antimonial compounds using recombinant Leishmania strains with reduced or absent biopterin transporter gene (BT1) alleles. No difference in susceptibility to Pentostam or Glucantime was observed in both wild type and BT1-knock out strains. However, pterin or folate supplementation resulted in reversal of Glucantime but not Pentostam susceptibility in both wild type and BT1-knock out strains. The reversal of Glucantime susceptibility by pterins in BT1-knock out strains suggests that the effect may be exerted independently of biopterin transporter, possibly by blocking Glucantime uptake

The Leishmania donovani LD1 locus gene ORFG encodes a biopterin transporter (BT1)

We have previously described two genes, ORFF and ORFG, from the LD1 locus near one telomere of chromosome 35, which are frequently amplified in Leishmania isolates. In Leishmania donovani LSB-51.1, gene conversion of the rRNA gene locus on chromosome 27 with these two genes resulted in their over-expression, because of their transcription by the RNA polymerase I-mediated rRNA promoter. The predicted ORFG protein has substantial sequence homology to the ESAG10 gene product from the Trypanosoma brucei VSG expression site and both are putative membrane proteins. Using successive rounds of gene replacement of the three ORFG genes in L. donovani LSB-51.1, ORFG null mutants were obtained. These mutant cell lines show a direct relationship between ORFG mRNA, protein expression levels and active transport of biopterin into the cells. Transformation of the null mutant with a plasmid containing ORFG restores biopterin transport activity. In addition, the null mutants are unable to grow in the absence of supplemental biopterin. Thus, ORFG encodes a biopterin transporter and has been renamed BT1

Inhibition of VEGF or TGF- Signaling Activates Endothelium and Increases Leukocyte Rolling

Objective Motivated by the central roles that VEGF and TGF-β play in the assembly and maintenance of the vasculature, we examined the impact of systemic VEGF or TGF-β signal inhibition on endothelial activation as detected by leukocyte-endothelial interactions. Methods and Results VEGF or TGF-β inhibition, accomplished using adenovirus expression of soluble Flt1 (Ad-sFlt1) or soluble endoglin (Ad-sEng), resulted in a significant increase in the number of leukocytes rolling along the mesenteric venous endothelium and a significant decrease in rolling velocity in Ad-sEng mice. Neutralization of VEGF or TGF-β resulted in endothelial surface expression of P-selectin and impaired peripheral vasodilatation. Neither inhibition of VEGF nor TGF-β was associated with platelet or leukocyte activation, as detected by the activation markers platelet P-selectin and the active integrin - αIIbβIII, or by leukocyte expression of L-selectin. Soluble VCAM-1 and E-selectin were increased in sEng-expressing mice, indicating higher levels of these adhesion receptors. Conclusions VEGF or TGF-β neutralization results in impaired endothelium-mediated vasodilatation and elevated expression of surface adhesion molecules, resulting in increased leukocyte adhesion. These results indicate an essential role for both VEGF and TGF-β in maintaining the endothelium in a non-activated state and have implications for therapeutic approaches that neutralize VEGF or TGF-β

Immunization with recombinant LD1 antigens protects against experimental leishmaniasis

The genes, ORFF and BT1 (previously ORFG), are part of the multigenic LD1 locus on chromosome 35 which is frequently amplified in Leishmania. BT1 encodes a biopterin transporter, while the function of the ORFF gene product is unknown, but it is localized to the nucleus. We show here that immunization of mice with recombinant ORFF and BT1 proteins, individually, or in combination, conferred partial protection against challenge with Leishmania donovani. Protection correlated with the production of antigen-specific antibodies and in vitro splenocyte proliferation. Thus, these antigens can be potential vaccine candidates against visceral leishmaniasis

Inhibition of VEGF or TGF-β Signaling Activates Endothelium and Increases Leukocyte Rolling

OBJECTIVE: Motivated by the central roles that VEGF and TGF-β play in the assembly and maintenance of the vasculature, we examined the impact of systemic VEGF or TGF-β signal inhibition on endothelial activation as detected by leukocyte-endothelial interactions. METHODS AND RESULTS: VEGF or TGF-β inhibition, accomplished using adenovirus expression of soluble Flt1 (Ad-sFlt1) or soluble endoglin (Ad-sEng), resulted in a significant increase in the number of leukocytes rolling along the mesenteric venous endothelium and a significant decrease in rolling velocity in Ad-sEng mice. Neutralization of VEGF or TGF-β resulted in endothelial surface expression of P-selectin and impaired peripheral vasodilatation. Neither inhibition of VEGF nor TGF-β was associated with platelet or leukocyte activation, as detected by the activation markers platelet P-selectin and the active integrin - αIIbβIII, or by leukocyte expression of L-selectin. Soluble VCAM-1 and E-selectin were increased in sEng-expressing mice, indicating higher levels of these adhesion receptors. CONCLUSIONS: VEGF or TGF-β neutralization results in impaired endothelium-mediated vasodilatation and elevated expression of surface adhesion molecules, resulting in increased leukocyte adhesion. These results indicate an essential role for both VEGF and TGF-β in maintaining the endothelium in a non-activated state and have implications for therapeutic approaches that neutralize VEGF or TGF-β

Comparison of Traditional and PCR Methods during Screening for and Confirmation of Aspiculuris tetraptera in a Mouse Facility

Pinworm detection in laboratory rodents typically is accomplished by using the tape test or various modifications of fecal flotation test to detect eggs. Direct examination of intestinal contents remains the ‘gold standard’ for pinworm detection, with the limitation of euthanasia of animals. Here, we compare traditional and real-time PCR methodologies during screening for and confirming the presence of Aspiculuris tetraptera. Two sets of pooled fecal samples collected from each of 521 microisolation cages in a mouse facility suspected to be pinworm-positive were tested by PCR and fecal flotation methods. The number of PCR-positive cages was 48 (9.2%) compared with 5 (0.96%) by the fecal flotation method. All of the cages determined to be positive by fecal flotation were positive by PCR. We evaluated 8 positive cages containing 26 mice from the screening group 5 wk later to confirm the initial findings; for 7 of these cages, PCR results from the initial screening were confirmed by fecal centrifugation concentration (FCC) or direct worm detection. Among the 26 mice, 4 were pinworm-positive by FCC, 5 by maceration, and 16 by PCR. All 4 mice positive by FCC were positive by PCR; PCR was positive for 7 of the 9 mice in which pinworms were detected by FCC or maceration. Our study demonstrates that real-time PCR for survival testing of mice for A. tetraptera effectively augments current detection methods for quarantine and routine health monitoring