Hb endocytosis in Leishmania is mediated through a 46-kDa protein located in the flagellar pocket. To understand the nature of the Hb receptor (HbR), we have purified the 46-kDa protein to homogeneity from Leishmania promastigote membrane. Purified HbR specifically binds Hb. The gene for HbR was cloned, and sequence analysis of the full-length HbR gene indicates the presence of hexokinase (HK) signature sequences, ATP-binding domain, and PTS-II motif. Four lines of evidence indicate that HbR in Leishmania is a hexokinase: 1) the recombinant HbR binds Hb, and the Hb-binding domain resides in the N terminus of the protein; 2) recombinant proteins and cell lysate prepared from HbR-overexpressing Leishmania promastigotes show enhanced HK activity in comparison with untransfected cells; 3) immunolocalization studies using antibodies against the N-terminal fragment (Ld-HbR-DeltaC) of Ld-HbR indicate that this protein is located in the flagellar pocket of Leishmania; and 4) binding and uptake of (125)I-Hb by Leishmania is significantly inhibited by anti-Ld-HbR-DeltaC antibody and Ld-HbR-DeltaC, respectively. Taken together, these results indicate that HK present in the flagellar pocket of Leishmania is involved in Hb endocytosis.

No abstract

Recent studies have shown that phagosome maturation depends on the balance between pro-inflammatory and anti-inflammatory cytokines, indicating that cytokine modulates phagosome maturation. However, the mechanism of cytokine-mediated modulation of intracellular trafficking remains to be elucidated. Here, we have shown that treatment of macrophages with IL-6 specifically induce the expression of Rab5 through the activation of extracellular signal-regulated kinase, whereas IL-12 exclusively upregulate the expression of Rab7 through the activation of p38 MAPK. We have cloned the 5'-flanking regions of the rab5c or rab7 into the promoterless reporter vector. Our results have shown that cells transfected with rab5c chimera are transactivated by IL-6, and IL-12 specifically transactivates cells containing rab7 chimera. Moreover, our results also show that IL-12 induces lysosomal transport, whereas IL-6 stimulates the fusion between early compartments in macrophages and accordingly modulates Salmonella trafficking and survival in macrophages. This is the first demonstration showing that cytokine differentially regulates endocytic trafficking by controlling the expression of appropriate Rab GTPase, and provides insight into the mechanism of cytokine-mediated regulation of intracellular trafficking.

Leishmaniasis is a severe infectious disease. Drugs used for leishmaniasis are very toxic, and no vaccine is available. We found that the hemoglobin receptor (HbR) of Leishmania was conserved across various strains of Leishmania, and anti-HbR antibody could be detected in kala-azar patients' sera. Our results showed that immunization with HbR-DNA induces complete protection against virulent Leishmania donovani infection in both BALB/c mice and hamsters. Moreover, HbR-DNA immunization stimulated the production of protective cytokines like interferon-γ (IFN-γ), interleukin-12 (IL-12), and tumor necrosis factor-α (TNF-α) with concomitant down-regulation of disease-promoting cytokines like IL-10 and IL-4. HbR-DNA vaccination also induced a protective response by generating multifunctional CD4(+) and CD8(+) T cells. All HbR-DNA-vaccinated hamsters showed sterile protection and survived during an experimental period of 8 months. These findings demonstrate the potential of HbR as a vaccine candidate against visceral leishmaniasis.

In our experience, TMJ injections using sonographic guidance is a safe, effective and accurate procedure.