Leishmania histone H1 overexpression delays parasite cell-cycle progression, parasite differentiation and reduces Leishmania infectivity in vivo.

Episomal expression of Leishmania histone H1 sense mRNAs in Leishmania major promastigotes was found previously to result in overexpression of this molecule and to reduce parasite infectivity in vitro. Herein, we evaluated the in vivo infectivity of these transfectants, in BALB/c mice, and showed that it is dramatically reduced. No lesions were observed in this group of mice and this was associated with an extremely low number of parasites both in the footpad and in the draining lymph nodes. Interestingly, the transfectants-reduced infectivity was associated with a delay in their cell-cycle progression and differentiation to axenic amastigotes, assessed in vitro. Therefore, the dramatic reduction in their infectivity may be attributed to the above-mentioned phenotypic modifications. As the metazoan linker histone H1(0) homologue is known to delay cell-cycle progression in mammalian cells we investigated whether its Leishmania counterpart, which possesses homology to its C-terminal region, when expressed in mammalian cells may also affect their cell-cycle progression. It was thus shown that Leishmania histone H1 expressed in COS7 and NIH 3T3 cells, delays cell-cycle progression in these cells too. The latter strengthens the phenotype observed in Leishmania and provides evidence that critical functions of histone H1 molecules are conserved throughout evolution.

Down-regulation of gp63 level in Leishmania amazonensis promastigotes reduces their infectivity in BALB/c mice.

Episomal expression of the major surface glycoprotein (gp63) sense and antisense mRNAs in Leishmania amazonensis was found previously to modulate the expression of this molecule as well as its infection of macrophages in vitro. Here, we evaluated the in vivo infectivity of these transfectants in BALB/c mice. Antisense downregulation of gp63 renders this parasite sensitive to complement-mediated lysis and less infective to mice, as indicated by a delay in lesion development and a significant reduction in lesion size and parasite loads at the site of inoculation and in the draining lymph nodes (DLNs). CD4+ cells at the site of inoculation decreased in number more rapidly and were 2-fold less numerous than those in controls by week 4. The number of IFN-gamma-positive cells was higher, while IL-10 positive cells were undetectable. In DLNs, CD4+ cells were higher in number, and the profile of cytokine-positive cells followed essentially the same patterns--found at the site of inoculation. These results suggest that the downregulation of gp63 increases extracellular lysis of the mutants by complement, in the in vivo environment, and reduces their infection of macrophages, resulting in a type 1 immune response seen at the site of inoculation and DLNs.

Sand fly specificity of saliva-mediated protective immunity in Leishmania amazonensis-BALB/c mouse model.

Immune response of BALB/c mice to the salivary antigens of sand flies was found to vary with different species used, i.e. Phlebotomus papatasi, Phlebotomus sergenti and Lutzomyia longipalpis. Exposure of mice to bites of these sand flies elicits production of antibodies, which are largely specific to different saliva antigens previously identified as unique to the respective fly species. When immunized intradermally (i.d.) with salivary gland lysates (SGL) of L. longipalpis, BALB/c mice developed partial protective immunity against challenges in the contralateral ears with Leishmania amazonensis plus the gland lysates. Preimmunization of these mice with the lysates from the other two species was ineffective, further indicative of the specificity of saliva-mediated immune response. The partial protective immunity observed is significant, although it is not as dramatic as reported previously in a different sand fly-mouse model. There is a correlation of this immunity with a lower number of mononuclear and polymorphonuclear phagocytes at the site of parasite inoculation. Vector species-specificity of this immunity implies its elicitation by unique saliva antigen-an issue which requires attention when designing saliva-based vaccines against leishmaniasis.