Unresolved questions.

<p>Unresolved questions.</p

Amino acid sequence alignments of the <i>L. infantum</i> Li1040 protein with its homologue proteins.

<p>The amino acid sequence of <i>L. infantum</i> (Li) (LinJ32_V3.1040) Li1040 protein was aligned with homologue proteins from <i>L. donovani</i>, (Ld); <i>L. major</i> (Lm) (LmjF32.0985): <i>L. braziliensis</i> (Lb), (LbrM32.1080); <i>T. cruzi</i> (Tc) (TC00.1047053511725.280); <i>T. brucei</i> (Tb) (Tb11.01.5840) and mouse Tsg101 protein (Tsg101). The amino acid sequences of <i>L. infantum</i> and <i>L. donovani</i> Li1040 proteins are identical. The Vps23_core domains are highlighted.</p

The <i>L. major</i> transfectants expressing the <i>L. donovani</i> Li1040 ortholog gene (Li1040) displayed increased virulence in visceral infection in mice.

<p><i>L. major</i> expressing the <i>L. donovani</i> Li1040 ortholog with tag or no tag and <i>L. major</i> containing the control pLA2tag vector (Neo) were used to infect BALB/c mice by tail vein injection. The liver and spleen parasite burden levels were determined after 4, 6, 8 and 10 weeks after infection. Replica experiments are shown in Panels A and B. The effect of Li1040 protein with no tag on <i>L. major</i> visceral infection was also examined and compared with Li1040 protein with the A2 tag (Panel C).</p

<i>Leishmania</i> Genes Used in This Study.

<p><i>Leishmania</i> Genes Used in This Study.</p

Comparison of Li1040 loci in <i>L. infantum</i> and <i>L. major</i>.

<p>Blast searching <i>L. major</i> shotgun sequences and Southern blot analysis indicated that the Li1040 ortholog gene is present in <i>L. major</i>. The earlier initial version of the <i>L. major</i> genome sequences contained only one of the flanking 384 bp repeat sequences, which flanks the Li1040 ortholog gene in <i>L. infantum</i> and <i>L. major</i>. Open reading frame: ORF.</p

Over expression of <i>L.major</i> Li1040 ortholog gene (Lm0985) in <i>L. major</i>.

<p>A. Western blot showing the expression levels of Li1040 and Lm0985 proteins (both with A2 tag) in transfected <i>L. major</i> promastigotes. The liver (B) and spleen (C) parasite burdens of mice infected with <i>L. major</i> over expressing Li1040 or Lm0985, and <i>L. major</i> containing control vector (Neo) were determined after 4, 6, 8 and 10 weeks after infection.</p

Gene expression analysis of the endogenous Li1040 gene ortholog in wild-type <i>L. donovani</i> and <i>L. major</i>.

<p>Total RNA was extracted from <i>L. donovani</i> and <i>L. major</i> and subjected to Northern blot analysis with the <i>L. donovani</i> Li1040 gene ortholog or the α-tubulin gene as the probes. Lane 1: <i>L. donovani</i> promastigotes (P); lane 2: <i>L. donovani</i> shifted to amastigote (A) culture conditions for 6 hours; lane 3: <i>L. major</i> promastigotes (P); lane 4: <i>L. major</i> shifted to amastigote (A) culture for 6 hours. The X-ray film exposure times (0.8, 2 or 7 days) are indicated.</p

<i>L. major</i> transfectants containing <i>L. donovani</i> Li1040 ortholog were selected after <i>in vivo</i> infection and <i>in vitro</i> culture selections.

<p>A. Diagram of the pLA2tag vector used to express and detect <i>L. donovani</i> gene products in <i>L. major</i>. B. Expression of <i>L. donovani</i> genes in <i>L. major</i> and <i>in vivo</i> and <i>in vitro</i> selection of <i>L. major</i> transfectants. Individual <i>L. donovani</i> genes were cloned into the expression vector pLA2tag, expressed in <i>L. major</i> and detected by Western blot analysis with anti-A2 epitope tag antibodies (Lanes 1–8). For <i>in vivo</i> selection, the <i>L. major</i> transfectants shown in lanes 1–8 were pooled in equal numbers, injected into the tail vein of BALB/c mice, and 6 weeks later were isolated from the spleen and subjected to Western blot analysis with anti-A2 monoclonal antibodies (Lane 9). For <i>in vitro</i> selection, the <i>L. major</i> transfectants shown in lanes 1–8 were pooled and placed in amastigote growth conditions (pH5.5 and 37°C) for 3 days and then subjected to Western blot analysis with anti-A2 monoclonal antibodies (lane 10). Note: 6 out of these 7 <i>L. donovani</i> genes were expressed in <i>L. major</i> at the expected size indicated by arrows. The <i>L. major</i> transfectants expressing the <i>L. donovani</i> Li1040 ortholog became dominant after both <i>in vivo</i> and <i>in vitro</i> selections (Lanes 9 &10).</p

The cellular localization of <i>L. donovani</i> Li1040 ortholog protein in <i>L. donovani</i> and <i>L. major</i>.

<p>A. Western blot showing expression of the GFP-Li1040 fusion protein in transfected <i>L. donovani</i> and <i>L. major</i> promastigotes. B. Cellular localization of GFP-Li1040 fusion protein in <i>L. donovani</i> (Top) and <i>L. major</i> (Middle) promastigotes determined by GFP fluorescence. C. Cellular localization of the expressed Li1040-A2tag protein in transfected <i>L. major</i> promastigotes determined by indirect immunofluoresence with the anti-A2 monoclonal antibody.</p

<i>Leishmania</i> genes implicated in the development of visceral disease.

<p><i>Leishmania</i> genes implicated in the development of visceral disease.</p