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

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

The <i>L. donovani</i> Li1040 ortholog gene effect on cutaneous infection and proliferation in culture.

<p>A. Footpad infection levels in BALB/c mice with <i>L. major</i> expressing the <i>L. donovani</i> Li1040 ortholog gene and control <i>L. major</i> (neo). B. <i>In vitro</i> growth curves of <i>L. major</i> promastigotes expressing the <i>L. donovani</i> Li1040 ortholog gene or the control vector (neo).</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

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

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

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

Additional file 6: of A microbiome case-control study of recurrent acute otitis media identified potentially protective bacterial genera

Figure S3. Beta diversity PCoA in the nasopharynx of cases and controls, sorted by other covariates. Case and control nasopharyngeal samples shown in Fig. 3 are coloured by other covariates. NA refers to samples where the covariate was not applicable or was missing (not given or recorded “unknown”) and the number represents individual samples. (PDF 564 kb