Fig 1.
Lesions in BALB/c and C57BL/6 mice infected with L. (L.) amazonensis promastigotes from LV79 and PH8 strains.
A. Lesion areas of BALB/c and C57BL/6 measured weekly during 12 weeks. T-test between PH8 and LV79 in the same mouse strain, **: p<0.01, ***: p<0.001. B. Area under curve for BALB/c mice infected with LV79 and PH8. C. Area under curve for C57BL/6 mice infected with LV79 and PH8, T-test, **: p<0.01. D. Images of BALB/c lesion 12 weeks p.i. with PH8 (upper) and LV79 (lower) promastigotes. E. Parasite recovery from PH8 and LV79 lesions from three independent infections in BALB/c mice. T-test.
Fig 2.
Lesions in BALB/c mice infected with L. (L.) amazonensis promastigotes from LV79 and PH8 strains for 12 weeks.
HE staining of BALB/c mice footpads infected with PH8 (A) and LV79 (B) with necrosis area (*). Parasite labeling in PH8 (C) and LV79 (D) lesions by IHQ using anti-Leishmania serum and anti-rabbit HRP antibody. In E, higher magnification of IHQ of PH8 lesion, in F, negative control with secondary antibody but no anti-Leishmania serum. A, B, C, D and F 400X magnification, E with 1000x magnification.
Fig 3.
Comparison between PH8 and LV79 lesion-derived amastigotes regarding infective characteristics.
A. Estimation of amastigote viability by MTT. Three independent experiments, T test, **:p<0.01. B. Trypsin-like activity of amastigote extracts. One experiment (representative of two) with technical triplicates, T test, ***:p<0.001. C. Density of promastigote cultures, indicating efficiency of conversion of amastigotes to promastigotes after 4 days in culture (one experiment with five technical replicates) T test, ***:p<0.001. D. Lesion development graph and E. the respective area under curve after infection with lesion-derived amastigotes from PH8, LV79 and normalized numbers (using viability percentages) of LV79- named as LV79 normalized. Results from three independent experiments, ANOVA followed by Tukey post test, **:p<0.01, ***:p<0.001 (6 weeks: ** for PH8 x LV79, *** for PH8 x LV79norm, 7 and 8 weeks: *** for PH8 x LV79 and LV79norm). F. Representative image in the last day of infection of BALB/c: I, infected with PH8 amastigotes, II, infected with LV79 amastigotes and III, infected with normalized numbers of LV79 amastigotes.
Fig 4.
Protein comparison in PH8 and LV79 lesion-derived amastigotes.
A. Venn diagram showing the overlap between all identified proteins in PH8 and LV79. B. Heat map representing log2 fold changes of the quantitative data (green—lowest abundance and red—highest abundance) of the differentially expressed proteins (T test, p <0.05). C. Main component analysis based on all proteins identified in LV79 and/or PH8. D. Heat map representing log2 fold changes of the quantitative data of all proteins identified. The first number (1 or 2) after strain name (1, 2 or 3) indicates infection experiment, the second corresponds to the technical replicate.
Table 1.
Proteins with different abundance between LV79 and PH8.
Protein names and uniport ID, fold change relative to PH8, and strain showing higher abundance (ED = exclusively detected). Proteins with fold changes of 2 or more in LV79/PH8 and in PH8/LV79 are depicted in bold font [39].
Fig 5.
Validation of proteome data by Western blot.
Western blot images and corresponding graphs showing expression of A. GP63, B. CPx in three samples of amastigotes of PH8 and LV79 strains. Statistical analysis by T test, **:p<0.01.