Enhanced entry of virulent AG83 promastigotes having increased metacyclogenesis and higher distribution of surface 9-O-AcSA with macrophages. (a) Decreased infectivity (%) of virulent promastigotes after de-O-acetylation and desialylation. The infection assays were performed using macrophage : promastigote ratio 1 : 10, for 2 h at 37°C using untreated (white square), esterase (black square), and sialidase (lined square) treated AG83, GE1 promastigotes as described in [69]. The reduction in infectivity (%) of de-O-acetylated virulent strains was compared against untreated control. In parallel, UR6 promastigotes with minimal sialic acids were similarly treated. ^# denotes P < 0.01 for AG83 and * denotes P < 0.05 for GE1. (b) Reduced phagocytic index of virulent promastigotes of L. donovani after de-O-acetylation (black square) and desialylation (lined square) as compared to untreated controls (white square). Similar experimental conditions were used as described in legends of Figure 4(a) [69]. In parallel desialylated promastigotes were also used. ^## denotes P < 0.01 for AG83 and ** denotes P < 0.01 for GE1. (c) Photomicrographs demonstrating enhanced entry of virulent L. donovani promastigotes within macrophages. Virulent AG83 and avirulent UR6 promastigotes were treated with esterase and sialidase for the assay under optimized conditions and the results were compared with untreated promastigotes by confocal microscopy. Column 1, phase photomicrograph. Column 2, detection of propidium iodide-stained fluorescence. Column 3, overlap of 1 and 2. (d) Increased proportion of metacyclic promastigotes in stationary, phase of virulent AG83 as compared to UR6. Promastigotes of logarithmic, stationary and metacyclic (after purification by PNA-negative agglutination) stages of AG83 and UR6 was assessed by flow cytometry to demonstrate the percent of metacyclics (FSC^low, R1 population) in as represented in FSC versus SSC plots. (e) Increased distribution of 9-O-AcSA on metacyclic promastigotes of AG83 as compared to UR6. Flow cytometric analysis of metacyclic promastigotes obtained from stationary phase cultures of AG83 and UR6 after subsequent enrichment through PNA-negative selection were incubated with FITC-Achatinin-H to detect the presence of 9-O-AcSA as described in [69]. (reproduced and adapted from [69] with permission of the publishers and the Cambridge University Press).