The role of phosphoglycans in Leishmania-sand fly interactions

Abstract

Leishmania promastigotes synthesize an abundance of phosphoglycans, either attached to the cell surface through phosphatidylinositol anchors (lipophosphoglycan, LPG) or secreted as protein-containing glycoconjugates. These phosphoglycans are thought to promote the survival of the parasite within both its vertebrate and invertebrate hosts. The relative contributions of different phosphoglycan-containing molecules in Leishmania-sand fly interactions were tested by using mutants specifically deficient in either total phosphoglycans or LPG alone. Leishmania donovani promastigotes deficient in both LPG and protein-linked phosphoglycans because of loss of LPG2 (encoding the Golgi GDP-Man transporter) failed to survive the hydrolytic environment within the early blood-fed midgut. In contrast, L. donovani and Leishmania major mutants deficient solely in LPG expression because of loss of LPG1 (involved in biosynthesis of the core oligosaccharide LPG domain) had only a slight reduction in the survival and growth of promastigotes within the early blood-fed midgut. The ability of the LPG1-deficient promastigotes to persist in the midgut after blood meal excretion was completely lost, and this defect was correlated with their inability to bind to midgut epithelial cells in vitro. For both mutants, when phosphoglycan expression was restored to wild-type levels by reintroduction of LPG1 or LPG2 (as appropriate), then the wild-type phenotype was also restored. We conclude, first, that LPG is not essential for survival in the early blood-fed midgut but, along with other secreted phosphoglycan-containing glycoconjugates, can protect promastigotes from the digestive enzymes in the gut and, second, that LPG is required to mediate midgut attachment and to maintain infection in the fly during excretion of the digested blood meal.

Figure 1

Structure of PG-containing glycoconjugates from wild-type and LPG1- and LPG2-deficient mutants. Structures enclosed within the dashed boxes designate those domains specifically affected by the respective mutations in either LPG1 or LPG2. PI, phosphatidylinositol; GIPL, glycoinositol phospholipid; GPI, glycophosphatidylinositol; sAP, secreted acid phosphatase; PPG, proteophosphoglycan.

Figure 2

Agglutination profiles of L. major wild-type (LV39) and LPG1-targeted null mutants with mAb WIC79.3 (A) and PNA lectin (B). LmLV39, closed squares; Lmajlpg1⁻+LPG1, open circles; Lmajlpg1⁻, open squares. Agglutination profiles of L. donovani wild-type (1S) and mutant promastigotes with mAb CA7AE (C) and PNA (D). Ld1S, open squares; LdR2D2, open diamonds; LdR2D2+LPG1, open circles; LdC3PO, open triangles; LdC3PO+LPG2, cross-hatched squares.

Figure 3

Infection of P. papatasi sand flies with L. major LPG1-targeted null mutants. (A) Flies were fed on mouse blood containing 2 × 10⁶ promastigotes per ml and were dissected on day 2 (stippled bars) or day 7 (solid bars). (B) Flies were fed on mouse blood containing 10⁷ promastigotes per ml, and midguts were dissected on day 3 (stippled bars) and on day 5, at which time midguts were grouped according to those that still retained blood (light solid bars) and those that had no detectable blood (dark solid bars). Graphs show the means ± SEM of promastigotes per midgut from a single experiment (10–12 flies per group). The percentages of flies positive for parasites in each group are shown.

Figure 4

Infection of P. argentipes sand flies with L. donovani R2D2 and C3P0 mutants. Flies were fed on mouse blood containing 2 × 10⁶ promastigotes per ml, and midguts were dissected on day 2 (stippled bars) and day 5 (solid bars) after feeding. Graphs show the means ± SEM of promastigotes per midgut, from a pool of three separate experiments (22–30 individuals flies) for day 2 and two separate experiments (14–19 total flies) for day 5. The percentages of flies positive for parasites in each group are shown.

Figure 5

Infection of P. argentipes sand flies with L. donovani LPG2-targeted null mutants. Flies were fed on mouse blood containing 2 × 10⁶ promastigotes per ml, and midguts were dissected on day 2 (stippled bars) and day 5 (solid bars) after feeding. Graphs show the means ± SEM of promastigotes per midgut from a single experiment (13–19 flies per group). The percentages of flies positive for parasites in each group are shown.