Binding of Leishmania infantum Lipophosphoglycan to the Midgut Is Not Sufficient To Define Vector Competence in Lutzomyia longipalpis Sand Flies

Abstract

The major surface lipophosphoglycan (LPG) of Leishmania parasites is critical to vector competence in restrictive sand fly vectors in mediating Leishmania attachment to the midgut epithelium, considered essential to parasite survival and development. However, the relevance of LPG for sand flies that harbor multiple species of Leishmania remains elusive. We tested binding of Leishmania infantum wild-type (WT), LPG-defective (Δlpg1 mutants), and add-back (Δlpg1 + LPG1) lines to sand fly midguts in vitro and their survival in Lutzomyia longipalpis sand flies in vivoLe. infantum WT parasites attached to the Lu. longipalpis midgut in vitro, with late-stage parasites binding to midguts in significantly higher numbers than were seen with early-stage promastigotes. Δlpg1 mutants did not bind to Lu. longipalpis midguts, and this was rescued in the Δlpg1 + LPG1 lines, indicating that midgut binding is mediated by LPG. When Lu. longipalpis sand flies were infected with the Le. infantum WT or Le. infantum Δlpg1 or Le. infantum Δlpg1 + LPG1 line of the BH46 or BA262 strains, the BH46 Δlpg1 mutant, but not the BA262 Δlpg1 mutant, survived and grew to numbers similar to those seen with the WT and Δlpg1 + LPG1 lines. Exposure of BH46 and BA262 Δlpg1 mutants to blood-engorged midgut extracts led to mortality of the BA262 Δlpg1 but not the BH46 Δlpg1 parasites. These findings suggest that Le. infantum LPG protects parasites on a strain-specific basis early in infection, likely against toxic components of blood digestion, but that it is not necessary to prevent Le. infantum evacuation along with the feces in the permissive vector Lu. longipalpisIMPORTANCE It is well established that the presence of LPG is sufficient to define the vector competence of restrictive sand fly vectors with respect to Leishmania parasites. However, the permissiveness of other sand flies with respect to multiple Leishmania species suggests that other factors might define vector competence for these vectors. In this study, we investigated the underpinnings of Leishmania infantum survival and development in its natural vector, Lutzomyia longipalpis We found that LPG-mediated midgut binding persists in late-stage parasites. This observation is of relevance for the understanding of vector-parasite molecular interactions and suggests that only a subset of infective metacyclic-stage parasites (metacyclics) lose their ability to attach to the midgut, with implications for parasite transmission dynamics. However, our data also demonstrate that LPG is not a determining factor in Leishmania infantum retention in the midgut of Lutzomyia longipalpis, a permissive vector. Rather, LPG appears to be more important in protecting some parasite strains from the toxic environment generated during blood meal digestion in the insect gut. Thus, the relevance of LPG in parasite development in permissive vectors appears to be a complex issue and should be investigated on a strain-specific basis.

Keywords: LPG; Leishmania; parasite binding; parasite survival; sand fly; sand fly midgut; vector competence.

FIG 1

In vitro binding of Leishmania major and Leishmania infantum to midgut epithelia of natural sand fly vectors. (A and B) binding of Le. major (A) and Le. infantum LLM-320 (B) parasites, harvested from 3-day-old cultures, to open midguts of Phlebotomus papatasi and Lutzomyia longipalpis sand flies, respectively. n = 2. (C) Binding of Le. infantum parasites from cultures harvested at day 4 to unopened and opened Lu. longipalpis midguts. n = 2. Unopen midguts were intact; open midguts were cut longitudinally along the anterior-posterior axis. Unfed midguts were used. *, statistically significant at P < 0.05.

FIG 2

Temporal and stage-specific binding of Leishmania infantum parasites to unfed Lutzomyia longipalpis midguts. (A to E) Le. infantum (MCAN/BR/09/52, LAB). (A) Binding of parasites harvested on different days of culture. n = 2. 3d, day 3; 4d, day 4; 5d, day 5; 6d, day 6. (B) Growth curve in culture. n = 3. (C) Metacyclic emergence on day 4, day 5, day 6, and day 7 of culture. n = 3. (D and E) Differential binding of ESP and LSP from cultures harvested on days 4 (D) and 5 (E). n = 2. (F and G) Differential binding of ESP and LSP of Le. infantum BH46 wild-type strain from cultures harvested on days 4 (F) and 5 (G). n = 2. ESP, early-stage parasites; LSP, late-stage parasites. Midguts were opened longitudinally along the anterior-posterior axis. *, statistically significant at P < 0.05. (H and I) Numbers of BH46 wild-type parasites stained by anti-LPG antibody in metacyclic-enriched samples (light green) and procyclic-enriched samples (dark green) for the 4-day-old (H) and 5-day-old (I) parasite cultures. FITC, fluorescein isothiocyanate.

FIG 3

Immunostaining of LPG on the surface of different Leishmania infantum BH46 developmental stages. (A to L) Wild-type parasites were stained with green fluorescent protein (GFP)-conjugated anti-LPG CA7AE antibody. (A to H) Seven-day-old cultures were harvested, and parasites were sorted into metacyclic (A to D) and procyclic (E to H) promastigotes in a Ficoll gradient. (I to L) Staining of 3-day-old culture parasites. Bar = 5 μm. Green = LPG. Blue = DAPI (nuclear DNA). Gray pictures = DIC (differential interference contrast).

FIG 4

Binding of Leishmania infantum BH46 wild-type, Δlpg1, and Δlpg1 + LPG1 lines to Lutzomyia longipalpis midguts dissected 5 days after blood feeding. (A to D) Cultures of Le. infantum BH46 WT (wild-type), −/− (Δlpg1), and add-back (Δlpg1 + LPG1) lines were harvested on days 3 (A), 5 (B), 6 (C), and 7 (D) and incubated with midguts of sand flies 5 days after blood feeding. Midguts were opened longitudinally along the anterior-posterior axis. The WT strain was also incubated with intact (unopened) midguts. n = 2. *, statistically significant at P < 0.05.

FIG 5

Lutzomyia longipalpis midgut infection with Leishmania infantum BH46 wild-type, Δlpg1, and Δlpg1 + LPG1 parasites. (A to J) Upon infection with 5 million parasites per ml, parasite load and infection prevalence were assessed on days 3 (A and B), 6 (C and D), 9 (E and F), 12 (G and H), and 15 (I and J) postinfection (Pi), respectively. Data represent WT (wild-type), −/− (Δlpg1), and add-back (Δlpg1 + LPG1) lines. Low, 500 to 5,000 parasites/midgut; Moderate, 5,000 to 10,000 parasites/midgut; Heavy, >10,000 parasites/midgut. n = 2. *, statistically significant at P < 0.05.

FIG 6

Lutzomyia longipalpis midgut infection with Leishmania infantum BA262 wild-type, Δlpg1, and Δlpg1 + LPG1 parasites. (A to J) Upon infection with 5 million parasites per ml, parasite load and infection prevalence were assessed on days 2 (A and B), 3 (C and D), 6 (E and F), 8 (G and H), 12 (I and J), and 15 (K and L) after infection, respectively. Data represent WT (wild-type), −/− (Δlpg1), and add-back (Δlpg1 + LPG1) lines. Low, 500 to 5,000 parasites/midgut; Moderate, 5,000 to 10,000 parasites/midgut; Heavy, >10,000 parasites/midgut. n = 2. *, statistically significant at P < 0.05.

FIG 7

Leishmania infantum survival after incubation with extracts of blood-engorged Lutzomyia longipalpis midguts in vitro. (A) BH46 WT (wild-type), −/− (Δlpg1), and add-back (Δlpg1 + LPG1) lines were exposed in vitro to either a PBS control (CTR) or extracts of engorged midguts dissected at 24 h or 48 h post-blood meal. (B) BA262 WT (wild-type), −/− (Δlpg1), and add-back (Δlpg1 + LPG1) parasites were exposed in vitro to either a PBS control (CTR) or extracts of engorged midguts dissected at 24 h or 48 h post-blood meal. n = 2. *, statistically significant at P < 0.05.