Transmission of cutaneous leishmaniasis by sand flies is enhanced by regurgitation of fPPG

Abstract

Sand flies are the exclusive vectors of the protozoan parasite Leishmania, but the mechanism of transmission by fly bite has not been determined nor incorporated into experimental models of infection. In sand flies with mature Leishmania infections the anterior midgut is blocked by a gel of parasite origin, the promastigote secretory gel. Here we analyse the inocula from Leishmania mexicana-infected Lutzomyia longipalpis sand flies. Analysis revealed the size of the infectious dose, the underlying mechanism of parasite delivery by regurgitation, and the novel contribution made to infection by filamentous proteophosphoglycan (fPPG), a component of promastigote secretory gel found to accompany the parasites during transmission. Collectively these results have important implications for understanding the relationship between the parasite and its vector, the pathology of cutaneous leishmaniasis in humans and also the development of effective vaccines and drugs. These findings emphasize that to fully understand transmission of vector-borne diseases the interaction between the parasite, its vector and the mammalian host must be considered together.

Figure 1

Sand flies make a significant contribution to transmission and egest exacerbation factors. Comparison of lesions developing from subcutaneous syringe injections of 10³ sand fly-egested metacyclic promastigotes (open circles) with those developing from single sand fly bites (closed circles), a, in BALB/c mice and b, in CBA/Ca mice. Data for individual fly bite in BALB/c and CBA/Ca mice were derived from 16 and 10 mice, respectively. Comparison of footpad lesions developing from injection of 10³ sand fly-egested metacyclic promastigotes in 20 μl medium alone (control, open circles) or in 20 μl egestion medium (squares), c, in BALB/c mice and d, in CBA/Ca mice. Lesion development was monitored by subtracting the width of the uninfected foot from the contralateral, infected foot, measured with a Vernier caliper. Final parasite loads in these mice were (± s.e.m) 1·71 x 10⁸ (± 0·44), 2·43 x 10⁸ (± 0·58) and 2·47 x 10⁸ (± 0·65) for BALB/c mice infected by control injection, fly bite and co-inoculation with egestion medium, respectively, and 2·0 x 10⁴ (± 1·12), 4·09 x 10⁶ (± 0·62), and 5·31 x 10⁶ (± 0·40) for corresponding groups of CBA/Ca mice. Asterisks represent statistically significant differences using an unpaired two-tailed Student's t-test (P< 0.05). Error bars represent 1 s.e.m.

Figure 2

The fPPG component of Leishmania PSG is egested by infected sand flies. a, Stacking gel immunoblot probed with monoclonal antibody AP3, specific for phosphoglycan terminal mannose oligosaccharide caps, of PSG (lane 1), control medium (lane 2), control medium incubated with cultured metacyclic promastigotes (lane 3), egestion medium from uninfected sand flies (lane 4), and egestion medium from infected sand flies (lanes 5-8), corresponding to the four egestion experiments in Table 1, respectively. b, Stacking gel immunoblot probed with AP3 of infected gut homogenates from flies sampled on day 0 (lane 1) and thereafter at daily intervals (lanes 2-9), together with a blood-fed uninfected control sample (lane 10). Each lane represents the equivalent of one sand fly. c, Composition of wild type PSG as revealed by Stains-All (lane 1), silver staining (lane 2), immunoblots with monoclonal antibodies raised against Leishmania phosphoglycans: AP3, (lane 3), LT6 (lane 4) and LT15 (lane 5), both specific for the galactose(Gal)-mannose(Man)-phosphate disaccharide repeat units, WIC108.3 (lane 6), recognizing both unsubstituted and substituted phosphosaccharides and hydroflouric acid dephosphorylated fPPG antisera (lane 7), that specifically recognizes the polypeptide backbone of fPPG, . Each lane contained 1 μg of PSG, except lane 2 that contained 5 μg, and molecular mass markers are as indicated and the arrow indicated the interface between the stacking and resolving gels. Composition of PSG produced by L. mexicana mutants as indicated, each probed with AP3.

Figure 3

PSG enhances L. mexicana disease progression more than saliva. a, c, e, Comparison of lesions developing from injection of 10³ cultured metacyclic promastigotes (control, open circles), + 1 μg saliva (a, triangles), + 1 μg PSG (c, filled circles), + 1 μg saliva and 1 μg PSG (e, diamonds) in BALB/c mice. b, d, f, An identical set of experiments, but performed in CBA/Ca mice. Lesions were monitored as before and upon termination processed for their total parasite loads: BALB/c control 3·85 x 10⁸ (± 0·49 s.e.m.); +saliva 1·81 x 10⁸ (± 0·39); +PSG 4·44 x 10⁸ (± 0·39); +saliva/PSG 2·69 x 10⁸ (± 0·72); CBA/Ca control 4·24 x 10⁵ (± 2·75); +saliva 1·13 x 10⁴ (± 0·79); +PSG 1·1 x 10⁶ (± 0·29); +saliva/PSG 4·09 x 10⁵ (± 2·03). Asterisks represent statistically significant differences using an unpaired two-tailed t-test (P< 0.05). Error bars indicate 1 s.e.m.

Figure 4

The fPPG fraction of PSG is responsible for enhancement of Leishmania infectivity. a-i, Comparison of lesion development in resistant CBA/Ca mice injected with 10³ L. mexicana metacyclic promastigotes (controls, open circles) with mice co-injected with parasites and various components of PSG. Asterisks represent statistically significant differences compared to controls, using a two-tailed t-test (P< 0.05). a, Parasites resuspended in 20 μl control medium, 20 μl egestion medium (filled squares), or 20 μl PSG-depleted egestion medium (open squares). Hash symbols indicate statistically significant differences between egestion medium and its depleted equivalent. b,c, After ultracentrifugation of PSG, parasites co-injected with whole PSG (filled squares), fPPG pellet (half-filled squares) (b) and supernatant fractions (open squares) (c). d, e, After SDS-PAGE separation, parasites co-injected with fPPG fraction (half-filled squares), all combined gel fractions (filled squares) (d) or the resolving gel components of PSG, i.e. lacking fPPG (open squares) (e). Mild acid hydrolysis (pH2, 60 0C, 1 hr) was used to selectively hydrolyse the acid labile phosphodiester linkages between the Gal-Man-phosphate repeat units of PSG. f,g, Using phosphoglycan-deficient lpg2^-/- L. mexicana , parasites co-injected with PSG (filled squares), heat-treated PSG (half-filled squares) (f),or deglycosylated PSG (open squares) (g). h, i, Effect of synthetic phosphoglycan [Gal(β1-4)Man(α)-PO₃H.NH3]₁₀ -OH.NH₃ (half-filled squares) (h) and synthetic peptide [APSSSSSAPSSSS] (filled squares) (i), representing the disaccharide repeat core and the protein backbone characteristic of Leishmania PGs and fPPG, respectively,. Final parasite loads reflected lesion development. Error bars indicate 1 s.e.m.