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

MATTHEW E. ROGERS1, THOMAS ILG2,*, ANDREI V. NIKOLAEV3, MICHAEL A. J. FERGUSON3 & PAUL A. BATES1

1 Liverpool School of Tropical Medicine, University of Liverpool, Pembroke Place, Liverpool L3 5QA, UK
2 Max-Planck-Institut fur Biologie, Abteilung Membranbiochemie, Corrensstrasse 38, 72076 Tubingen, Germany
3 Division of Biological Chemistry and Molecular Microbiology, School of Life Sciences, The Wellcome Trust Biocentre, University of Dundee, Dundee DD1 5EH, UK
* Present address: Intervet Innovation GmbH, Zur Propstei, 55270 Schwabenheim, Germany
Correspondence and requests for materials should be addressed to P.A.B. (pbates@liv.ac.uk).

Sand flies are the exclusive vectors of the protozoan parasite Leishmania1, 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 gel2, 3. 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.
Leishmaniasis is a parasitic disease that now infects some 12 million people worldwide, causing severe morbidity and mortality4. Infection is initiated by distinct life cycle stages, metacyclic promastigotes, that are introduced into the skin by fly bite along with sand fly saliva5-7. Leishmania are known to express various 'virulence factors' in the sand fly, which may facilitate transmission to, and infection of, the mammalian host8-12. However, despite these discoveries our knowledge of parasite molecules that facilitate sand fly transmission is still limited. Furthermore, a number of key issues of transmission remain unresolved, such as the true infective dose, the mechanism of parasite delivery and the biological consequences of these upon infection. Importantly, in all Leishmania–vector combinations examined so far, a gel-like plug—the parasite-derived promastigote secretory gel (PSG)—blocks the anterior parts of the midgut coincident with the accumulation of metacyclic promastigotes2, 3. An important structural component of PSG is filamentous proteophosphoglycan (fPPG), an unusual mucin-like glycoprotein unique to Leishmania13, 14. Here we address these issues regarding transmission and reveal a novel contribution made by L. mexicana PSG to the infection process.
To begin to understand the nature of the infective inoculum, we determined the number and composition of L. mexicana parasites delivered during transmission (see Methods). We adapted a membrane feeding system to collect parasites egested by infected sand flies, revealing an average of 1,086 parasites delivered per bite, highly enriched in metacyclic promastigotes (86–98%) (Table 1). The only previous investigations quantifying egested parasites have been made using microcapillary forced feeding15, 16. When this method was employed on L. mexicana-infected sand flies an average of 105 promastigotes was collected per sand fly (n = 19), significantly (P < 0.005) underestimating the size of the average infective dose egested under voluntary feeding conditions. To address the origin of the average 103 parasites delivered during transmission, we quantified the foregut populations in sand flies. The average foregut population in pre-fed flies was 118 promastigotes (n = 10), and in post-fed flies was 53 promastigotes (n = 13) per sand fly. These populations are considerably smaller than the number of promastigotes that were actually egested. Therefore, the main source of egested parasites (> 90%) lies behind the pharynx, thus clearly demonstrating active regurgitation of parasites from the oesophagus and behind, and not merely inoculation of the foregut population.
Next we investigated the efficiency of sand fly transmission by comparing infections caused by a single fly bite with those generated by a syringe inoculation. From the above results, a syringe inoculum of 103 metacyclic promastigotes was used to simulate natural transmission, and BALB/c and CBA/Ca mice expressed non-healing and healing phenotypes, respectively (Fig. 1a, b). However, when individual mice were infected by single fly bites different outcomes were quantitatively (BALB/c) and qualitatively (CBA/Ca) observed. BALB/c mice exhibited greatly accelerated lesion development, these appearing four to five weeks earlier than syringe inoculation and rapidly increasing in size without healing (Fig. 1a), whereas CBA/Ca mice developed non-healing chronic lesions (Fig. 1b). By analysing Leishmania infection using single fly bites we provide definitive evidence that delivery by sand flies makes a contribution to Leishmania infection beyond simple injection of parasites.
Figure 1 Sand flies make a significant contribution to transmission and egest exacerbation factors. Full legend High resolution image and legend (60k)
One plausible explanation for the results described above is that sand flies egest 'exacerbation factors' along with metacyclic promastigotes, which assist the establishment of the parasite in the mammalian host. Such factors could be parasite- and/or sand fly-derived. We exploited our method to collect metacyclic promastigotes from sand flies, and investigated whether any such exacerbation factors were co-egested along with the parasites. Egestion medium (with parasites removed) or control medium were mixed with 103 metacyclic promastigotes and inoculated into mice. BALB/c mice showed significantly enhanced lesion development and increased parasite burden when metacyclic promastigotes were co-inoculated with egestion medium (Fig. 1c). In CBA/Ca mice the infection was enhanced and the outcome was altered by egestion medium (Fig. 1d). The lesions quickly reached a large size, then decreased slightly after peaking but did not resolve, thus behaving like those produced by sand fly bites (Fig. 1b). Again there was a significant increase in parasite burden.
This led us to consider the identity of potential exacerbation factors. Previous work has shown that co-inoculation of parasites and sand fly salivary gland homogenate by syringe can exacerbate leishmaniasis6, 7, although the interpretation of these data in the context of natural transmission has been called into question1, 17, 18. We confirmed, as expected, the presence of saliva in the egestion medium (Supplementary Fig. 1). We also considered the additional possibility that PSG is egested along with metacyclic promastigotes and may itself contribute to disease exacerbation. Egestion of PSG seems likely because we have shown here that metacyclic promastigotes are regurgitated from behind the pharynx, where PSG accumulates2, 3. We performed immunoblotting on the egestion medium of infected flies and this revealed the presence of fPPG (Fig. 2a), the only component of PSG detected in egestion medium. Analysis of sand fly gut homogenates showed that fPPG accumulated as the infections developed, reaching a peak on day 7 when flies were used in transmission experiments (Fig. 2b). The composition of PSG was further investigated by SDS–polyacrylamide gel electrophoresis (SDS–PAGE) and immunoblotting (Fig. 2c). The main component of PSG was confirmed to be fPPG. We also detected other parasite-secreted glycans of lower molecular mass in PSG, together with a limited number of minor protein components. No lipophosphoglycan (LPG; 10–50 kDa) was detected in PSG or was free in the lumen of infected sand fly midguts, as predicted for a parasite surface glycoconjugate that mediates attachment to the midgut9, 10 (Fig. 2c; Supplementary Fig. 2). We then infected sand flies with various L. mexicana null mutants selectively deficient in the following specific phosphoglycans: secreted acid phosphatase (sAP)19, PPG220 and LPG21. Analysis of PSG from these flies also indicated fPPG to be the dominant component (Fig. 2c). Mutants that cannot synthesize any phosphoglycans22 (lpg2-/-) were unable to synthesize PSG or survive in sand flies. These data provide evidence for the existence of a second potential exacerbation factor, the parasite PSG, in addition to sand fly saliva.
Figure 2 The fPPG component of Leishmania PSG is egested by infected sand flies. Full legend High resolution image and legend (43k)
Comparison of the disease-enhancing properties of saliva and PSG in BALB/c mice showed that saliva did not significantly exacerbate L. mexicana infection with 103 metacyclic promastigotes (Fig. 3a), and the final parasite burdens were actually lower than controls. However, in CBA/Ca mice saliva did cause moderate disease exacerbation, but these lesions also resolved with time (Fig. 3b). In contrast, PSG caused substantial disease exacerbation in both BALB/c and CBA/Ca mice (Fig. 3c, d), and in the latter prevented healing of the lesions, which is characteristic of those generated by sand fly bite (Fig. 1b) and co-injection with egestion medium (Fig. 1d). In both mouse strains parasite burdens in PSG-co-inoculated mice were higher than in their respective controls. Because both saliva and PSG are delivered by sand flies during transmission by bite, we also examined the effect of co-inoculation of both upon infection (Fig. 3e, f). In both BALB/c and CBA/Ca mice an intermediate course of lesion development resulted, indicating that the large disease exacerbation effect of PSG and the smaller effect of saliva, rather than acting synergistically, seemed to antagonize each other. Similarly, the parasite burdens in these mice were also intermediate. The antagonism between saliva and PSG presumably reflects differences in the underlying immunological mechanisms involved. These results show that PSG increased both the pathogenicity and survival of L. mexicana.
Figure 3 PSG enhances L. mexicana disease progression more than saliva. Full legend High resolution image and legend (53k)
Next we considered the identity of the active ingredient(s) in PSG. First phosphoglycans (but not saliva) were adsorbed from egestion medium by binding to DEAE-Sepharose, which consequently eliminated the exacerbatory properties of the egestion medium (Fig. 4a; Supplementary Fig. 1). Second, when PSG was ultracentrifuged the exacerbation effect segregated with the fPPG pellet fraction (Fig. 4b, c; Supplementary Fig. 3a). These data are consistent with the negative charge and high molecular weight of fPPG13, 14. Third, PSG was fractionated by SDS–PAGE, which showed fPPG retained by the stacking gel to be the active fraction (Fig. 4d, e; Supplementary Fig. 3b). Fourth, PSGs from sap1/2-/-, ppg2-/- and lpg1-/- L. mexicana null mutants19-21 were co-inoculated with metacyclic promastigotes into CBA/Ca mice and were each found to exacerbate infections to the same extent as wild-type PSG (Supplementary Fig. 3d–f). By eliminating naturally occurring sAP and PPG2, or the possibility of contaminative LPG molecules within PSG, the only known molecule secreted by L. mexicana that possesses all of these properties, and the only molecule that we could detect in egestion medium is the fPPG component of PSG. From these data we cannot completely eliminate the existence of another bioactive component that makes a minor contribution to the exacerbation effect. However, based on the evidence, we conclude that the main active ingredient of PSG is fPPG.
Figure 4 The fPPG fraction of PSG is responsible for enhancement of Leishmania infectivity. Full legend High resolution image and legend (87k)
The protein backbone of fPPG includes a repetitive serine-rich motif that is very heavily glycosylated13, 14. Therefore, lastly we investigated whether disease exacerbation was associated with the peptide and/or glycan moieties of fPPG. For these experiments we used wild-type and also phosphoglycan-deficient lpg2-/- parasites22. The glycans of fPPG were removed by mild acid hydrolysis and such deglycosylated fPPG completely lost its exacerbatory properties (Fig. 4f, g; Supplementary Fig. 3g–i). Furthermore, a chemically synthesized Leishmania phosphoglycan23 proved to be highly effective at exacerbating lpg2-/- L. mexicana infections (Fig. 4h; Supplementary Fig. 3j), whereas the serine-rich repeat motif, synthesized to mimic the backbone of L. mexicana fPPG13, 14, did not influence infections at all (Fig. 4i). Therefore, these data show that the glycan moieties of the egested fPPG were responsible for the exacerbation of infection.
Thus a new picture of the transmission of this vector-borne disease has emerged, showing how sand flies act as vectors of leishmaniasis. Proof of regurgitation as the mechanism of natural parasite delivery led to the discovery of a new component of the infective inoculum, the PSG. This can be added to the existing part that PSG plays in creating a 'blocked fly' that experiences difficulty in feeding, leading to multiple and longer feeding attempts and more opportunities for transmission2, 3, 24. The active ingredient in PSG that led to long-term disease exacerbation was identified as fPPG, showing the advantage of secreted phosphoglycans for transmission. Thus this report demonstrates that L. mexicana uses secreted fPPG to manipulate both insect vector and mammalian host. This dual role of fPPG ensures efficient transmission and proves to be a highly beneficial survival strategy. Given the selection pressures in the evolution of infectious diseases, it is not surprising that Leishmania has found intriguing and novel ways to secure its own transmission.
Methods
Female L. longipalpis were infected by feeding on 2 106 L. mexicana lesion amastigotes per ml rabbit blood3. L. longipaplis is not the natural vector of L. mexicana but has been shown to support the complete development of the parasite and transmit the infection to mice through biting3. For the generation of mutant PSG, flies were infected with 6 106 sap1/2-/- (ref. 19), ppg2-/- (ref. 20) and lpg1-/- (ref. 21) amastigotes/promastigotes in their first passage, to retain their infectivity. This modification was required because these mutants did not infect flies as efficiently as wild-type parasites. Flies were maintained for 7–9 days to allow mature infections to develop. To obtain egested metacyclics, groups of 50 L. mexicana-infected flies then had the opportunity to feed through a chick skin on promastigote culture medium3. Individual flies that alighted on the apparatus and began feeding were observed, immediately removed from the cage when they retracted their mouthparts, and kept in a separate container. After 1 h the feeder was withdrawn and the egestion medium processed to reveal the number of egested parasites regurgitated by sand flies in 2 ml of egestion medium, which were concentrated by centrifugation and counted. The sand flies were dissected and morphological analysis of parasites was performed3. Foregut populations were regarded as parasites located anterior of the oesophagus–pharynx junction. Microcapillary forced feeding was performed as described25.
Groups of 8–10 mice were infected either by individual sand fly bite or by subcutaneous inoculation of 20 µl containing metacyclic promastigotes, with or without test materials (such as PSG and saliva), in the upper surface of the right hind foot. Sand flies with 7-day-old infections were introduced singly into a cage with an anaesthetized mouse that was screened from the fly except for its right hind leg. Flies were removed from the cage when they had taken a single feeding attempt and were then checked for infection by microscopy. Lesion development was monitored by measuring the swelling of the foot with Vernier calipers. At the end of experiments mice were humanely killed, their feet removed and parasite burdens determined by homogenization and counting.
Cultured metacyclic promastigotes were generated as described26, except that Grace's culture medium (Invitrogen), supplemented with 10% (v/v) fetal calf serum and adjusted to pH 5.5, was used. Such L. mexicana cultured metacyclic promastigotes were of equal infectivity to those egested by sand flies (Supplementary Information Fig. 4), and were used in experiments in which higher numbers of parasites were required.
High molecular weight PPGs were analysed by SDS–PAGE using an enlarged 4% polyacrylamide stacking gel, as described13, and by immunoblotting with monoclonal antibodies AP3, LT6, LT15, WIC108.3 and anti-HF-deglycosylated fPPG13, 27. Infected gut homogenates were prepared as described3, centrifuged to remove cells and debris, and the supernatant mixed with an equal volume of 2 gel sample buffer. Proteophosphoglycans were concentrated by adsorption from 1 ml volumes of egestion medium by incubation with 20% (v/v) DEAE-Sepharose (Pharmacia), beads were washed in PBS (10 mM sodium phosphate, 145 mM sodium chloride, pH 7.2) and proteophosphoglycans were eluted into 50 µl gel sample buffer.
Salivary glands were obtained by dissection of 6–8-day-old female flies in PBS on cooled slides and transferred to a clean slide. This was followed by puncture of individual glands with fine entomological needles in 5 µl PBS. The saliva released was centrifuged and stored at -70 °C. Crude whole salivary gland homogenate or sonicate was deliberately not used to avoid contamination of saliva with salivary gland epithelial cell components1. Salivary gland homogenate was found to produce significantly more disease exacerbation than saliva (Supplementary Information Fig. 5). PSG plugs were isolated from the midguts of day 7 infected flies3, and PSG was separated from the parasites by dispersing in PBS (5 µl per plug) followed by four rounds of centrifugation (10,000 g, 5 min) and retention of supernatant, then stored at -70 °C. Saliva and PSG were quantified using the BCA (bicinchoninic acid) protein assay (Pierce). Sand flies contained, on average, 0 97 µg saliva and 0 86 µg PSG per fly.
Filamentous PPG was fractionated from PSG by non-reducing SDS–PAGE, followed by resuspension in culture medium. Twelve PSG plugs were separated (three plugs four lanes) and the stacking gel (fPPG fraction) was removed from and processed separately to the resolving gel (other PPGs and protein fraction). Equal volumes of gel, including a control piece of acrylamide (control fraction), were washed in PBS then homogenized in 1 ml culture medium using a Teflon coated pestle until it could be injected freely using a 21-gauge needle. After centrifugation (10,000 g, 5 min) the supernatants were combined with parasites to infect mice. Mild acid hydrolysis (pH 2, 60 °C, 1 h) was performed as described28. Synthetic phosphoglycan was synthesized as described23 and the peptide mimicking the fPPG backbone was prepared commercially (Pepsyn). Each were used at 1 µg per infection.
Supplementary information accompanies this paper.
Received 3 February 2004; accepted 18 May 2004

References
1. Sacks, D. L. & Kamhawi, S. Molecular aspects of parasite-vector and vector-host interactions in leishmaniasis. Annu. Rev. Microbiol. 55, 453–483 (2001) | Article | PubMed | ISI | ChemPort |
2. Stierhof, Y.-D. et al. Filamentous proteophosphoglycan secreted by Leishmania promastigotes forms gel-like three-dimensional networks that obstruct the digestive tract of infected sandfly vectors. Eur. J. Cell Biol. 78, 675–689 (1999) | PubMed | ISI | ChemPort |
3. Rogers, M. E., Chance, M. L. & Bates, P. A. The role of promastigote secretory gel in the origin and transmission of the infective stage of Leishmania mexicana by the sandfly Lutzomyia longipalpis. Parasitol. 124, 495–507 (2002) | Article | ISI | ChemPort |
4. UNDP/World Bank/WHO. Leishmaniasis http://www.who.int/tdr/diseases/leish/diseaseinfo.htm (2004).
5. Sacks, D. L. & Perkins, P. V. Identification of an infective stage of Leishmania promastigotes. Science 223, 1417–1419 (1984) | PubMed | ISI | ChemPort |
6. Titus, R. G. & Ribeiro, J. M. Salivary gland lysates from the sand fly Lutzomyia longipalpis enhance Leishmania infectivity. Science 239, 1306–1308 (1988) | PubMed | ISI | ChemPort |
7. Belkaid, Y. et al. Development of a natural model of cutaneous leishmaniasis: powerful effects of vector saliva and saliva preexposure on the long-term outcome of Leishmania major infection in the mouse ear dermis. J. Exp. Med. 188, 1941–1953 (1998) | Article | PubMed | ISI | ChemPort |
8. Schlein, Y., Jacobson, R. L. & Messer, G. Leishmania infections damage the feeding mechanism of the sandfly vector and implement parasite transmission by bite. Proc. Natl Acad. Sci. USA 89, 9944–9948 (1992) | PubMed | ChemPort |
9. Pimenta, P. F. et al. Stage-specific adhesion of Leishmania promastigotes to the sandfly midgut. Science 256, 1812–1815 (1992) | PubMed | ISI | ChemPort |
10. Sacks, D. L. et al. The role of phosphoglycans in Leishmania-sand fly interactions. Proc. Natl Acad. Sci. USA 97, 406–411 (2000) | Article | PubMed | ChemPort |
11. Späth, G. F. et al. Lipophosphoglycan is a virulence factor distinct from related glycoconjugates in the protozoan parasite Leishmania major. Proc. Natl Acad. Sci. USA 97, 9258–9263 (2000) | Article | PubMed | ISI | ChemPort |
12. Späth, G. F. et al. Persistence without pathology in phosphoglycan-deficient Leishmania major. Science 301, 1241–1243 (2003) | Article | PubMed | ISI | ChemPort |
13. Ilg, T. et al. Purification and structural characterization of a filamentous, mucin-like proteophosphoglycan secreted by Leishmania parasites. J. Biol. Chem. 271, 21583–21596 (1996) | Article | PubMed | ISI | ChemPort |
14. Ilg, T. Proteophosphoglycans of Leishmania. Trends Parasitol. 16, 489–497 (2000) | Article | ChemPort |
15. Warburg, A. & Schlein, Y. The effect of post-bloodmeal nutrition of Phlebotomus papatasi on the transmission of Leishmania major. Am. J. Trop. Med. Hyg. 35, 926–930 (1986) | PubMed | ISI | ChemPort |
16. Saraiva, E. M. B. et al. Changes in lipophosphoglycan and gene expression associated with the development of Leishmania major in Phlebotomus papatasi. Parasitol. 111, 275–287 (1995) | ISI | ChemPort |
17. Kamhawi, S., Belkaid, Y., Modi, G., Rowton, E. & Sacks, D. L. Protection against cutaneous leishmaniasis resulting from bites of uninfected sand flies. Science 290, 1351–1354 (2000) | Article | PubMed | ISI | ChemPort |
18. Castro-Sousa, F. et al. Dissociation between vasodilation and Leishmania-enhancing effects of sand fly saliva and maxadilan. Mem. Inst. Oswaldo Cruz 96, 997–999 (2001) | PubMed | ISI | ChemPort |
19. Wiese, M., Ilg, T., Lottspeich, F. & Overath, P. Ser/Thr-rich repetitive motifs as targets for phosphoglycan modifications in Leishmania mexicana secreted acid phosphatase. EMBO J. 14, 1067–1074 (1995) | PubMed | ISI | ChemPort |
20. Gopfert, U. et al. Proteophosphoglycans of Leishmania mexicana. Molecular characterization of the Leishmania mexicana ppg2 gene encoding the protephosphoglycans aPPG and pPPG2 that are secreted by amastigotes and promastigotes. Biochem. J. 344, 787–795 (1999) | Article | PubMed | ISI | ChemPort |
21. Ilg, T. Lipophosphoglycan is not required for infection of macrophages or mice by Leishmania mexicana. EMBO J. 19, 1953–1962 (2000) | Article | PubMed | ISI | ChemPort |
22. Ilg, T., Demar, M. & Harbecke, D. Phosphoglycan repeat-deficient Leishmania mexicana parasites remain infectious to macrophages and mice. J. Biol. Chem. 276, 4988–4997 (2001) | Article | PubMed | ISI | ChemPort |
23. Nikolaev, A. V., Chudek, J. A. & Ferguson, M. A. J. The chemical synthesis of Leishmania donovani phosphoglycan via polycondensation of a glycobiosyl hydrogen phosphonate monomer. Carbohydr. Res. 272, 179–189 (1995) | Article | PubMed | ISI | ChemPort |
24. Beach, R., Kiilu, G. & Leeuwenburg, J. Modification of sandfly biting behaviour by Leishmania leads to increased parasite transmission. Am. J. Trop. Med. Hyg. 34, 278–282 (1985) | PubMed | ISI | ChemPort |
25. Hertig, M. & McConnell, E. Experimental infection of Panamanian Phlebotomus sandflies with Leishmania. Exp. Parasitol. 14, 92–106 (1963) | Article | PubMed | ISI | ChemPort |
26. Bates, P. A. & Tetley, L. Leishmania mexicana: induction of metacyclogenesis by cultivation of promastigotes at acidic pH. Exp. Parasitol. 76, 412–423 (1993) | Article | PubMed | ISI | ChemPort |
27. Ilg, T., Harbecke, D., Wiese, M. & Overath, P. Monoclonal antibodies directed against Leishmania secreted acid phosphatase and lipophosphoglycan. Eur. J. Biochem. 217, 603–615 (1993) | PubMed | ISI | ChemPort |
28. Bates, P. A., Hermes, I. & Dwyer, D. M. Golgi-mediated post-translational processing of secretory acid phosphatase by Leishmania donovani promastigotes. Mol. Biochem. Parasitol. 39, 247–256 (1990) | Article | PubMed | ISI | ChemPort |
Acknowledgements. The technical assistance of D. Moor and J. Archer is acknowledged. We thank M. Hajmova and P. Volf for assistance with the forced feeding technique and M. Wiese and P. Overath for antibodies and Leishmania mutants. This work received financial support from the UNDP/World Bank/WHO Special Programme for Research and Training in Tropical Diseases (TDR) and the Wellcome Trust, UK.
Competing interests statement. The authors declare that they have no competing financial interests.

© 2004 Nature Publishing Group
Privacy Policy