New insight into the interaction between Leishmania and sandfly midgut

Our research reveals a new mode of binding that explains how Leishmania can exploit almost any sandfly in the world as a vector. This interaction is essential for the survival of the parasite in the midgut of the sandfly.

In the sand fly, Leishmania turn into a number of flagellates promastigote forms, each having specific roles to play in colonizing the vector for further transmission. However, surviving in a sand fly is not easy.

The problem

As Leishmania live exclusively in the gut of sandflies, this can present unique problems for the parasite. First, they must resist the hostile proteolytic environment of blood meal during its digestion and survive attacks from oxidative radicals. Then they have to come out of the digested blood meal, which is locked in a chitinous peritrophic matrix, before the sand fly defecates. Third, they must resist the loss of the sand fly when it defecates and make its way to the mouthparts of the sandfly to develop into its infectious form, the metacyclic promastigote. Here, they face the ultimate challenge of making their way through the skin of the vertebrate host against the flow of an incoming meal of fresh blood without being drawn back into the midgut.

Leishmania life cycle. Attribution: LadyofHats, Public Domain, via Wikimedia Commons

Midgut attachment

One of the most critical steps to survive in a sand fly is to resist defecation with the digested blood meal.

Leishmania attaching to microvilli of the midgut of sand fly. Wikimedia Commons; attribution, Rod Dillon, Liverpool School of Tropical Medicine.

To do this Leishmania become anchored between the microvilli that line the midgut in a process involving the “adhesin” parasite lipophosphoglycan (LPG). This glycolipid covers the entire surface of the promastigotes, which undergoes a significant change when the parasites differentiate into an infectious metacyclic promastigote. This prevents reattachment to the gut, giving them the best chance of being transmitted.

Epidemiologically, sandflies fall into two categories – they are either restrictive or permissive. In restrictive sandfly species, attachment is provided by the LPG of nectomonad and leptomonad promastigotes in the early to middle phase of Leishmania development binding to a lectin expressed by the intestine; resulting in the selective transmission of a single Leishmania species. In contrast, most sandflies are more permissive and can harbor a wide range of Leishmania species by an unknown mechanism. However, one clue to their attachment stands out: Permissive sand flies line their gut with mucus that contains an abundance of a particular sugar, N-acetyl galactosamine (GalNAc).

Atomic force microscopy

Our collaborative work between parasitologists, biophysicists and synthetic chemists, published in the Royal Society of Chemistry, explains how force spectroscopy was used to probe the surface of nectomonad and metacyclic promastigotes of Leishmania mexicana with tips covered with a GalNAc imitator. We measure Leishmania adhesion using an atomic force microscope (AFM), capable of measuring the adhesion of a few picoNewtons (pN). The AFM contains a small spike which is attached to its controller via a cantilever bracket.

Image of a Leishmania promastigote using force spectroscopy. The color of each pixel represents the adhesion of the AFM tip to the parasite. In this image, the tip was covered in sugars. The chemistry for doing this is remarkable. The tips have a nominal radius of about 20 nm; the 2 micron scale bar is 100 times larger than that! The regions of strong adhesion to the sugar-coated tip of this parasite appear to be confined to a central line, which we believe could be significant. According to Hall et al. 2020.

When the tip comes in contact with a surface, the overhang bends a bit. This flexion can be measured, and from this the calculated interaction force and a nanoscale map of surface adhesion can be constructed for each parasite.

Using AFM, we tested the hypothesis that LPG might still be involved in the attachment to the permissive guts of the sand fly through glycan-glycan interactions – i.e. not involving lectins. We found that there was a direct interaction between this GalNAc and LPG which was limited to the intestinal promastigote stages and could be blocked by the introduction of excess GalNAc. This novel mode of binding is comparable to the attachment of pili from pathogenic intestinal bacteria to the mucus of the human intestinal epithelium and offers a new model to study the competence of sandflies for Leishmania and their transmission.

Transmission blockade

Data collected from these experiences and previously published studies on Leishmania infections in sandflies allowed us to mathematically model the likely performance of a form of vaccine blocking transmission based on this mechanism. We found that such a vaccine could work well, although if the vaccine was not effective enough it would make the problem worse. Further research is needed to unravel this relationship in order to guide the search for the most appropriate vaccine candidates.

A new hypothesis for the competence of permissive vectors of the sand fly?

At this time, it is not known to what extent midgut adhesion contributes to vector competence for Leishmania transmission. But the discovery that Leishmania can use their LPG to bind directly to sugars is a big step forward in our understanding of the interplay between Leishmania parasites and sandflies and possibly the vertebrate host after transmission.

Looking to the future, what excites us most is the enormous potential that biophysics and interdisciplinary research have to offer to explore vector-parasite and vector-parasite-host interactions.

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