Our research uncovered a new type of binding that explains how Leishmania can use almost every sand fly in the world as a vector. This interaction is crucial for the survival of the parasites in the midgut of the sand fly.

Inside the sand fly, Leishmania transform into a number of flagella promastigote forms, each of which plays a specific role in colonizing the vector for onward transmission. However, surviving in a sand fly is not easy.

The problem

How Leishmania live exclusively in the sand fly gut. This can present some unique problems for the parasite. First, they must withstand the hostile proteolytic environment of the blood meal when it is digested and survive attack by oxidative radicals. Next, they need to get out of the digested blood meal, which is trapped in a chitin-containing peritrophic matrix, before the sand fly faeces. Third, they must resist getting lost from the sand fly in defecation and find their way to the sand fly’s mouth parts to develop into its infectious form, the metacyclic promastigote. Here they face the final challenge of getting into the skin of the vertebrate host against the flow of an incoming meal with fresh blood without being washed back into the midgut.

Leishmania life cycle. Attribution: LadyofHats, public domain, via Wikimedia Commons

Midgut attachment

One of the most important steps to surviving in a sand fly is not to get emptied with the digested blood meal.

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

In order to do this Leishmania They anchor themselves between the microvilli that line midgut tissue in a process that involves the “adhesin” parasite, lipophosphoglycan (LPG). This glycolipid covers the entire surface of promastigotes, which undergo significant modification when the parasites differentiate into the infectious metacyclic promastigote form. This prevents them from clinging to the intestines again and gives them the best chance of being transmitted.

Epidemiologically, sand flies fall into two categories – they are either restrictive or permissive. In restrictive sandfly species, nectomonad and leptomonad promastigotes are deposited by LPG in the early to middle phase of Leishmania Developmental linkage to a lectin expressed in the intestine; which leads to selective transmission of only one Leishmania Species. In contrast, most sand flies are more permissive and can accommodate a wide range of species Leishmania Species through an unknown mechanism. However, there is an indication of their attachment: Permitted sand flies line their intestines with a mucus that contains an abundance of a certain sugar, N-acetylgalactosamine (GalNAc).

Atomic force microscopy

Our collaboration between parasitologists, biophysicists and synthetic chemists, published in the Royal Society of Chemistry, explains how force spectroscopy was used to study the surface of nectomonads and metacyclic promastigotes Leishmania mexicana with tips coated with a GalNAc mimetic. We measured Leishmania Adhesion using an atomic force microscope (AFM) which can measure the adhesion of a few picoNewtons (pN). The AFM contains a tiny tip that is attached to the controller via a cantilever bracket.

Force spectroscopy image of a Leishmania promastigote. The color of each pixel represents the adherence of the AFM tip to the parasite. In this picture the tip was coated with sugar. The chemistry for this is remarkable. The tips have a nominal radius of approx. 20 nm; The 2 micron scale is 100 times larger than this! The areas of high adhesion on the sugar-coated tip of this parasite appear to be confined to a center line that we believe to be significant. From Hall et al. 2020.

When the tip touches a surface, the boom flexes a little. This bending can be measured, and from this the interaction force can be calculated and a nanoscale map of the surface adhesion can be created for each parasite.

With AFM, we tested the hypothesis that LPG may still be involved in attachment to acceptable sand fly guts through glycan-glycan interactions – that is, without lectins. We found that there was a direct interaction between this GalNAc and LPG that was limited to the well-adherent promastigote stages and could be blocked by the introduction of excess GalNAc. This new type of binding is comparable to the attachment of pili of well-pathogenic bacteria to the mucus of human intestinal epithelia and offers a new model for investigating the competence of sandflies for Leishmania and their transmission.

Transmission blockage

Data is collected from these experiments and previously published studies over Leishmania Infections in sand flies allowed us to mathematically model the likely performance of some form of vaccine that blocks transmission based on this mechanism. We found that such a vaccine can perform well, although this would make the problem worse if the vaccine wasn’t effective enough. More research is needed to distinguish this relationship and guide the search for the most suitable vaccine candidates.

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

So far it is unclear to what extent midgut adhesion contributes to vector competence for Leishmania transmission. But discovering that Leishmania Using their LPG to bind directly to sugars is a huge advancement in our understanding of the interaction between Leishmania Parasites and sand flies and possibly the vertebrate host after transmission.

What excites us most about the future is the enormous potential of biophysics and interdisciplinary research to study vector-parasite and vector-parasite-host interactions.


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