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Micro-CT visualization of a promastigote secretory gel (PSG) and parasite plug in the digestive tract of the sand fly Lutzomyia longipalpis infected with Leishmania mexicana.

ABSTRACT: Leishmaniasis is a debilitating disease of the tropics, subtropics and southern Europe caused by Leishmania parasites that are transmitted during blood feeding by phlebotomine sand flies (Diptera: Psychodidae). Using non-invasive micro-computed tomography, we were able to visualize the impact of the laboratory model infection of Lutzomyia longipalpis with Leishmania mexicana and its response to a second blood meal. For the first time we were able to show in 3D the plug of promastigote secretory gel (PSG) and parasites in the distended midgut of whole infected sand flies and measure its volume in relation to that of the midgut. We were also able to measure the degree of opening of the stomodeal valve and demonstrate the extension of the PSG and parasites into the pharynx. Although our pilot study could only examine a few flies, it supports the hypothesis that a second, non-infected, blood meal enhances parasite transmission as we showed that the thoracic PSG-parasite plug in infected flies after a second blood meal was, on average, more than twice the volume of the plug in infected flies that did not have a second blood meal.

SUBMITTER: Hall MJR

PROVIDER: S-EPMC8396784 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:BackgroundImmunity to the sand fly salivary protein SALO (Salivary Anticomplement of Lutzomyia longipalpis) protected hamsters against Leishmania infantum and L. braziliensis infection and, more recently, a vaccine combination of a genetically modified Leishmania with SALO conferred strong protection against L. donovani infection. Because of the importance of SALO as a potential component of a leishmaniasis vaccine, a plan to produce this recombinant protein for future scale manufacturing as well as knowledge of its structural characteristics are needed to move SALO forward for the clinical path.Methodology/principal findingsRecombinant SALO was expressed as a soluble secreted protein using Pichia pastoris, rSALO(P), with yields of 1g/L and >99% purity as assessed by SEC-MALS and SDS-PAGE. Unlike its native counterpart, rSALO(P) does not inhibit the classical pathway of complement; however, antibodies to rSALO(P) inhibit the anti-complement activity of sand fly salivary gland homogenate. Immunization with rSALO(P) produces a delayed type hypersensitivity response in C57BL/6 mice, suggesting rSALO(P) lacked anti-complement activity but retained its immunogenicity. The structure of rSALO(P) was solved by S-SAD at Cu-Kalpha to 1.94 Å and refined to Rfactor 17%. SALO is ~80% helical, has no appreciable structural similarities to any human protein, and has limited structural similarity in the C-terminus to members of insect odorant binding proteins. SALO has three predicted human CD4+ T cell epitopes on surface exposed helices.Conclusions/significanceThe results indicate that SALO as expressed and purified from P. pastoris is suitable for further scale-up, manufacturing, and testing. SALO has a novel structure, is not similar to any human proteins, is immunogenic in rodents, and does not have the anti-complement activity observed in the native salivary protein which are all important attributes to move this vaccine candidate forward to the clinical path.

Project description:BackgroundPhlebotomine sand flies transmit the haemoflagellate Leishmania, the causative agent of human leishmaniasis. The Leishmania promastigotes are confined to the gut lumen and are exposed to the gut microbiota within female sand flies. Here we study the colonisation resistance of yeast and bacteria in preventing the establishment of a Leishmania population in sand flies and the ability of Leishmania to provide colonisation resistance towards the insect bacterial pathogen Serratia marcescens that is also pathogenic towards Leishmania.MethodsWe isolated microorganisms from wild-caught and laboratory-reared female Lutzomyia longipalpis, identified as Pseudozyma sp. Asaia sp. and Ochrobactrum intermedium. We fed the females with a sugar meal containing the microorganisms and then subsequently fed them with a bloodmeal containing Leishmania mexicana and recorded the development of the Leishmania population. Further experiments examined the effect of first colonising the sand fly gut with L. mexicana followed by feeding with, Serratia marcescens, an insect bacterial pathogen. The mortality of the flies due to S. marcescens was recorded in the presence and absence of Leishmania.ResultsThere was a reduction in the number of flies harbouring a Leishmania population that had been pre-fed with Pseudozyma sp. and Asaia sp. or O. intermedium. Experiments in which L. mexicana colonised the sand fly gut prior to being fed an insect bacterial pathogen, Serratia marcescens, showed that the survival of flies with a Leishmania infection was significantly higher compared to flies without Leishmania infection.ConclusionsThe yeast and bacterial colonisation experiments show that the presence of sand fly gut microorganisms reduce the potential for Leishmania to establish within the sand fly vector. Sand flies infected with Leishmania were able to survive an attack by the bacterial pathogen that would have killed the insect and we concluded that Leishmania may benefit its insect host whilst increasing the potential to establish itself in the sand fly vector. We suggest that the increased ability of the sand fly to withstand a bacterial entomopathogen, due to the presence of the Leishmania, may provide an evolutionary pressure for the maintenance of the Leishmania-vector association.

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Micro-CT visualization of a promastigote secretory gel (PSG) and parasite plug in the digestive tract of the sand fly Lutzomyia longipalpis infected with Leishmania mexicana.

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