Leishmania HASP and SHERP Genes are Required for In Vivo Differentiation, Parasite Transmission and Virulence Attenuation in the Host

Differentiation of extracellular Leishmania promastigotes within their sand fly vector, termed metacyclogenesis, is considered to be essential for parasites to regain mammalian host infectivity. Metacyclogenesis is accompanied by changes in the local parasite environment, including secretion of complex glycoconjugates within the promastigote secretory gel and colonization and degradation of the sand fly stomodeal valve. Deletion of the stage-regulated HASP and SHERP genes on chromosome 23 of Leishmania major is known to stall metacyclogenesis in the sand fly but not in in vitro culture. Here, parasite mutants deficient in specific genes within the HASP/SHERP chromosomal region have been used to investigate their role in metacyclogenesis, parasite transmission and establishment of infection. Metacyclogenesis was stalled in HASP/SHERP mutants in vivo and, although still capable of osmotaxis, these mutants failed to secrete promastigote secretory gel, correlating with a lack of parasite accumulation in the thoracic midgut and failure to colonise the stomodeal valve. These defects prevented parasite transmission to a new mammalian host. Sand fly midgut homogenates modulated parasite behaviour in vitro, suggesting a role for molecular interactions between parasite and vector in Leishmania development within the sand fly. For the first time, stage-regulated expression of the small HASPA proteins in Leishmania (Leishmania) has been demonstrated: HASPA2 is expressed only in extracellular promastigotes and HASPA1 only in intracellular amastigotes. Despite its lack of expression in amastigotes, replacement of HASPA2 into the null locus background delays onset of pathology in BALB/c mice. This HASPA2-dependent effect is reversed by HASPA1 gene addition, suggesting that the HASPAs may have a role in host immunomodulation

Discovery of essential kinetoplastid-insect adhesion proteins and their function in Leishmania -sand fly interactions

Leishmania species, members of the kinetoplastid parasites, cause leishmaniasis, a neglected tropical disease, in millions of people worldwide. Leishmania has a complex life cycle with multiple developmental forms, as it cycles between a sand fly vector and a mammalian host; understanding their life cycle is critical to understanding disease spread. One of the key life cycle stages is the haptomonad form, which attaches to insect tissues through its flagellum. This adhesion, conserved across kinetoplastid parasites, is implicated in having an important function within their life cycles and hence in disease transmission. Here, we discover the kinetoplastid-insect adhesion proteins (KIAPs), which localise in the attached Leishmania flagellum. Deletion of these KIAPs impairs cell adhesion in vitro and prevents Leishmania from colonising the stomodeal valve in the sand fly, without affecting cell growth. Additionally, loss of parasite adhesion in the sand fly results in reduced physiological changes to the fly, with no observable damage of the stomodeal valve and reduced midgut swelling. These results provide important insights into a comprehensive understanding of the Leishmania life cycle, which will be critical for developing transmission-blocking strategies

Whole cell reconstructions of Leishmania mexicana through the cell cycle

The unicellular parasite Leishmania has a precisely defined cell architecture that is inherited by each subsequent generation, requiring a highly coordinated pattern of duplication and segregation of organelles and cytoskeletal structures. A framework of nuclear division and morphological changes is known from light microscopy, yet this has limited resolution and the intrinsic organisation of organelles within the cell body and their manner of duplication and inheritance is unknown. Using volume electron microscopy approaches, we have produced three-dimensional reconstructions of different promastigote cell cycle stages to give a spatial and quantitative overview of organelle positioning, division and inheritance. The first morphological indications seen in our dataset that a new cell cycle had begun were the assembly of a new flagellum, the duplication of the contractile vacuole and the increase in volume of the nucleus and kinetoplast. We showed that the progression of the cytokinesis furrow created a specific pattern of membrane indentations, while our analysis of sub-pellicular microtubule organisation indicated that there is likely a preferred site of new microtubule insertion. The daughter cells retained these indentations in their cell body for a period post-abscission. By comparing cultured and sand fly derived promastigotes, we found an increase in the number and overall volume of lipid droplets in the promastigotes from the sand fly, reflecting a change in their metabolism to ensure transmissibility to the mammalian host. Our insights into the cell cycle mechanics of Leishmania will support future molecular cell biology analyses of these parasites

Peritrophic matrix of Phlebotomus duboscqi and its kinetics during Leishmania major development

Light microscopy of native preparations, histology, and electron microscopy have revealed that Phlebotomus duboscqi belongs to a class of sand fly species with prompt development of the peritrophic matrix (PM). Secretion of electron-lucent fibrils, presumably chitin, starts immediately after the ingestion of a blood meal and, about 6 h later, is followed by secretion of amorphous electron-dense components, presumably proteins and glycoproteins. The PM matures in less than 12 h and consists of a thin laminar outer layer and a thick amorphous inner layer. No differences have been found in the timing of the disintegration of the PM in females infected with Leishmania major. In both groups of females (infected and uninfected), the disintegration of the PM is initiated at the posterior end. Although parasites are present at high densities in the anterior part of the blood meal bolus, they escape from the PM at the posterior end only. These results suggest that L. major chitinase does not have an important role in parasite escape from the PM. Promastigotes remain in the intraperitrophic space until the PM is broken down by sand-fly-derived chitinases and only then migrate anteriorly. Disintegration of the PM occurs simultaneously with the morphological transformation of parasites from procyclic forms to long nectomonads. A novel role is ascribed to the anterior plug, a component of the PM secreted by the thoracic midgut; this plug functions as a temporary barrier to stop the forward migration of nectomonads to the thoracic midgut

Vývoj leishmanií v přenašečích rodu Phlebotomus

DISSERTATION ABSTRACT Development of Leishmania parasites in vector sandflies (Diptera: Phlebotominae) Jovana Sádlová Introduction. The work is dealing with two topics: (1) factors affecting development of Leishmania parasites in sandfly vectors and (2) mechanism of Leishmania transmission to the vertebrate host. It includes a review on Leishmania - sandfly - host interactions, four papers published in scientific journals and one manuscript submitted for publication. (1) Within the sandfly gut, Leishmania promastigotes encounter a number of factors which affect their development. They are usually well-adapted to the habitat inside the gut of their natural vectors, but characters of the parasites crucial for the development in the vector have not been elucidated. The main cell-surface molecules of promastigotes, lipophosphoglycan (LPG) and metalloprotease gp63, are known as the most important virulence factors influencing Leishmania interactions with the vertebrate host. However, their role in the vector part of Leishmania life-cycle remains to be clarified. The idea of the work is based on the fact that there are differences in the courses of sandfly infections with various strains and lines of L. major. Therefore, a detailed characterization of individual strains/lines may lead to the recognition of a..

Vývoj leishmanií v přenašečích rodu Phlebotomus

DISSERTATION ABSTRACT Development of Leishmania parasites in vector sandflies (Diptera: Phlebotominae) Jovana Sádlová Introduction. The work is dealing with two topics: (1) factors affecting development of Leishmania parasites in sandfly vectors and (2) mechanism of Leishmania transmission to the vertebrate host. It includes a review on Leishmania - sandfly - host interactions, four papers published in scientific journals and one manuscript submitted for publication. (1) Within the sandfly gut, Leishmania promastigotes encounter a number of factors which affect their development. They are usually well-adapted to the habitat inside the gut of their natural vectors, but characters of the parasites crucial for the development in the vector have not been elucidated. The main cell-surface molecules of promastigotes, lipophosphoglycan (LPG) and metalloprotease gp63, are known as the most important virulence factors influencing Leishmania interactions with the vertebrate host. However, their role in the vector part of Leishmania life-cycle remains to be clarified. The idea of the work is based on the fact that there are differences in the courses of sandfly infections with various strains and lines of L. major. Therefore, a detailed characterization of individual strains/lines may lead to the recognition of a..