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1.  Stage-Specific Adhesion of Leishmania Promastigotes to Sand Fly Midguts Assessed Using an Improved Comparative Binding Assay 
The binding of Leishmania promastigotes to the midgut epithelium is regarded as an essential part of the life-cycle in the sand fly vector, enabling the parasites to persist beyond the initial blood meal phase and establish the infection. However, the precise nature of the promastigote stage(s) that mediate binding is not fully understood.
Methodology/Principal Findings
To address this issue we have developed an in vitro gut binding assay in which two promastigote populations are labelled with different fluorescent dyes and compete for binding to dissected sand fly midguts. Binding of procyclic, nectomonad, leptomonad and metacyclic promastigotes of Leishmania infantum and L. mexicana to the midguts of blood-fed, female Lutzomyia longipalpis was investigated. The results show that procyclic and metacyclic promastigotes do not bind to the midgut epithelium in significant numbers, whereas nectomonad and leptomonad promastigotes both bind strongly and in similar numbers. The assay was then used to compare the binding of a range of different parasite species (L. infantum, L. mexicana, L. braziliensis, L. major, L. tropica) to guts dissected from various sand flies (Lu. longipalpis, Phlebotomus papatasi, P. sergenti). The results of these comparisons were in many cases in line with expectations, the natural parasite binding most effectively to its natural vector, and no examples were found where a parasite was unable to bind to its natural vector. However, there were interesting exceptions: L. major and L. tropica being able to bind to Lu. longipalpis better than L. infantum; L. braziliensis was able to bind to P. papatasi as well as L. major; and significant binding of L. major to P. sergenti and L. tropica to P. papatasi was observed.
The results demonstrate that Leishmania gut binding is strictly stage-dependent, is a property of those forms found in the middle phase of development (nectomonad and leptomonad forms), but is absent in the early blood meal and final stages (procyclic and metacyclic forms). Further they show that although gut binding may be necessary for parasite establishment, in several vector-parasite pairs the specificity of such in vitro binding alone is insufficient to explain overall vector specificity. Other significant barriers to development must exist in certain refractory Leishmania parasite-sand fly vector combinations. A re-appraisal of the specificity of the Leishmania-sand fly relationship is required.
Author Summary
Many infectious diseases such as leishmaniasis are transmitted to people by biting insects, in this case by female sand flies. To control this and similar diseases we need to understand why particular species of sand fly transmit particular species of Leishmania. One important feature of the Leishmania parasite-sand fly interaction is the ability of the parasite to bind to the midgut wall of the fly, as it is within the gut that the parasite lives. Here we have studied the specificity of this interaction and report two main findings. The first is that only specific stages in the parasite life-cycle are capable of binding to the gut. The second is that, providing these life-cycle stages are analysed, parasite species that can be transmitted by particular sand flies are always capable of binding to their guts, but in some cases they are also capable of binding to non-transmitting sand fly species. This shows that gut binding by parasites is necessary but not sufficient to explain transmission. This research advances our understanding of Leishmania biology, but also shows us that there are further aspects that need to be investigated before we can fully understand the Leishmania-sand fly relationship.
PMCID: PMC2935393  PMID: 20838647
2.  Ecotin-like serine peptidase inhibitor ISP1 of Leishmania major plays a role in flagellar pocket dynamics and promastigote differentiation 
Cellular Microbiology  2012;14(8):1271-1286.
Leishmania ISPs are ecotin-like natural peptide inhibitors of trypsin-family serine peptidases, enzymes that are absent from the Leishmania genome. This led to the proposal that ISPs inhibit host serine peptidases and we have recently shown that ISP2 inhibits neutrophil elastase, thereby enhancing parasite survival in murine macrophages. In this study we show that ISP1 has less serine peptidase inhibitory activity than ISP2, and in promastigotes both are generally located in the cytosol and along the flagellum. However, in haptomonad promastigotes there is a prominent accumulation of ISP1 and ISP2 in the hemidesmosome and for ISP2 on the cell surface. An L. major mutant deficient in all three ISP genes (Δisp1/2/3) was generated and compared with Δisp2/3 mutants to elucidate the physiological role of ISP1. In in vitro cultures, the Δisp1/2/3 mutant contained more haptomonad, nectomonad and leptomonad promastigotes with elongated flagella and reduced motility compared with Δisp2/3 populations, moreover it was characterized by very high levels of release of exosome-like vesicles from the flagellar pocket. These data suggest that ISP1 has a primary role in flagellar homeostasis, disruption of which affects differentiation and flagellar pocket dynamics.
PMCID: PMC3440592  PMID: 22486816

Results 1-2 (2)