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

Chemical and biological control of phlebotominae sand flies

Phlebotominae sand flies (Diptera: Phlebotominae) are important vectors of leishmaniasis. Control measures are complicated by the fact that sand fly breeding sites and resting places are generally hard to find. Measures used to control adult sand flies include the use of chemical insecticides for insecticide-treated bednets or curtains, residual spraying of dwellings, eventually the space-spraying. Domestic dogs as reservoir host of visceral leishmaniosis can be protected by dog-collars impregnated with insecticides or by spot on application of insecticides. Chemical insecticides may be toxic for non-target organisms and the increase of insecticide-resistance of some sand flies populations is possible. Therefore, new methods of biological control should be tested; entomopathogenic organisms (Bacillus sphaericus, B. thuringiensis, Beauveria bassiana), pheromone-baited traps and noxious plants could be used. Hormone TMOF, inhibitor of trypsin biosynthesis, was successfully tested in mosquitos. Based on similarity of mosquitos and sand flies, the effect of this peptide on blood digestion and egg development is tested in Phlebotomus and Lutzomyia species

Bloodmeal digestion of phlebotomine sand flies and its effect on Leishmania development

Leishmania development in their vectors is closely connected with bloodmeal digestion. This thesis focuses on factors affecting bloodmeal digestion, egg development and Leishmania infection within the sand fly gut. First, we compared the effect of mammalian (rabbit) and avian (chicken) blood on digestion and eggs development in Phlebotomus duboscqi. Sand flies fed on chickens had twice lower protein concentrations in the midgut and significantly lower trypsin activity compared to those fed on rabbits. The highest differences in the trypsin activity were observed during first 24 hours post bloodmeal. In addition, females fed on chickens had slower eggs development and their eggs were 10 % smaller compared to those fed on rabbits. In the second part of the thesis we tested the effect of mosquito hormone TMOF on the trypsin activity and eggs development of Lutzomyia longipalpis. Rabbit blood with TMOF (28 mg/ml) was presented to the females via a membrane feeding system. Sand flies fed on blood with TMOF had 15 - 35 % less trypsin activity than control females fed on only rabbit blood. In addition, females fed on blood with TMOF had developed 30 % less eggs and their eggs were 12 - 24 % smaller compared to control group. However the effect of TMOF we observed was lower than that described previously..

Leishmania development in sand flies during bloodmeal digestion

This thesis is focused on the bloodmeal digestion of phlebotomine sand flies and its effects on Leishmania development within their midguts. In the first part, we studied various parameters of bloodmeal digestion in four sand fly species differing in susceptibility to Leishmania donovani to evaluate the effects on vector competence. Both proven vectors of L. donovani (Phlebotomus orientalis and P. argentipes) showed lower trypsin activity and slower formation of the peritrophic matrix (PM) than refractory species (P. papatasi and Sergentomyia schwetzi). Remarkably, P. orientalis and P. argentipes strikingly differed from each other in a time course of bloodmeal digestion. Phlebotomus orientalis females showed very slow bloodmeal digestion with low peaks of proteolytic activities and defecated around day five post bloodmeal. In contrast, P. argentipes females digested faster with a very high peak of chymotrypsin activity, their PM was present for only a short time and defecation was finished by day three post bloodmeal. We presume that the period between the degradation of the PM and defecation (i.e. time frame when Leishmania bind to the midgut to avoid expulsion with bloodmeal remnants), is one of crucial parameters affecting the establishment of Leishmania in the sand flies. In both natural..

Chemical and biological control of phlebotominae sand flies

Phlebotominae sand flies (Diptera: Phlebotominae) are important vectors of leishmaniasis. Control measures are complicated by the fact that sand fly breeding sites and resting places are generally hard to find. Measures used to control adult sand flies include the use of chemical insecticides for insecticide-treated bednets or curtains, residual spraying of dwellings, eventually the space-spraying. Domestic dogs as reservoir host of visceral leishmaniosis can be protected by dog-collars impregnated with insecticides or by spot on application of insecticides. Chemical insecticides may be toxic for non-target organisms and the increase of insecticide-resistance of some sand flies populations is possible. Therefore, new methods of biological control should be tested; entomopathogenic organisms (Bacillus sphaericus, B. thuringiensis, Beauveria bassiana), pheromone-baited traps and noxious plants could be used. Hormone TMOF, inhibitor of trypsin biosynthesis, was successfully tested in mosquitos. Based on similarity of mosquitos and sand flies, the effect of this peptide on blood digestion and egg development is tested in Phlebotomus and Lutzomyia species

Leishmania development in sand flies during bloodmeal digestion

This thesis is focused on the bloodmeal digestion of phlebotomine sand flies and its effects on Leishmania development within their midguts. In the first part, we studied various parameters of bloodmeal digestion in four sand fly species differing in susceptibility to Leishmania donovani to evaluate the effects on vector competence. Both proven vectors of L. donovani (Phlebotomus orientalis and P. argentipes) showed lower trypsin activity and slower formation of the peritrophic matrix (PM) than refractory species (P. papatasi and Sergentomyia schwetzi). Remarkably, P. orientalis and P. argentipes strikingly differed from each other in a time course of bloodmeal digestion. Phlebotomus orientalis females showed very slow bloodmeal digestion with low peaks of proteolytic activities and defecated around day five post bloodmeal. In contrast, P. argentipes females digested faster with a very high peak of chymotrypsin activity, their PM was present for only a short time and defecation was finished by day three post bloodmeal. We presume that the period between the degradation of the PM and defecation (i.e. time frame when Leishmania bind to the midgut to avoid expulsion with bloodmeal remnants), is one of crucial parameters affecting the establishment of Leishmania in the sand flies. In both natural..

Analysis of parasite infection intensities in the sand fly midgut.

<p>A) Rates and intensity of infections with representative parasite lines were scored by light microscopy at day 2, 5 or 6, 9 and 12 PBM. Infections were considered according to infected sand fly species and scored as light (<100 parasites/gut), moderate (100–1000 parasites/gut), heavy (>1000 parasites/gut) and very heavy (>>1000 parasites/gut) as described in Materials and Methods. Sand fly infection experiments were repeated at least 3x for each line. Differences between lines were analysed by χ² test: in P. (P.) papatasi day 2 PBM, P = 0.083, χ² = 11.18, df = 6; day 5 PBM, P<0.001, χ² = 55.7, df = 12; day 9 PBM, P<0.001, χ² = 72.05, df = 12; day 12 PBM, P<0.001, χ² = 74.422, df = 12; and in P. (P.) duboscqi day 6 PBM, P = 0.028, χ² = 38.835, df = 24; and day 12 PBM, P<0.001, χ² = 55.95, df = 24. B) Light microscopic scoring was verified by qPCR analysis of parasite DNA content per midgut of 30 sand flies at day 12 PBM per parasite line shown in A). Data were normalized (log₁₀[x+1] transformation) and analysed by a non-parametric one-way ANOVA (Kruskal-Wallis test + Dunns pair wise post-test): in P. (P.) papatasi no significant difference was described (P = 0.1615); the same test showed a significant difference for tested L. (L.) major lines in P. (P.) duboscqi (P<0.001). A Dunn’s Multiple Comparison post-test showed significant difference in HASPB sKI vs. FVI, cDNA16 dKO, cDNA16 sKI, HASPA1 sKI, HASPA1/2 sKI (all P≤0.001) and HASPAs sKI (P≤0.05); SHERP sKI vs. FVI (P≤0.01), cDNA16 sKI (P≤0.05), HASPA1 sKI (P≤0.001) and HASPA1/2 sKI (P≤0.05), HASPA1 sKI vs. HASPA1/2 sKI (P≤0.05).</p

Analysis of cultured and midgut-derived metacyclic parasites by confocal imaging.

<p>Detection of HASPB and SHERP expression by immunofluorescent antibody labelling in the parental line (FVI), cDNA16 sKI, cDNA16 dKO, HASPB sKI and SHERP sKI mutants, collected from day 7 p.i. M199 culture and day 12 PBM midguts. DAPI (white) stained nucleus and kinetoplastid DNA; HASPB or SHERP (green) were detected by specific mononuclear antibodies (rabbit), which was tagged by a secondary 488 anti-rabbit antibody. Size bar = 10 μm.</p

Assessing the impact of different growth conditions on HASP and SHERP expression.

<p>A) Growth curves of parasite lines (FVI, cDNA16 dKO, cDNA16 sKI, HASPA2 sKI) without (-ve) or with day 6 (+Day 6) or day 12 (+Day 12) PBM filter-sterilized midgut homogenates in M199 medium. B) Immunoblots from two repeats of FVI, cDNA16 dKO, cDNA16 sKI grown in M199 with (+) or without (-) day 6 and 12 PBM midgut homogenates from uninfected blood-fed female sand flies. C) Fold difference of HASPA and HASPB expression in FVI and cDNA16 sKI grown with day 6 or 12 PBM midgut homogenates compared to no homogenates. cDNA16 dKO served as a negative control for HASPA and HASPB detection. D) Immunoblots of HASPA2 sKI and HASPB sKI grown with or without day 6 or 12 PBM midgut homogenates probed for HASPA and HASPB. NMT served in all blots as a loading control.</p

Quantitative PCR analysis of mRNA from cultured and midgut-derived parasites.

<p>mRNA was extracted from cultured and midgut-derived FVI, cDNA16 dKO, cDNA16 sKI, HASPAB sKI, SHERP sKI and HASPA2 sKI parasites at day 3, 5 and 7 p.i. and day 6, 9 and 12 PBM, respectively. Mean C_t values were normalized against the housekeeping gene NMT and ΔΔC_t values were generated against FVI day 6 PBM for midgut-derived and day 3 p.i. for culture-derived samples. Fold-differences for HASPAs, HASPB and SHERP mRNA were plotted.</p