Leishmaniasis is widely regarded as a vaccine-preventable disease, but the costs required to reach pivotal Phase 3 studies and uncertainty about which candidate vaccines should be progressed into human studies significantly limits progress in vaccine development for this neglected tropical disease. Controlled human infection models (CHIMs) provide a pathway for accelerating vaccine development and to more fully understand disease pathogenesis and correlates of protection. Here, we describe the isolation, characterization and GMP manufacture of a new clinical strain of Leishmania major. Two fresh strains of L. major from Israel were initially compared by genome sequencing, in vivo infectivity and drug sensitivity in mice, and development and transmission competence in sand flies, allowing one to be selected for GMP production. This study addresses a major roadblock in the development of vaccines for leishmaniasis, providing a key resource for CHIM studies of sand fly transmitted cutaneous leishmaniasis.
- MeSH
- fylogeneze MeSH
- hmyz - vektory parazitologie MeSH
- Leishmania major genetika růst a vývoj fyziologie MeSH
- leishmanióza kožní parazitologie přenos MeSH
- lidé MeSH
- modely nemocí na zvířatech MeSH
- myši inbrední BALB C MeSH
- paraziti genetika MeSH
- Psychodidae parazitologie MeSH
- sekvenování celého genomu MeSH
- zvířata MeSH
- Check Tag
- lidé MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
- Geografické názvy
- Izrael MeSH
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.
- MeSH
- antigeny protozoální metabolismus MeSH
- buněčná diferenciace fyziologie MeSH
- fluorescenční protilátková technika MeSH
- hmyz - vektory parazitologie MeSH
- imunoblotting MeSH
- interakce hostitele a parazita fyziologie MeSH
- Leishmania major růst a vývoj patogenita MeSH
- leishmanióza genetika přenos MeSH
- modely nemocí na zvířatech MeSH
- myši inbrední BALB C MeSH
- myši MeSH
- polymerázová řetězová reakce MeSH
- protozoální proteiny metabolismus MeSH
- Psychodidae parazitologie MeSH
- virulence fyziologie MeSH
- zvířata MeSH
- Check Tag
- myši MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
BACKGROUND: Infection caused by parasites from L. donovani complex can manifest as a serious visceral disease or a self-healing milder cutaneous form. The different tropism and pathology in humans is caused by the interaction between parasites, host and vector determinants but the mechanisms are not well understood. In Cukurova region in Turkey we previously identified a major focus of cutaneous leishmaniasis caused by L. donovani/infantum hybrids (CUK strain) and isolated this parasite from the locally abundant sand fly, Phlebotomus tobbi. Here, we present the first experimental study with P. tobbi. We tested the susceptibility of this species to various Leishmania under laboratory conditions, characterized glycoproteins in the P. tobbi midgut putatively involved in parasite-vector interaction and compared the development of the CUK strain in the sand fly with one other dermotropic and three viscerotropic strains belonging to the L. donovani complex. METHODS: Females of laboratory reared P. tobbi, P. perniciosus and Lutzomyia longipalpis were infected using membrane feeding on rabbit blood containing promastigotes of various Leishmania species with different tropisms. The individual guts were checked microscopically for presence and localization of Leishmania parasites; the number of parasites was assessed more precisely by qPCR. In addition, glycosylation of midgut proteins of P. tobbi was studied by lectin blotting of midgut lysate with lectins specific for terminal sugars of N-type and O-type glycans. RESULTS: High infection rates, heavy parasite loads and late-stage infection with colonization of the stomodeal valve were observed in P. tobbi infected by Leishmania major or L. infantum CUK hybrid. In parallel, lectin blotting revealed the presence of O-glycosylated proteins in the P. tobbi midgut. In P. perniciosus and L. longipalpis all five Leishmania strains tested developed well. In both vectors, significantly higher parasite numbers were detected by qPCR for dermotropic L. donovani from Cyprus, however, in all other parameters studied, including localization of infection and colonization of stomodeal valve, dermotropic and viscerotropic strains were not significantly different. CONCLUSIONS: We showed high susceptibility of P. tobbi to various Leishmania spp. This, together with the presence of O-glycosylated midgut proteins in their midguts demonstrate that P. tobbi is a permissive vector. Two dermotropic and three viscerotropic strains from the L. donovani complex developed late-stage infections in natural L. infantum vectors, P. perniciosus and L. longipalpis and none of the parameters studied seem to be linked with different tropism of parasites in the vertebrate host.
- MeSH
- gastrointestinální trakt parazitologie MeSH
- hmyz - vektory parazitologie MeSH
- králíci MeSH
- Leishmania infantum genetika růst a vývoj izolace a purifikace MeSH
- Leishmania major genetika růst a vývoj izolace a purifikace MeSH
- leishmanióza kožní epidemiologie parazitologie MeSH
- lidé MeSH
- Phlebotomus parazitologie MeSH
- Psychodidae parazitologie MeSH
- tropismus MeSH
- zvířata MeSH
- Check Tag
- králíci MeSH
- lidé MeSH
- ženské pohlaví MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
- srovnávací studie MeSH
- Geografické názvy
- Turecko MeSH
BACKGROUND: The development of pathogens transmitted by haematophagous invertebrate vectors is closely connected with the digestion of bloodmeals and is thus affected by midgut enzymatic activity. Some studies have demonstrated that avian blood inhibits Leishmania major infection in the Old World vector Phlebotomus papatasi; however, this effect has never been observed in the New World vectors of the genus Lutzomyia infected by other Leishmania species. Therefore, our study was focused on the effect of chicken blood on bloodmeal digestion and the development of Leishmania major in its natural vector Phlebotomus duboscqi, i.e. in a vector-parasite combination where the effect of blood is assumed. In addition, we tested the effect of avian blood on midgut trypsin activity and the influence of repeated feedings on the susceptibility of sand flies to Leishmania infection. METHODS: Phlebotomus duboscqi females were infected by rabbit blood containing L. major and either before or after the infection fed on chickens or mice. The individual guts were checked microscopically for presence and localization of Leishmania, parasite numbers were detected by Q-PCR. In addition, midgut trypsin activity was studied. RESULTS: Sand fly females fed on chicken blood had significantly lower midgut trypsin activity and delayed egg development compared to those fed on rabbits. On the other hand, there was no effect detected of avian blood on parasite development within the sand fly gut: similar infection rates and parasite loads were observed in P. duboscqi females infected by L. major and fed on chickens or mouse one or six days later. Similarly, previous blood feeding of sand flies on chickens or mice did not show any differences in subsequent Leishmania infections, and there was equal susceptibility of P. duboscqi to L. major infection during the first and second bloodmeals. CONCLUSION: In spite of the fact that avian blood affects trypsin activity and the oocyte development of sand flies, no effect of chicken blood was observed on the development of L. major in P. duboscqi. Our study unambiguously shows that sand fly feeding on avian hosts is not harmful to Leishmania parasites within the sand fly midgut.
- MeSH
- gastrointestinální trakt parazitologie MeSH
- králíci MeSH
- krev metabolismus MeSH
- krmivo pro zvířata MeSH
- kur domácí MeSH
- kvantitativní polymerázová řetězová reakce MeSH
- Leishmania major genetika růst a vývoj MeSH
- mikroskopie MeSH
- myši MeSH
- Phlebotomus parazitologie MeSH
- zvířata MeSH
- Check Tag
- králíci MeSH
- myši MeSH
- ženské pohlaví MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
The stage-regulated HASPB and SHERP proteins of Leishmania major are predominantly expressed in cultured metacyclic parasites that are competent for macrophage uptake and survival. The role of these proteins in parasite development in the sand fly vector has not been explored, however. Here, we confirm that expression of HASPB is detected only in vector metacyclic stages, correlating with the expression of metacyclic-specific lipophosphoglycan and providing the first definitive protein marker for this infective sand fly stage. Similarly, SHERP is expressed in vector metacyclics but is also detected at low levels in the preceding short promastigote stage. Using genetically modified parasites lacking or complemented for the LmcDNA16 locus on chromosome 23 that contains the HASP and SHERP genes, we further show that the presence of this locus is essential for parasite differentiation to the metacyclic stage in Phlebotomus papatasi. While wild-type and complemented parasites transform normally in late-stage infections, generating metacyclic promastigotes and colonizing the sand fly stomodeal valve, null parasites accumulate at the earlier elongated nectomonad stage of development within the abdominal and thoracic midgut of the sand fly. Complementation with HASPB or SHERP alone suggests that HASPB is the dominant effector molecule in this process.
- MeSH
- antigeny protozoální biosyntéza MeSH
- esenciální geny MeSH
- geneticky modifikované organismy MeSH
- genový knockout MeSH
- Leishmania major růst a vývoj MeSH
- Phlebotomus parazitologie MeSH
- protozoální proteiny biosyntéza MeSH
- stanovení celkové genové exprese MeSH
- testy genetické komplementace MeSH
- zvířata MeSH
- Check Tag
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
- MeSH
- feces parazitologie MeSH
- finanční podpora výzkumu jako téma MeSH
- hmyz - vektory parazitologie MeSH
- králíci MeSH
- Leishmania major izolace a purifikace růst a vývoj MeSH
- Leishmania tropica izolace a purifikace růst a vývoj MeSH
- Phlebotomus parazitologie MeSH
- zvířata MeSH
- Check Tag
- králíci MeSH
- zvířata MeSH
- MeSH
- galaktosamin farmakologie MeSH
- interakce hostitele a parazita MeSH
- Leishmania major růst a vývoj účinky léků MeSH
- lektiny antagonisté a inhibitory MeSH
- Psychodidae parazitologie MeSH
- trávicí systém parazitologie účinky léků MeSH
- vztah mezi dávkou a účinkem léčiva MeSH
- zvířata MeSH
- Check Tag
- zvířata MeSH
