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

Étude moléculaire des glycoprotéines d'attachement des Henipavirus (identification et caractérisation des domaines responsables de l'interaction avec les récepteurs cellulaires, éphrineB2 et éphrineB3)

Les Henipavirus, le virus Nipah (VN) et le virus Hendra (VH), sont des virus zoonotiques émergents, responsables de pathologies chez l homme et les animaux. Leur virulence élevée et l absence de traitements disponibles ont conduit à leur classification en pathogène de niveau 4. L entrée de ces virus dans la cellule hôte est sous contrôle de deux glycoprotéines virales, la protéine d attachement (G) et la protéine de fusion (F). Nous avons identifié plusieurs résidus de la protéine G du VN, importants pour la liaison au récepteur cellulaire, l éphrine B2. Ces résidus forment un site contigu, localisé à la surface sur le haut de la tête globulaire de la protéine d attachement du VN. Nous avons également montré que le site de liaison à l éphrine B2 sur la protéine G du VH se trouvait dans une localisation similaire et impliquait en grande majorité les mêmes résidus. Nous avons également étudié le rôle potentiel de ces résidus dans la liaison à l éphrine B3, un récepteur alternatif du VN. Nos résultats indiquent que le VH pourrait utiliser l éphrine B3 comme récepteurThe Henipaviruses - Nipah (NiV) and Hendra (HeV) - are recently emergent zoonotic paramyxoviruses, responsible for pathologies in the man and the animals. Their high virulence and the absence of available treatments led to their classification into P4 pathogens. The entry of these viruses in the host cell is under control of two glycoproteins, the attachment glycoprotein (G) and the fusion protein (F). We identified several residues of the attachment glycoprotein of the NiV (NiV-G), potentially participating in receptor-binding, the ephrinB2. Ours results suggest that a receptor-binding site localizes on the top surface of the NiV-G globular head. We also showed that the site responsible for ephrinB2 interaction on the globular head of the HeV attachment glycoprotein (HeV-G) is in an identical location to that predicted for NiV-G. We also tested the attachment glycoprotein residues of the HeV and NiV for ephrinB3 interaction, an alternate receptor of the NiV. Our results indicate that the HeV could use ephrinB3 as receptor.LYON1-BU.Sciences (692662101) / SudocSudocFranceF

Exosome Secretion by the Parasitic Protozoan Leishmania within the Sand Fly Midgut

Despite several studies describing the secretion of exosomes by Leishmania in vitro, observation of their formation and release in vivo has remained a major challenge. Herein, we show that Leishmania constitutively secretes exosomes within the lumen of the sand fly midgut through a mechanism homologous to the mammalian pathway. Through egestion experiments, we demonstrate that Leishmania exosomes are part of the sand fly inoculum and are co-egested with the parasite during the insect’s bite, possibly influencing the host infectious process. Indeed, co-inoculation of mice footpads with L. major plus midgut-isolated or in-vitro-isolated L. major exosomes resulted in a significant increase in footpad swelling. Notably, co-injections produced exacerbated lesions through overinduction of inflammatory cytokines, in particular IL-17a. Our data indicate that Leishmania exosomes are an integral part of the parasite’s infectious life cycle, and we propose to add these vesicles to the repertoire of virulence factors associated with vector-transmitted infections

Phenotypic adaptations of Leishmania donovani to recurrent miltefosine exposure and impact on sand fly infection

© The Author(s) 2020.[Background]: Since the introduction of miltefosine (MIL) as first-line therapy in the kala-azar elimination programme in the Indian subcontinent, treatment failure rates have been increasing. Since parasite infectivity and virulence may become altered upon treatment relapse, this laboratory study assessed the phenotypic effects of repeated in vitro and in vivo MIL exposure.[Methods]: Syngeneic Leishmania donovani lines either or not exposed to MIL were compared for drug susceptibility, rate of promastigote multiplication and metacyclogenesis, macrophage infectivity and behaviour in the sand fly vector, Lutzomyia longipalpis.[Results]: Promastigotes of both in vitro and in vivo MIL-selected strains displayed a slightly reduced drug susceptibility that was associated with a reduced MIL-accumulation linked to a lower copy number (disomic state) of chromosome 13 harboring the miltefosine transporter (LdMT) gene. In vitro selected promastigotes showed a lower rate of metacyclogenesis whereas the in vivo derived promastigotes displayed a moderately increased growth rate. Repeated MIL exposure did neither influence the parasite load nor metacyclogenesis in the sand fly vector.[Conclusions]: Recurrent in vitro and in vivo MIL exposure evokes a number of very subtle phenotypic and genotypic changes which could make promastigotes less susceptible to MIL without attaining full resistance. These changes did not significantly impact on infection in the sand fly vector.Funding was available through the Research Fund Flanders (G051812N, 12I0317N and 1189617N) and a research fund of the University of Antwerp (TT-ZAPBOF 33049 and TOP-BOF 35017). LR was supported by Redes de Investigación Cooperativa RETICS-FEDER RD16/0027/0010.Peer reviewe

Evidence of a Potential Receptor-Binding Site on the Nipah Virus G Protein (NiV-G): Identification of Globular Head Residues with a Role in Fusion Promotion and Their Localization on an NiV-G Structural Model

As a preliminary to the localization of the receptor-binding site(s) on the Nipah virus (NiV) glycoprotein (NiV-G), we have undertaken the identification of NiV-G residues that play a role in fusion promotion. To achieve this, we have used two strategies. First, as NiV and Hendra virus (HeV) share a common receptor and their cellular tropism is similar, we hypothesized that residues functioning in receptor attachment could be conserved between their respective G proteins. Our initial strategy was to target charged residues (which can be expected to be at the surface of the protein) conserved between the NiV-G and HeV-G globular heads. Second, we generated NiV variants that escaped neutralization by anti-NiV-G monoclonal antibodies (MAbs) that neutralize NiV both in vitro and in vivo, likely by blocking receptor attachment. The sequencing of such “escape mutants” identified NiV-G residues present in the epitopes to which the neutralizing MAbs are directed. Residues identified via these two strategies whose mutation had an effect on fusion promotion were localized on a new structural model for the NiV-G protein. Our results suggest that seven NiV-G residues, including one (E533) that was identified using both strategies, form a contiguous site on the top of the globular head that is implicated in ephrinB2 binding. This site commences near the shallow depression in the center of the top surface of the globular head and extends to the rim of the barrel-like structure on the top loops of β-sheet 5. The topology of this site is strikingly similar to that proposed to form the SLAM receptor site on another paramyxovirus attachment protein, that of the measles virus hemagglutinin

Phenotypic adaptations of Leishmania donovani to recurrent miltefosine exposure and impact on sand fly infection

© The Author(s) 2020.[Background]: Since the introduction of miltefosine (MIL) as first-line therapy in the kala-azar elimination programme in the Indian subcontinent, treatment failure rates have been increasing. Since parasite infectivity and virulence may become altered upon treatment relapse, this laboratory study assessed the phenotypic effects of repeated in vitro and in vivo MIL exposure.[Methods]: Syngeneic Leishmania donovani lines either or not exposed to MIL were compared for drug susceptibility, rate of promastigote multiplication and metacyclogenesis, macrophage infectivity and behaviour in the sand fly vector, Lutzomyia longipalpis.[Results]: Promastigotes of both in vitro and in vivo MIL-selected strains displayed a slightly reduced drug susceptibility that was associated with a reduced MIL-accumulation linked to a lower copy number (disomic state) of chromosome 13 harboring the miltefosine transporter (LdMT) gene. In vitro selected promastigotes showed a lower rate of metacyclogenesis whereas the in vivo derived promastigotes displayed a moderately increased growth rate. Repeated MIL exposure did neither influence the parasite load nor metacyclogenesis in the sand fly vector.[Conclusions]: Recurrent in vitro and in vivo MIL exposure evokes a number of very subtle phenotypic and genotypic changes which could make promastigotes less susceptible to MIL without attaining full resistance. These changes did not significantly impact on infection in the sand fly vector.Funding was available through the Research Fund Flanders (G051812N, 12I0317N and 1189617N) and a research fund of the University of Antwerp (TT-ZAPBOF 33049 and TOP-BOF 35017). LR was supported by Redes de Investigación Cooperativa RETICS-FEDER RD16/0027/0010.Peer reviewe

A New Model of Progressive Visceral Leishmaniasis in Hamsters by Natural Transmission via Bites of Vector Sand Flies

Background. Visceral leishmaniasis (VL) is transmitted by sand flies. Protection of needle-challenged vaccinated mice was abrogated in vector-initiated cutaneous leishmaniasis, highlighting the importance of developing natural transmission models for VL. Methods. We used Lutzomyia longipalpis to transmit Leishmania infantum or Leishmania donovani to hamsters. Vector-initiated infections were monitored and compared with intracardiac infections. Body weights were recorded weekly. Organ parasite loads and parasite pick-up by flies were assessed in sick hamsters. Results. Vector-transmitted L. infantum and L. donovani caused ≥5-fold increase in spleen weight compared with uninfected organs and had geometric mean parasite loads (GMPL) comparable to intracardiac inoculation of 10(7)–10(8) parasites, although vector-initiated disease progression was slower and weight loss was greater. Only vector-initiated L. infantum infections caused cutaneous lesions at transmission and distal sites. Importantly, 45.6%, 50.0%, and 33.3% of sand flies feeding on ear, mouth, and testicular lesions, respectively, were parasite-positive. Successful transmission was associated with a high mean percent of metacyclics (66%–82%) rather than total GMPL (2.0 × 10(4)–8.0 × 10(4)) per midgut. Conclusions. This model provides an improved platform to study initial immune events at the bite site, parasite tropism, and pathogenesis and to test drugs and vaccines against naturally acquired VL