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Immunity to LuloHya and Lundep, the salivary spreading factors from Lutzomyia longipalpis, protects against Leishmania major infection.

ABSTRACT: Salivary components from disease vectors help arthropods to acquire blood and have been shown to enhance pathogen transmission in different model systems. Here we show that two salivary enzymes from Lutzomyia longipalpis have a synergist effect that facilitates a more efficient blood meal intake and diffusion of other sialome components. We have previously shown that Lundep, a highly active endonuclease, enhances parasite infection and prevent blood clotting by inhibiting the intrinsic pathway of coagulation. To investigate the physiological role of a salivary hyaluronidase in blood feeding we cloned and expressed a recombinant hyaluronidase from Lu. longipalpis. Recombinant hyaluronidase (LuloHya) was expressed in mammalian cells and biochemically characterized in vitro. Our study showed that expression of neutrophil CXC chemokines and colony stimulating factors were upregulated in HMVEC cells after incubation with LuloHya and Lundep. These results were confirmed by the acute hemorrhage, edema and inflammation in a dermal necrosis (dermonecrotic) assay involving a massive infiltration of leukocytes, especially neutrophils, in mice co-injected with hemorrhagic factor and these two salivary proteins. Moreover, flow cytometry results showed that LuloHya and Lundep promote neutrophil recruitment to the bite site that may serve as a vehicle for establishment of Leishmania infection. A vaccination experiment demonstrated that LuloHya and Lundep confer protective immunity against cutaneous leishmaniasis using the Lu. longipalpis-Leishmania major combination as a model. Animals (C57BL/6) immunized with LuloHya or Lundep showed minimal skin damage while lesions in control animals remained ulcerated. This protective immunity was abrogated when B-cell-deficient mice were used indicating that antibodies against both proteins play a significant role for disease protection. Rabbit-raised anti-LuloHya antibodies completely abrogated hyaluronidase activity in vitro. Moreover, in vivo experiments demonstrated that blocking LuloHya with specific antibodies interferes with sand fly blood feeding. This work highlights the relevance of vector salivary components in blood feeding and parasite transmission and further suggests the inclusion of these salivary proteins as components for an anti-Leishmania vaccine.

SUBMITTER: Martin-Martin I

PROVIDER: S-EPMC5953502 | biostudies-literature | 2018 May

REPOSITORIES: biostudies-literature

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Immunity to LuloHya and Lundep, the salivary spreading factors from Lutzomyia longipalpis, protects against Leishmania major infection.

Martin-Martin Ines I Chagas Andrezza Campos AC Guimaraes-Costa Anderson B AB Amo Laura L Oliveira Fabiano F Moore Ian N IN DeSouza-Vieira Thiago S TS Sanchez Elda E EE Suntravat Montamas M Valenzuela Jesus G JG Ribeiro Jose M C JMC Calvo Eric E

PLoS pathogens 20180503 5

Salivary components from disease vectors help arthropods to acquire blood and have been shown to enhance pathogen transmission in different model systems. Here we show that two salivary enzymes from Lutzomyia longipalpis have a synergist effect that facilitates a more efficient blood meal intake and diffusion of other sialome components. We have previously shown that Lundep, a highly active endonuclease, enhances parasite infection and prevent blood clotting by inhibiting the intrinsic pathway o ...[more]

PMID: 29723281

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Project description:BackgroundLeishmania transmission occurs in the presence of insect saliva. Immunity to Phlebotomus papatasi or Lutzomyia longipalpis saliva or salivary components confers protection against an infection by Leishmania in the presence of the homologous saliva. However, immunization with Lutzomyia intermedia saliva did not protect mice against Leishmania braziliensis plus Lu. intermedia saliva. In the present study, we have studied whether the immunization with Lu. longipalpis saliva or a DNA plasmid coding for LJM19 salivary protein would be protective against L. braziliensis infection in the presence of Lu. intermedia saliva, the natural vector for L. braziliensis.Methodology/principal findingsImmunization with Lu. longipalpis saliva or with LJM19 DNA plasmid induced a Delayed-Type Hypersensitivity (DTH) response against Lu. longipalpis as well as against a Lu. intermedia saliva challenge. Immunized and unimmunized control hamsters were then intradermally infected in the ears with L. braziliensis in the presence of Lu. longipalpis or Lu. intermedia saliva. Animals immunized with Lu. longipalpis saliva exhibited smaller lesion sizes as well as reduced disease burdens both at lesion site and in the draining lymph nodes. These alterations were associated with a significant decrease in the expression levels of IL-10 and TGF-β. Animals immunized with LJM19 DNA plasmid presented similar findings in protection and immune response and additionally increased IFN-γ expression.Conclusions/significanceImmunization with Lu. longipalpis saliva or with a DNA plasmid coding LJM19 salivary protein induced protection in hamsters challenged with L. braziliensis plus Lu. intermedia saliva. These findings point out an important role of immune response against saliva components, suggesting the possibility to develop a vaccine using a single component of Lu. longipalpis saliva to generate protection against different species of Leishmania, even those transmitted by a different vector.

Project description:The microbial consortium associated with sandflies has gained relevance, with its composition shifting throughout distinct developmental stages, being strongly influenced by the surroundings and food sources. The bacterial components of the microbiota can interfere with Leishmania development inside the sandfly vector. Microbiota diversity and host-microbiota-pathogen interactions regarding New World sandfly species have yet to be thoroughly studied, particularly in Lutzomyia longipalpis, the primary vector of visceral leishmaniasis in Brazil.The native microbiota of different developmental stages and physiological conditions of Lu. longipalpis (Lapinha Cave), was described by culturing and 16s rRNA gene sequencing. The 16s rRNA sequencing of culture-dependent revealed 13 distinct bacterial genera (Bacillus, Enterococcus, Erwinia, Enterobacter, Escherichia, Klebsiella, Lysinibacillus, Pseudocitrobacter, Providencia, Pseudomonas, Serratia, Staphylococcus and Solibacillus). The in vitro and in vivo effects of each one of the 13 native bacteria from the Lu. longipalpis were analyzed by co-cultivation with promastigotes of L.i. chagasi, L. major, L. amazonensis, and L. braziliensis. After 24 h of co-cultivation, a growth reduction observed in all parasite species. When the parasites were co-cultivated with Lysinibacillus, all parasites of L. infantum chagasi and L. amazonensis died within 24 hours. In the in vivo co-infection of L.chagasi, L. major and L. amazonensis with the genera Lysinibacillus, Pseudocitrobacter and Serratia it was possible to observe a significant difference between the groups co-infected with the bacterial genera and the control group.These findings suggest that symbiont bacteria (Lysinibacillus, Serratia, and Pseudocitrobacter) are potential candidates for paratransgenic or biological control. Further studies are needed to identify the nature of the effector molecules involved in reducing the vector competence for Leishmania.

Project description:BackgroundPhlebotomine sand flies transmit the haemoflagellate Leishmania, the causative agent of human leishmaniasis. The Leishmania promastigotes are confined to the gut lumen and are exposed to the gut microbiota within female sand flies. Here we study the colonisation resistance of yeast and bacteria in preventing the establishment of a Leishmania population in sand flies and the ability of Leishmania to provide colonisation resistance towards the insect bacterial pathogen Serratia marcescens that is also pathogenic towards Leishmania.MethodsWe isolated microorganisms from wild-caught and laboratory-reared female Lutzomyia longipalpis, identified as Pseudozyma sp. Asaia sp. and Ochrobactrum intermedium. We fed the females with a sugar meal containing the microorganisms and then subsequently fed them with a bloodmeal containing Leishmania mexicana and recorded the development of the Leishmania population. Further experiments examined the effect of first colonising the sand fly gut with L. mexicana followed by feeding with, Serratia marcescens, an insect bacterial pathogen. The mortality of the flies due to S. marcescens was recorded in the presence and absence of Leishmania.ResultsThere was a reduction in the number of flies harbouring a Leishmania population that had been pre-fed with Pseudozyma sp. and Asaia sp. or O. intermedium. Experiments in which L. mexicana colonised the sand fly gut prior to being fed an insect bacterial pathogen, Serratia marcescens, showed that the survival of flies with a Leishmania infection was significantly higher compared to flies without Leishmania infection.ConclusionsThe yeast and bacterial colonisation experiments show that the presence of sand fly gut microorganisms reduce the potential for Leishmania to establish within the sand fly vector. Sand flies infected with Leishmania were able to survive an attack by the bacterial pathogen that would have killed the insect and we concluded that Leishmania may benefit its insect host whilst increasing the potential to establish itself in the sand fly vector. We suggest that the increased ability of the sand fly to withstand a bacterial entomopathogen, due to the presence of the Leishmania, may provide an evolutionary pressure for the maintenance of the Leishmania-vector association.

Dataset Information

Immunity to LuloHya and Lundep, the salivary spreading factors from Lutzomyia longipalpis, protects against Leishmania major infection.

Publications

Immunity to LuloHya and Lundep, the salivary spreading factors from Lutzomyia longipalpis, protects against Leishmania major infection.

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