Discovery of markers of exposure specific to bites of Lutzomyia longipalpis, the vector of Leishmania infantum chagasi in Latin America

Abstract

Background: Sand flies deliver Leishmania parasites to a host alongside salivary molecules that affect infection outcomes. Though some proteins are immunogenic and have potential as markers of vector exposure, their identity and vector specificity remain elusive.

Methodology/principal findings: We screened human, dog, and fox sera from endemic areas of visceral leishmaniasis to identify potential markers of specific exposure to saliva of Lutzomyia longipalpis. Human and dog sera were further tested against additional sand fly species. Recombinant proteins of nine transcripts encoding secreted salivary molecules of Lu. longipalpis were produced, purified, and tested for antigenicity and specificity. Use of recombinant proteins corresponding to immunogenic molecules in Lu. longipalpis saliva identified LJM17 and LJM11 as potential markers of exposure. LJM17 was recognized by human, dog, and fox sera; LJM11 by humans and dogs. Notably, LJM17 and LJM11 were specifically recognized by humans exposed to Lu. longipalpis but not by individuals exposed to Lu. intermedia.

Conclusions/significance: Salivary recombinant proteins are of value as markers of vector exposure. In humans, LJM17 and LJM11 emerged as potential markers of specific exposure to Lu. longipalpis, the vector of Leishmania infantum chagasi in Latin America. In dogs, LJM17, LJM11, LJL13, LJL23, and LJL143 emerged as potential markers of sand fly exposure. Testing these recombinant proteins in large scale studies will validate their usefulness as specific markers of Lu. longipalpis exposure in humans and of sand fly exposure in dogs.

Figure 1. Lutzomyia longipalpis salivary proteins recognized by human, dog and fox sera from the visceral leishmaniasis endemic areas of São Luis and Teresina.

A, Silver staining of a Bis-Tris NuPAGE gel of salivary proteins from Lu. longipalpis. B, Western Blot of salivary proteins of Lu. longipalpis separated on a Tris-Glycine gel using a representative reactive sera from either human (São Luis), dog or fox (Teresina).

Figure 2. Detection of salivary proteins from different species of sand flies by Western Blot.

A, Two representative human sera from São Luis, a VL- endemic area where Lutzomyia longipalpis predominates, were screened against salivary proteins from Lu. longipalpis (LL), Lu. intermedia (LI), Lu. verrucarum (LV), and Phlebotomus perniciosus (PPe). B, Western blot analysis of salivary proteins from Lu. longipalpis using human sera from Canoa, a cutaneous leishmaniasis endemic area where Lu. intermedia predominates (CL endemic area; left bottom panel), or from São Luis where Lu. longipalpis predominates (VL endemic area). Sera from individuals from a non-endemic area were used as negative controls (CTL–). The profile of salivary proteins from Lu. intermedia recognized by a representative human serum (out of six tested ) from the Canoa endemic area (CL endemic area; right bottom panel).

Figure 3. Total IgG response in dogs from endemic areas and dogs experimentally exposed to Lutzomyia longipalpis bites.

Dog sera from Teresina, an area endemic for visceral leishmaniasis where Lu. longipalpis predominates, (Dog-VL endemic area) and sera from dogs experimentally exposed to Lu. longipalpis bites (Dog – Experimentally exposed) were screened against salivary proteins from Lu. longipalpis (LL), Lu. intermedia (LI), Lu. verrucarum (LV), and Phlebotomus perniciosus (PPe) by Western Blot.

Figure 4. Production of recombinant sand fly salivary proteins.

A, Flowchart describing the expression, purification and detection of recombinant sand fly salivary proteins from Lutzomyia longipalpis. B, Chromatogram of the purification of the LJM17 recombinant protein by HPLC-metal affinity chromatography. C, Detection of positive fractions for the LJM17 recombinant protein using sera from mice immunized with LJM17 plasmid. D, Silver-stained SDS-PAGE of LJM17 recombinant protein before (1) and after (2) HPLC purification.

Figure 5. Detection of Lutzomyia longipalpis recombinant salivary proteins by human and dog sera from visceral leishmaniasis endemic areas.

Human and dog sera from São Luis and Teresina respectively, were used to screen recombinant salivary proteins LJM17, LJM11, LJM111, LJL13, LJL23, LJM04, and LJL143 by Western Blot (+). Human and dog sera from a non-endemic area were used as negative controls (–).

Figure 6. Detection of the recombinant salivary proteins LJM17 and LJM11 by human sera from endemic areas.

Western Blot with LJM17 or LJM11 recombinant proteins were screened with sera from humans in Canoa where Lutzomyia intermedia predominates ( Human – CL endemic area) or with sera from São Luis area where Lu. longipalpis predominates (Human – VL endemic area). Sera from individuals from a non-endemic area were used as negative controls (CTL–).