RNA-sequencing of the Nyssomyia neivai sialome: a sand fly-vector from a Brazilian endemic area for tegumentary leishmaniasis and pemphigus foliaceus

Abstract

Leishmaniasis encompasses a spectrum of diseases caused by a protozoan belonging to the genus Leishmania. The parasite is transmitted by the bite of sand flies, which inoculate the promastigote forms into the host's skin while acquiring a blood meal. Nyssomyia neivai is one of the main vectors of tegumentary leishmaniasis (TL) in Brazil. Southeastern Brazil is an endemic region for TL but also overlaps with an endemic focus for pemphigus foliaceus (PF), also known as Fogo Selvagem. Salivary proteins of sand flies, specifically maxadilan and LJM11, have been related to pemphigus etiopathogenesis in the New World, being proposed as an environmental trigger for autoimmunity. We present a comprehensive description of the salivary transcriptome of the N. neivai, using deep sequencing achieved by the Illumina protocol. In addition, we highlight the abundances of several N. neivai salivary proteins and use phylogenetic analysis to compare with Old- and New-World sand fly salivary proteins. The collection of protein sequences associated with the salivary glands of N. neivai can be useful for monitoring vector control strategies as biomarkers of N. neivai, as well as driving vector-vaccine design for leishmaniasis. Additionally, this catalog will serve as reference to screen for possible antigenic peptide candidates triggering anti-Desmoglein-1 autoantibodies.

Figure 1

Molecular phylogenetic analysis and sequence alignment of Nyssomyia neivai 15 protein family. (A) The evolutionary history was inferred by using the Maximum Likelihood method. The tree with the highest log likelihood is shown. The tree is drawn to scale, with branch lengths measured in number of substitutions per site. A discrete Gamma distribution was used to model evolutionary rate differences among sites [5 categories (+ G, parameter = 4.0108)]. The rate variation model allowed for some sites to be evolutionarily invariable ([+ I], 2.61% sites). All positions with less than 95% site coverage were eliminated. Evolutionary analyses were conducted in MEGA7. (B) Multiple alignments of SP15 from Nyssomyia neivai with Nyssomyia intermedia and Lutzomyia longipalpis SP15 proteins using Muscle. Black shading represents identical amino acids, light gray shading represents similar amino acids.

Figure 2

Molecular phylogenetic analysis and sequence alignment of Nyssomyia neivai 15 protein family. (A) Multiple alignments of SP13 from Nyssomyia neivai with other SP13 sand fly salivary proteins using Muscle. Black shading represents identical amino acids, light gray shading represents similar amino acids.

Figure 3

Molecular phylogenetic analysis and sequence alignment of Nyssomyia neivai C-type lectin protein family. (A) The evolutionary history was inferred by using the Maximum Likelihood method. The tree with the highest log likelihood is shown. A discrete Gamma distribution was used to model evolutionary rate differences among sites [5 categories (+ G, parameter = 3.6037)]. The tree is drawn to scale, with branch lengths measured in number of substitutions per site. All positions with less than 95% site coverage were eliminated. Evolutionary analyses were conducted in MEGA7. (B) Multiple alignments of C-type lectins from Brazilian sand flies using Muscle. Black shading represents identical amino acids, light gray shading represents similar amino acids.

Figure 4

Molecular phylogenetic analysis and sequence alignment of Nyssomyia neivai Maxadilan-simile protein family. (A) The evolutionary history was inferred by using the Maximum Likelihood method. The tree with the highest log likelihood is shown. The tree is drawn to scale, with branch lengths measured in number of substitutions per site. All positions with less than 95% site coverage were eliminated. Evolutionary analyses were conducted in MEGA7. (B) Multiple alignments of Maxadilan and N. neivai (JAV08462.1) proteins using Muscle. Black shading represents identical amino acids, light gray shading represents similar amino acids.

Figure 5

Molecular phylogenetic analysis and sequence alignment of ML-domain protein family of Nyssomyia neivai. (A) The evolutionary history was inferred by using the Maximum Likelihood method. The tree with the highest log likelihood is shown. The tree is drawn to scale, with branch lengths measured in number of substitutions per site. All positions with less than 95% site coverage were eliminated. Evolutionary analyses were conducted in MEGA7. (B) Multiple alignments of ML-domain from Nyssomyia neivai with South American sand flies ML-domain proteins using Muscle. Black shading represents identical amino acids, light gray shading represents similar amino acids. (C) Alignment of Nyssomyia neivai JAV08582.1, N. intermedia AFP99241.1 and B. olmeca ANW11447.1 from the ML-domain protein family using Muscle. Black shading represents identical amino acids, light gray shading represents similar amino acids.

Figure 6

Molecular phylogenetic analysis and sequence alignment Yellow-related protein family of Nyssomyia neivai. (A) The evolutionary history was inferred by using the Maximum Likelihood method. The tree with the highest log likelihood is shown. The tree is drawn to scale, with branch lengths measured in number of substitutions per site. A discrete Gamma distribution was used to model evolutionary rate differences among sites [5 categories (+ G, parameter = 1.6114)]. All positions with less than 95% site coverage were eliminated. Evolutionary analyses were conducted in MEGA7. (B) Multiple alignments of Yellow-related protein from Nyssomyia neivai with Nyssomyia intermedia and Lutzomyia longipalpis Yellow-related protein using Muscle. Black shading represents identical amino acids, light gray shading represents similar amino acids.