A Deep Insight into the Sialome of Rhodnius neglectus, a Vector of Chagas Disease

Abstract

Background: Triatomines are hematophagous insects that act as vectors of Chagas disease. Rhodnius neglectus is one of these kissing bugs found, contributing to the transmission of this American trypanosomiasis. The saliva of hematophagous arthropods contains bioactive molecules responsible for counteracting host haemostatic, inflammatory, and immune responses.

Methods/principal findings: Next generation sequencing and mass spectrometry-based protein identification were performed to investigate the content of triatomine R. neglectus saliva. We deposited 4,230 coding DNA sequences (CDS) in GenBank. A set of 636 CDS of proteins of putative secretory nature was extracted from the assembled reads, 73 of them confirmed by proteomic analysis. The sialome of R. neglectus was characterized and serine protease transcripts detected. The presence of ubiquitous protein families was revealed, including lipocalins, serine protease inhibitors, and antigen-5. Metalloproteases, disintegrins, and odorant binding protein families were less abundant.

Conclusions/significance: The data presented improve our understanding of hematophagous arthropod sialomes, and aid in understanding hematophagy and the complex interplay among vectors and their vertebrate hosts.

Fig 1. Phylogram of lipocalin containing triabin domain from R. neglectus SG transcriptome.

Phylogenetic tree derived from the alignment of R. neglectus CDS and other triatomine lipocalin sequences as described in Methods section. The bar at the bottom represents 20% amino acid substitution. The colored circles indicate each species whose sequences were used: blue, R. neglectus sequences from SG transcriptome; red, R. prolixus; yellow, Triatoma dimidiata; green, Triatoma brasiliensis; dark green, Triatoma matogrossensis; dark blue, T. pallidipennis; purple, Triatoma protacta; magenta, Triatoma infestans; gray, Dipetalogaster maxima.

Fig 2. Phylogram of lipocalin containing nitrophorin domain from R. neglectus SG transcriptome.

Phylogenetic tree derived from the alignment of R. neglectus CDS and R. prolixus nitrophorin sequences as described in Methods section. The bar at the bottom represents 20% amino acid substitution. The colored circles indicate each species whose sequences were used: blue, R. neglectus sequences from SG transcriptome and red, R. prolixus sequences from NCBI.

Fig 3. Phylogram of Antigen-5 proteins from R. neglectus SG transcriptome.

Phylogenetic tree derived from the alignment of R. neglectus CDS and other insect antigen-5 sequences as described in Methods section. The bar represents 10% amino acid substitution.

Fig 4. Kazal-type members from R. neglectus SG transcriptome.

ClustalW alignment of Kazal-type domain-containing members from R. neglectus salivary transcriptome (RN_5563 and RN_549) and other insect kazal-type sequences, identified as described in Methods section. The alignment indicates conserved residues in black and similar residues in gray background, the six conserved cysteines (boxes) and the blue bar indicates the signal peptide indicative of secretion.

Fig 5. Phylogram of Serine Protease inhibitors from R. neglectus SG transcriptome.

Phylogenetic tree derived from the alignment of R. neglectus CDS and other insect sequences as described in Methods section. The bar at the bottom represents 20% amino acid substitution.

Fig 6. Phylogram of serpin proteins from R. neglectus SG transcriptome.

Phylogenetic tree derived from the alignment of R. neglectus CDS and other insect sequences as described in Methods section. The bar represents 20% amino acid substitution.

Fig 7. The secreted metalloprotease from R. neglectus SG transcriptome.

(A) ClustalW alignment of the secreted metalloprotease from R. neglectus salivary transcriptome (RN_21266) and other metalloproteases sequences, identified as described in Methods section. The alignment indicates conserved residues in black and similar residues in gray background. The blue bar indicates the signal peptide indicative of secretion. The boxes limit the family signature sequences showing the determinant residues (black asterisk). (B) Phylogenetic tree derived from the alignment of R. neglectus CDS and other metalloproteases sequences as described in Methods section. The bar represents 10% amino acid substitution.

Fig 8. The ADAMTS sequence from R. neglectus SG transcriptome.

ClustalW alignment of the ADAMTS sequence from R. neglectus salivary transcriptome (RN_11351) and other insect ADAMTS sequences, identified as described in Methods section. The alignment indicates conserved residues in black and similar residues in gray background. The bar indicates the signal peptide indicative of secretion. The boxes limit the family signature sequences showing the determinant residues (black asterisk). The symbols (black hash) above indicate the conserved cysteines.

Fig 9. Serine proteases from R. neglectus SG transcriptome.

(A) ClustalW alignment of a serine protease from R. neglectus SG transcriptome (RN_1189) and other serine proteases members, identified as described in Methods section. The alignment indicates conserved residues in black and similar residues in gray background. The blue bar indicates the signal peptide indicative of secretion. The symbols above the residues indicate (black circle) cleavage site and (black triangle) active site showing the HDS triad. (B) Phylogenetic tree was built from the alignment of R. neglectus CDS and other insect sequences as described in Methods section The bar at the bottom represents 20% amino acid substitution. The colored circles identify the sequences used: blue, R. neglectus sequences from SG transcriptome; red, Hemiptera order; green, Hymenoptera order; magenta, Dictyoptera order.

Fig 10. The secreted OBP family from R. neglectus SG transcriptome.

ClustalW alignment of secreted OBPs from R. neglectus SG transcriptome (RN_5213, RN_3440 and RN_18954) and other members from the OBP family, identified as described in Methods section. The alignment indicates conserved residues in black and similar residues in gray background. The blue bar indicates the signal peptide indicative of secretion and the boxes, the six conserved cysteines.