Abstract
BackgroundCulex quinquefasciatus is a hematophagous insect from the Culicidae family that feeds on the blood of humans, dogs, birds and livestock. This species transmits a wide variety of pathogens between humans and animals. The midgut environment is the first location of pathogen-vector interactions for blood-feeding mosquitoes and the expression of specific peptidases in the early stages of feeding could influence the outcome of the infection. Trypsin-like serine peptidases belong to a multi-gene family that can be expressed in different isoforms under distinct physiological conditions. However, the confident assignment of the trypsin genes that are expressed under each condition is still a challenge due to the large number of trypsin-coding genes in the Culicidae family and most likely because they are low abundance proteins.MethodsWe used zymography for the biochemical characterization of the peptidase profile of the midgut from C. quinquefasciatus females fed on sugar. Protein samples were also submitted to SDS-PAGE followed by liquid chromatography–tandem mass spectrometry (LC–MS/MS) analysis for peptidase identification. The peptidases sequences were analyzed with bioinformatics tools to assess their distinct features.ResultsZymography revealed that trypsin-like serine peptidases were responsible for the proteolytic activity in the midgut of females fed on sugar diet. After denaturation in SDS-PAGE, eight trypsin-like serine peptidases were identified by LC-MS/MS. These peptidases have structural features typical of invertebrate digestive trypsin peptidases but exhibited singularities at the protein sequence level such as: the presence of different amino acids at the autocatalytic motif and substrate binding regions as well as different number of disulfide bounds. Data mining revealed a group of trypsin-like serine peptidases that are specific to C. quinquefasciatus when compared to the culicids genomes sequenced so far.ConclusionWe demonstrated that proteomics approaches combined with bioinformatics tools and zymographic analysis can lead to the functional annotation of trypsin-like serine peptidases coding genes and aid in the understanding of the complexity of peptidase expression in mosquitoes.
Highlights
Culex quinquefasciatus is a hematophagous insect from the Culicidae family that feeds on the blood of humans, dogs, birds and livestock
The coupling of zymography, proteomic approaches and bioinformatic analyses, as performed here, shows to be a powerful approach in exploring the presence of active peptidases, which helps in the identification of genes that are expressed at the protein level in a specific tissue
We identified and characterized trypsin peptidases that are expressed in the midgut of C. quinquefasciatus
Summary
Culex quinquefasciatus is a hematophagous insect from the Culicidae family that feeds on the blood of humans, dogs, birds and livestock This species transmits a wide variety of pathogens between humans and animals. Despite having anthropophilic and endophilic habits, adult females exhibit high plasticity in their feeding behavior that characterizes this species as an opportunistic insect that feeds on the blood of humans, dogs, birds and livestock. This feature makes this species important in the zoonotic transmission of a wide variety of pathogens between humans and animals [1, 2]. This species plays an important role as a vector of helminths such as the causative agent of lymphatic filariasis, Wuchereria bancrofti, and the dog heartworm, Dirofilaria immitis [3,4,5,6,7,8]
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