In-depth characterization of trypsin-like serine peptidases in the midgut of the sugar fed Culex quinquefasciatus

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.

Genome-wide identification and expression profiling of serine proteases and homologs in the diamondback moth, Plutella xylostella (L.)

“…Clade I contained 11 P. xylostella trypsin genes, which were clustered with those in C. quinquefasciatus and N. lugens . In this clade, CqTrys 1, 4, and 5 have been reported to be constitutively expressed in the midgut of females, indicating their potential roles in digestion [ 36 ]. NlTry2 and NlTry5 are highly expressed in the midgut [ 11 ].…”

Juvenile hormone affects the splicing of Culex quinquefasciatus early trypsin messenger RNA

“…Borges‐Veloso et al () characterized trypsin‐like enzymes expressed in the gut of sugar fed Cx. quinquefasciatus .…”

“…Thus, it appears that an unknown mechanism yet to be deciphered is responsible for the control of the mosquito early and late trypsins. -Veloso et al (2015) characterized trypsin-like enzymes expressed in the gut of sugar fed Cx.…”

Differential expression of trypsin-3 and phosrestin II genes in the main malaria vector, Anopheles darlingi, from the Brazilian Amazon region

“…Transcriptome analysis of A. aquasalis females fed with sugar showed the expression of trypsin-type peptidases (Costa-da-Silva et al 2014). Muller et al (1995 reported that A. gambiae expressed a set of seven trypsin genes, including antryp1 and antryp2, which are induced after feeding w i t h blood, while antryp3, antryp4, and antryp7 are constitutively expressed and undetectable after feeding with blood (Borges-Veloso et al 2015). Understanding of the physiology and presence of specific trypsins in mosquito tissues that are involved in host-pathogen interactions may provide relevant findings for the development of alternative and more effective control measures for the vectors (Shahabuddin and Costero, 2001;Chertemps et al 2010).…”