Expression profiling and comparative analyses of seven midgut serine proteases from the yellow fever mosquito, Aedes aegypti

Abstract

Aedes aegypti utilizes blood for energy production, egg maturation and replenishment of maternal reserves. The principle midgut enzymes responsible for bloodmeal digestion are endoproteolytic serine-type proteases within the S1.A subfamily. While there are hundreds of serine protease-like genes in the A. aegypti genome, only five are known to be expressed in the midgut. We describe the cloning, sequencing and expression profiling of seven additional serine proteases and provide a genomic and phylogenetic assessment of these findings. Of the seven genes, four are constitutively expressed and three are transcriptionally induced upon blood feeding. The amount of transcriptional induction is strongly correlated among these genes. Alignments reveal that, in general, the conserved catalytic triad, active site and accessory catalytic residues are maintained in these genes and phylogenetic analysis shows that these genes fall within three distinct clades; trypsins, chymotrypsins and serine collagenases. Interestingly, a previously described trypsin consistently arose with other serine collagenases in phylogenetic analyses. These results suggest that multiple gene duplications have arisen within the S1.A subfamily of midgut serine proteases and/or that A. aegypti has evolved an array of proteases with a broad range of substrate specificities for rapid, efficient digestion of bloodmeals.

Fig. 1

mRNA expression profiles of the midgut serine proteases. Relative levels of gene transcripts are presented as the ratio of gene transcript abundance relative to ribosomal protein S7 RNA abundance. Relative mRNA levels were measured in unfed (U) midguts and at 3, 6, 12, 24, 36, 48, 72, 96 and 120 h post-bloodmeal. Data are included for AaLT (A), AaSP I (B), AaSP group I (C), AaSP VI (D) and AaSP VII (E). Statistically homogeneous (p > 0.05) groupings appear under a common letter and were identified with pairwise t-tests among the different time points. Values in (C) were not significantly different.

Fig. 2

Transcriptional control of midgut serine proteases is correlated. The average transcription levels of AaLT, AaSP I, AaSP VI and AaSP VII plotted against one another. Each point is labeled as to whether it was measured in unfed (U) midguts or at 3, 6, 12, 24, 36, 48, 72, 96, and 120 h post-bloodmeal. The line on each graph represents the linear regression model obtained and Pearson’s correlation coefficients and associated probability values are listed on each graph. Numbers within figures define the timepoints post-bloodmeal. The values for timepoints 96 and 120 are not labeled within (A) because they overlap.

Fig. 3

Protein alignment of serine proteases. Protein alignment of all serine proteases used in this analysis from positions ~170–220. Numbering is based on bovine α-chymotrypsinogen. Residues of importance are represented as follows; (*) Ser195 catalytic triad residue, (◆) accessory catalytic residues, (▲) the third and final disulfide bridge and (Δ) Asp194 where position 1 (Ile/Val) becomes buried following activation of the mature peptide.

Fig. 4

Maximum Parsimony analysis of serine protease codons. Aligned codons were used for this analysis. Clade A corresponds with the known trypsins, clade B represents a unique group of chymotrypsin-like serine proteases including A. gambiae CHYMO, AaJA15 and AaSP group I, clade C.1 contains the known chymotrypsins and clade C.2 the serine collagenases. Bootstrap analysis was performed with 1000 replicates.

Fig. 5

Genome annotation of A. aegypti midgut serine proteases. Each of the serine proteases were mapped to their respective supercontigs and oriented. The supercontigs were further analyzed for the presence of putative serine protease sequences and their positional relation to the characterized serine proteases schematically represented. Not represented are AaET, AaCHYMO and AaSP I which are found on supercontigs 284, 76 and 255, respectively.