Next Generation Sequencing Identifies Five Major Classes of Potentially Therapeutic Enzymes Secreted by Lucilia sericata Medical Maggots

Abstract

Lucilia sericata larvae are used as an alternative treatment for recalcitrant and chronic wounds. Their excretions/secretions contain molecules that facilitate tissue debridement, disinfect, or accelerate wound healing and have therefore been recognized as a potential source of novel therapeutic compounds. Among the substances present in excretions/secretions various peptidase activities promoting the wound healing processes have been detected but the peptidases responsible for these activities remain mostly unidentified. To explore these enzymes we applied next generation sequencing to analyze the transcriptomes of different maggot tissues (salivary glands, gut, and crop) associated with the production of excretions/secretions and/or with digestion as well as the rest of the larval body. As a result we obtained more than 123.8 million paired-end reads, which were assembled de novo using Trinity and Oases assemblers, yielding 41,421 contigs with an N50 contig length of 2.22 kb and a total length of 67.79 Mb. BLASTp analysis against the MEROPS database identified 1729 contigs in 577 clusters encoding five peptidase classes (serine, cysteine, aspartic, threonine, and metallopeptidases), which were assigned to 26 clans, 48 families, and 185 peptidase species. The individual enzymes were differentially expressed among maggot tissues and included peptidase activities related to the therapeutic effects of maggot excretions/secretions.

Figure 1

Gene ontology analysis of the L. sericata transcriptome. All identified contigs (41,421) were categorized using three GO terms: (a) biological process; (b) cellular component; (c) molecular function.

Figure 2

Proportional representation of each peptidase class in the L. sericata transcriptome. Among 577 clusters identified as peptidases, 557 clusters were assigned to 5 peptidase classes (serine peptidases (270), metallopeptidases (202), cysteine peptidases (45), threonine peptidases (25), and aspartic peptidases (15)) and 20 clusters remained unassigned.

Figure 3

Gene ontology analysis of L. sericata peptidases. All identified peptidase clusters (577 in total) were categorized using three GO terms: (a) biological process, (b) molecular function, and (c) cellular component. Analysis of (a) biological process and (b) molecular function was performed at level three, whereas analysis of (c) cellular component was performed at level two.

Figure 4

Partial amino acid sequence alignment of A1 peptidases found in the L. sericata transcriptome. Amino acid sequences of A1 aspartic peptidases were aligned using MAFFT [199]. Only one cluster (LST_LS005572) contained a polyproline loop (underlined). Asterisk (∗) indicates positions which have a single, fully conserved residue. Colon (:) indicates conservation between groups of strongly similar properties, scoring > 0.5 in the Gonnet PAM 250 matrix. Period (.) indicates conservation between groups of weakly similar properties, scoring =< 0.5 in the Gonnet PAM 250 matrix.

Figure 5

Phylogenetic analysis of L. sericata caspases. The phylogenetic analysis of eight identified caspases in the transcriptome of L. sericata. For the analysis the sequences were compared with Bombyx mori (BOMMO), UniProtKB [E0D2V3; E9JEH0; E0D2V3; E9JEH0; Q2HZ05; Q8I9V7], Tribolium castaneum (TRICA), UniProtKB [D6WD84; D6WD86; D6WFK4; D6WIV9; D6WJH5; D6X3B3], Musca domestica (MUSDO), UniProtKB [T1P9A0; T1PAQ7; T1PFC3; T1PK82; T1PN44; B5AK94], Bactrocera cucurbitae (BACCU), UniProtKB [A0A0A1WTD1; A0A0A1X1L7; A0A0A1X8B8; A0A0A1XL94; A0A0A1XM78; A0A0A1XQW9; A0A0A1XRT4], D. melanogaster (Strica, UniProtKB [Q7KHK9]; Dronc, UniProtKB [Q9XYF4]; Dredd, UniProtKB [Q8IRY7]; Damm, UniProtKB [O44252]; Drice, UniProtKB [O01382]; Dcp-1, UniProtKB [O02002]; Decay, UniProtKB [Q9VET9]), and Anopheles gambiae (agL1, agL2, ags1–ags14), UniProtKB [Q5TMK1; Q7Q2Q0; Q5TMS0; Q7PZJ9; Q7PZK0; Q7PV92; Q7Q802; Q7Q803; Q7Q4X6; Q7PWK2; Q7QGM9; Q7PSJ2; Q7Q801; Q7QGN0; Q7QGM8; Q7QKT2]. For the alignment MAFFT [199] was used.

Figure 6

Verification of digital gene expression analysis by quantitative RT-PCR. (a) The individual clusters encoding for aspartic (LST_LS005916), cysteine (LST_LS006048), metallo (LST_LS004632), threonine (LST_LS006843), and serine (LST_LS010066) peptidases are depicted on the left. Shown are log₂-transformed RPKM values (blue resembles lower-expressed genes, while red represents highly expressed genes). (b) The mRNA expression profiles of five enzymatic genes in various larval tissues were determined by qRT-PCR and normalized with the 60S acidic ribosomal protein P0 (RPLPO) and 40S ribosomal protein S3 (RPS3) genes. The expressions of individual genes were related to the maximum mRNA level of each gene set as 100%.