A C‐type lectin with a single carbohydrate‐recognition domain involved in the innate immune response of Tribolium castaneum

Abstract

C‐type lectins are one of the pattern‐recognition proteins involved in innate immunity in invertebrates. Although there are 16 C‐type lectin genes that have been identified in the genome of Tribolium castaneum, their functions and mechanisms in innate immunity remain unknown. Here, we identified one C‐type lectin orthologue, TcCTL6 (TC003708), by sequencing random clones from the cDNA library of the coleopteran beetle, T. castaneum. TcCTL6 contains a 654 bp open reading frame encoding a protein of 217 amino acids that includes a single carbohydrate‐recognition domain. The expression of TcCTL6 was significantly induced by Escherichia coli, Staphylococcus aureus and stimulation with carbohydrates, including lipopolysaccharide and peptidoglycan. A binding assay suggested that the recombinant TcCTL6 not only bound to lipopolysaccharide and peptidoglycan but also bound to Gram‐positive (S. aureus, Bacillus subtilis and Bacillus thuringiensis) and Gram‐negative bacteria (E. coli and Pseudomonas aeruginosa) in the presence of calcium ions. Furthermore, when TcCTL6 was knocked down by RNA interference, four antimicrobial peptides (attacin1, attacin2, coleoptericin1 and coleoptericin2) were significantly decreased. These results demonstrate that TcCTL6 plays a vital role in the immune response towards pathogen infection by influencing the expression of antimicrobial peptides and the agglutination of bacteria in the presence of calcium ions in T. castaneum.

A C‐type lectin with dual‐CRD from Tribolium castaneum is induced in response to bacterial challenge

“…31,32 2.2 cDNA cloning and sequence analysis Total RNA was isolated from pools of three 15-day-old larvae, and then 1 μg total RNA was converted to cDNA using a previously reported method. 29,31 The full-length cDNA sequence of TcCTL3 was obtained from Beetlebase (http://www.beetlebase.org/). The sequence of TcCTL3 was amplified by polymerase chain reaction (PCR) using primers TcCTL3-F and TcCTL3-R (Table 1) and re-sequenced for confirmation.…”

“…To measure TcCTL3 expression after lipopolysaccharide (LPS) and PGN stimulation, 15-day-old larvae were collected and separated into three groups. Approximately 60 synchronous larvae in each group were injected with LPS, PGN or physiological buffer 29 Pools of three randomly selected larvae were sampled for RNA isolation at 3, 6, 9, 12 and 24 h after injection to check the temporal expression profile of TcCTL3. TcCTL3 expression levels in the beetles stimulated with LPS or PGN were determined via qRT-PCR as described in section 2.3.…”

“…ELISA (enzyme-linked immunosorbent assay) was performed to explore whether rTcCTL3 could interact with the carbohydrates (LPS or PGN) using a previously reported method. 29 Briefly, LPS or PGN at 80 μg mL −1 (50 μL) were used to coat a 96-well microtiter plate (Corning, New York, USA), incubated overnight at 37°C and heated at 60°C for 30 min. The plate was blocked with 200 μL well -1 of 1 mg mL −1 BSA in TBS for 2 h at 37°C, 100 mL serial concentrations of rTcCTL3 were added to each well in the presence of BSA (0.1 mg mL −1 ) at 18°C for 3 h. After incubation, the plate was washed three times with TBS.…”

“…Five types of microorganisms, as mentioned earlier, were also used to detect the direct agglutination ability of rTcCTL3 according to a previously described method. 13,29 The equal volume of bacterial suspension was incubated with recombinant proteins (rTcCTL3, rCRD1 or rCRD2, 100 μg mL −1 ) at 28°C for 1 h, in the presence or absence of 10 mmol L -1 Ca 2+ . The recombinant methyl-CpG-binding domain (rTcMBD) with a His tag from T. castaneum was used as a negative control.…”

“…The dsRNA targeting GFP gene (as control) was synthesized as previously described. 29 To investigate the RNA interference (RNAi) efficiency, a total of 200 ng of ds-TcCTL3 in 200 nL was injected into the body cavity of each 13-day larva. Insects injected with an equal volume of ds-GFP were set as control groups.…”