Caspase-1 from the silkworm, Bombyx mori, is involved in Bombyx mori nucleopolyhedrovirus infection

2.1 Animals and real-time PCR analysis

Silkworms (NB, BC8, and 306 strains) were reared on fresh mulberry leaves at 25°C under 12:12-h light/dark cycles. Total RNA from B. mori larval tissues was extracted using the RNeasy® Mini Kit (Qiagen, Valencia, CA, USA). The extracted RNA was used to synthesize cDNA using the PrimeScript™ RT reagent Kit (Takara, Kyoto, Japan) following the manufacturer’s instructions. Semiquantitative PCR was performed according to the standard protocol using gene-specific primes and internal control Bm-rp49 primers. Gene expression was evaluated by real-time PCR as previously described [11]. Bm-rp49 primers were used as internal control.

2.2 Cloning of Bm-caspase-1 and sequence analysis

Tn-caspase-1 sequence was used as target sequence to identify the Bm-caspase-1 from B. mori expressed sequence tags (EST) database. Multiple sequence alignment was performed with BioEdit software (http://www.ebi.ac.uk/clustaw/). The phylogenetic tree was constructed in MEGA 5 software using neighbor-joining (NJ) method.

2.3 Bm-caspase-1 expression in E. coli

Gene-specific primers for Bm-caspase-1 (forward: 5′-CGCGGATCCATGGCTGATGAAGAAAAGAAAACC-3′; reverse: 5′-AGTAGCGGCCGCCAAACAAGAGAAGGCGTGTCAG-3′) containing BamHI and XhoI restriction sites were synthesized (Shanghai Shenggong, Shanghai, China). The full length of Bm-caspase-1 was amplified by PCR using pfu polymerase (Takara) and cloned into the pET-30a expression vector. After ligation, the recombinant vector was transformed into E. coli BL21. The expression plasmid was induced with 0.4 mM isopropyl β-d-1-thiogalactopyranoside (IPTG) at 28°C for 4 h.

Subsequently, the bacterial cells were centrifuged and resuspended in 50 mM phosphate buffer (pH 8.0). The bacterial cells were harvested by sonication and centrifugation and then they were boiled for 10 min. Protein samples were subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and then stained with Coomassie brilliant blue.

2.4 Caspase activity assay

Caspase activity was measured using a caspase activity kit according to the manufacturer’s protocol (Beyotime, Haimen, China). After IPTG induction, cell lysates were prepared and the supernatants were incubated in a 96-well plate with Ac-DEVD-pNA, Ac-LEHD-pNA, or Ac-LETD-pNA at 37°C overnight. The absorbance values of pNA were measured at 405 nm using plate reader (BioTek, Santa Barbara, CA, USA).

3.1 Isolation and characterization of Bm-caspase-1

The cDNA sequence showed high similarity to lepidopteran caspase-1 that was identified from the B. mori EST database using Trichoplusia ni caspase-1 sequence as a template. The open reading frame of this cDNA is 879 bp and encodes 293 amino acids with a putative molecular mass of 33 kDa (Figure 1). The Bm-caspase-1 protein sequence contained the highly conserved amino acid motif QACQG surrounding the catalytic site. It also contained two consensus motifs of caspase cleavage, DEGDA and TETDG (Figure 1). Protein sequence alignment results revealed that Bm-caspase-1 shared 83% identity with Manduca sexta, 81% identity with T. ni, 80% identity with Chilo suppressalis, and 79% identity with Helicoverpa armigera orthologues (Figure 2). As shown in Figure 2, the Bm-caspase-1 amino acid sequence shared a high level of identity among reported lepidopteran effector caspase-1 genes by multiple sequence alignment (Figure 2). All of these caspase-1 proteins displayed the highly conserved amino acid QACQG and two consensus caspase-cleavage motifs DEGDA and TETDG (Figure 2). Next, CLUSTALX and MEGA 5.0 phylogenetic analyses were performed to compare the Bm-caspase-1 with other species by the NJ method. As shown in Figure 3, Bm-caspase-1 was most closely related to Hm-caspase-1. The phylogenetic relationship analysis revealed that Bm-caspase-1 had closer orthologous relationships with other lepidopteran caspase-1 genes (Figure 3).

Figure 1:

Sequence analysis of Bm-caspase-1: open reading frame sequence and amino acid sequences of Bm-caspase-1. Filled boxes, the two cleavage sites, DEGDA and TETDG, used to generate the large and small subunits; black lines, pentapeptide QACQG active site.

Figure 2:

Amino sequence alignment of the Bm-caspase-1 with other lepidopteran caspase-1: multiple sequence alignment of caspase-1 from Manduca sexta (AEF30493), Trichoplusia ni (AAO17788), Chilo suppressalis (AFJ97219), Helicoverpa armigera (ABS18284), Euphydryas aurinia (AEF30498), Spodoptera exigua (AEK12771), Heliconius melpomene (ACU11588) and Spodoptera litura (BAM62940). Filled boxes, the two cleavage sites DEGDA and TETDG, used to generate the large and small subunits; black lines, pentapeptide QACQG active site.

Figure 3:

NJ phylogram showing the relationships between Bm-caspase-1 with respect to the other caspase-1. The phylogenetic tree was constructed in MEGA 5 software using the NJ method. ▲, Bm-caspase-1.

3.2 Expression and caspase activity analysis of Bm-caspase-1

We then expressed the complete ORF of Bm-caspase-1 in E. coli BL21 using the vector pET-30a. After induction with 0.5 mM IPTG for 3 h, the recombinant protein was detected using SDS-PAGE analysis (Figure 4A). Cell lysates were prepared from E. coli BL21 and centrifuged at 16,200 g for 10 min at 4°C. The resultant supernatants were examined using different synthetic caspase substrates. The results showed significant proteolytic activity for the caspase-3 Ac-DEVD-pNA substrate. However, only negligible proteolytic activity was observed for the caspase-8 Ac-LETD-pNA and caspase-9 Ac-LEHD-pNA substrates (Figure 4B). Similar results were also observed using purified proteins as shown in Figure 4C. The results demonstrated that Bm-caspase-1 protein expressed in E. coli BL21 functions as an effector caspase.

Figure 4:

Caspase activity assay of Bm-caspase-1 in Escherichia coli BL21 cells. (A) Protein samples were separated by SDS-PAGE and stained with Coomassie brilliant blue. Lane 1, proteins from BL21 cells transformed with pET30a plasmid. Lanes 2 and 3, proteins from BL21 cells transformed with pET-30a-Bm-caspase-1. (B, C) Supernatants from BL21 cells or purified proteins were incubated with Ac-DEVD-pNA, Ac-IEHD-pNA, or Ac-LEHD-pNA at 37°C overnight. The absorbance values of pNA were measured at 405 nm. Relative caspase activity was calculated from the pNA standard curve.

3.3 Expression profiles of Bm-caspase-1 in different tissues and developmental stages

Reverse transcription-PCR (RT-PCR) was used to determine the Bm-caspase-1 mRNA expression levels in different tissues and developmental stages. Bm-caspase-1 could be detected in all of the examined tissues including fat body, integument, head, midgut, silk gland, trachea, and hemocytes (Figure 5A). Next, the mRNA from day 1 of egg, pupae, silkworm moth, and first to fifth instars was isolated, and the Bm-caspase-1 mRNA expression levels were examined. RNA from RT-PCR revealed that Bm-caspase-1 was expressed in egg, pupae, silkworm moth, and first to fifth instars (Figure 5B). These results indicate that Bm-caspase-1 is universally expressed in different tissues and at different developmental stages.

Figure 5:

Transcriptional levels of Bm-caspase-1 in different tissues and developmental stages. (A) Bm-caspase-1 transcription levels in fat body, skin, head, midgut, silk gland, trachea, and hemocytes were measured by RT-PCR analysis. Rp49 was used as an internal control. (B) mRNA expression levels of Bm-caspase-1 in the egg, pupae, silkworm moth, and first to fifth instars were measured by RT-PCR.

3.4 Bm-caspase-1 mRNA expression levels increased during baculovirus infection

Previous studies have demonstrated that AcMNPV infection induced the expression and maturation of SI-caspase-1 in SL2 cells [12]. Silkworm larvae infected with BmNPV died with in 110 h post-infection [13]. However, no study has examined the caspase activity after infection by BmNPV in vivo. When fifth-instar B. mori larvae were infected with BmNPV, the mRNA expression levels of Bm-caspase-1 in the midgut from different strains were significantly increased from 24 to 72 h compared with the control (Figure 6A). Next, the caspase activity was analyzed during the same 24 to 72 h post-infection. As shown in Figure 6B, the caspase activity of infected silkworms increased greatly after 72 h post-infection, but was not heightened at the time of infection. Taken together, these results demonstrated that Bm-caspase-1 was likely involved in BmNPV infection of the silkworm.

Figure 6:

Bm-caspase-1 transcription levels and caspase-1 activity in the midgut of silkworms after BmNPV infection. (A) BmNPV-resistant strain NB, BC8, and susceptible strain 306 were infected with BmNPV for 24 h or 72 h. Then, the midguts were isolated and mRNA was extracted for RT-PCR analysis. Rp49 was used as an internal control. (B). Protein samples from the midgut in the BmNPV-resistant strain NB were isolated and supernatants were incubated with Ac-DEVD-pNA at 37°C overnight. The absorbance values of pNA were measured at 405 nm. Error bars and asterisks indicate standard deviation and statistical significance, respectively. *p<0.05.

3.5 Bm-caspase-1 transcriptional level up-regulated in BmNPV-resistant silkworms

The differences in the 50% lethal dosage (LD50) values among silkworm populations were defined to be the level of resistance to BmNPV infection. A silkworm strain named NB, with high resistance to BmNPV infection, was previously established. There was an approximately 1000-fold LD50 difference between NB and susceptible strain 306 after infection. A near-isogenic line named BC8 with resistance to BmNPV was also established [10], [14]. We then compared the mRNA expression levels of Bm-caspase-1 between the two strains. As shown in Figure 7A, the Bm-caspase-1 transcriptional level in NB was much higher than in the susceptible strain 306, even at 0 h infection. At 48 and 72 h post-infection, Bm-caspase-1 transcriptional levels were much higher in NB compared with the susceptible strain 306. Similar results were also observed in another resistant strain BC8 (Figure 7B). These results suggested that Bm-caspase-1 probably played a critical role in silkworm resistance against BmNPV infection.

Figure 7:

Relative expression levels of Bm-caspase-1 in the BmNPV-resistant strain NB, BC8, and susceptible strain 306. (A, B) The BmNPV-resistant strain NB, BC8, and susceptible strain 306 were infected with BmNPV for 48 or 72 h. Fat bodies in the silkworm were isolated, and mRNA was extracted for RT-PCR analysis. Rp49 was used as an internal control. Error bars and asterisks indicate standard deviation and statistical significance, respectively. *p<0.05.