Molecular characterization of a Niemann–Pick disease type C2 protein from the honeybee Apis cerana

    Molecular characterization of a Niemann-Pick disease type C2 protein from the honeybee Apis cerana Kwang Sik Lee, Hee Geun Park, Deng Yijie, Bo Yeon Kim, Seung Su Kyung, Yong Soo Choi, Hyung Joo Yoon, Mingshun Li, Byung Rae Jin PII: DOI: Reference:

S1226-8615(14)00065-X doi: 10.1016/j.aspen.2014.05.005 ASPEN 535

To appear in:

Journal of Asia-Pacific Entomology

Received date: Revised date: Accepted date:

9 April 2014 7 May 2014 14 May 2014

Please cite this article as: Lee, Kwang Sik, Park, Hee Geun, Yijie, Deng, Kim, Bo Yeon, Kyung, Seung Su, Choi, Yong Soo, Yoon, Hyung Joo, Li, Mingshun, Jin, Byung Rae, Molecular characterization of a Niemann-Pick disease type C2 protein from the honeybee Apis cerana, Journal of Asia-Pacific Entomology (2014), doi: 10.1016/j.aspen.2014.05.005

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Molecular characterization of a Niemann-Pick disease type C2 protein

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from the honeybee Apis cerana

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Kwang Sik Lee a, Hee Geun Park a, Deng Yijie b, Bo Yeon Kim a, Seung Su Kyung a,

a

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Yong Soo Choi c, Hyung Joo Yoon c, Mingshun Li b, Byung Rae Jin a,*

College of Natural Resources and Life Science, Dong-A University, Busan 604-714,

College of Life Science and Technology, Huazhong Agricultural University, Wuhan, P.

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R. China

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Republic of Korea

Department of Agricultural Biology, National Academy of Agricultural Science, Suwon,

Republic of Korea

* Corresponding author. Tel./fax: +82 51 200 7594 E-mail address: [email protected] (B.R. Jin)

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ABSTRACT

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Drosophila Niemann-Pick disease type C2 (NPC2) proteins play roles in sterol

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homeostasis, steroid biosynthesis, and innate immune signaling pathways. In this study,

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a bee (Apis cerana) NPC2a protein (AcNPC2a) that might function in innate immune

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reactions was identified. AcNPC2a consisted of 148 amino acids, which included six conserved cysteine residues. Recombinant AcNPC2a protein (expressed in baculovirus-

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infected insect cells) bound directly to live Escherichia coli, Bacillus thuringiensis, and

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Beauveria bassiana; however, AcNPC2a did not show antimicrobial activity against

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these microorganisms. Nevertheless, the expression of AcNPC2a was significantly induced in the fat body of A. cerana worker bees after injection with E. coli, B. thuringiensis, or B. bassiana. Our data suggest a role for AcNPC2a in innate immunity that is induced in response to microbial challenge and binds directly to the cell walls of bacteria and fungi. These findings provide insight into the role of AcNPC2a during the innate immune response following bacterial and fungal infection.

Keywords: Apis cerana; Honeybee; NPC2; Innate immunity; Insect

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Introduction

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Niemann-Pick type C (NPC) disease is a cholesterol homeostasis-related disorder in

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humans (Patterson, 2003). Mutations in either of the two human NPC genes, NPC1 and

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NPC2, cause a fatal neurodegenerative disease that is associated with abnormal

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cholesterol accumulation in cells. In Drosophila, NPCA1a regulates sterol homeostasis, similar to mammalian NPC1, and is required for the synthesis of the molting hormone

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ecdysone (Huang et al., 2005). Neuronal loss of Drosophila NPC1a causes cholesterol

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aggregation and age-progressive neurodegeneration (Phillips et al., 2008). The

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Drosophila NPC2 genes control sterol homeostasis and steroid biosynthesis, and mutation of Drosophila NPC2a, which is most similar in sequence to vertebrate NPC2, results in abnormal sterol distribution in many cells (Huang et al., 2007). Furthermore, Drosophila NPC2a functions redundantly with NPC2b in regulating sterol homeostasis and ecdysteroid biosynthesis (Huang et al., 2007). Two NPC1 (NPC1a and NPC1b) and eight NPC2 genes have been identified in Drosophila (Huang et al., 2005, 2007; Shi et al., 2012). Based on the numbers of cysteine residues in the protein sequences, the eight NPC2 genes in Drosophila are divided into three subgroups: 6 (NPC2a, -2b, -2c, and 2f), 7 (NPC2d and -2e), and 8 cysteine residues (NPC2g and -2h) (Shi et al., 2012).

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Insect ML (MD-2 (myeloid differentiation factor-2)-related lipid-recognition) genes,

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which are named NPC2- or MD-2-like genes in different species, have been identified:

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thirteen MD-2-like genes are found in the mosquito Anopheles gambiae, fifteen NPC2-

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like genes are found in the mosquito Aedes aegypti, eight MD-2-like genes are found in

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the beetle Tribolium castaneum, and at least four MD-2-like genes are found in the

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silkworm Bombyx mori (Shi et al., 2012). Silkworm NPC2 inhibited cellular proliferation and increased triglyceride accumulation in BmN4 cells (Adachi et al.,

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2014). Furthermore, mounting evidence indicates that NPC2 proteins in insects may

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function in innate immune reactions. An. gambiae MDL1 (AgMDL1) has a role in the

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immune response against Plasmodium falciparum (Dong et al., 2006). The tobacco hornworm Manduca sexta ML-1 (MsML-1) protein binds to lipopolysaccharide (LPS), which suggests its involvement in LPS-induced signaling (Ao et al., 2008). Thus, AgMDL-1 and MsML-1 may have functions in innate immune reactions. Additionally, the European hard tick Ixodes ricinus ML-domain-containing protein is involved in lipid binding and transport, pathogen recognition, and the immune response (Horáčková et al., 2010). A shrimp ML protein binds to LPS, and its transcriptional level is induced by LPS challenge (Liao et al., 2011). Drosophila NPC2 proteins bind bacterial cell wall components and may function in innate signal pathways (Shi et al., 2012). However,

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whether insect NPC2 proteins bind directly to the cell walls of bacteria or fungi and are

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induced in response to bacterial or fungal injection in vivo remains to be determined.

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Although the sequences of NPC2-like genes from bees, such as honeybees and

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bumblebees, are found in database searches, the roles of bee-derived NPC2 proteins

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remain relatively unexplored. Here, we report the molecular characterization of the bee

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(Apis cerana)-derived NPC2a protein (AcNPC2a). First, we cloned AcNPC2a cDNA and expressed recombinant AcNPC2a in baculovirus-infected insect cells. Recombinant

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AcNPC2a was assayed for microbial binding and antimicrobial activity. Next, we

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characterized the transcriptional induction of AcNPC2a in response to microbial

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challenge. We provide the first evidence that AcNPC2a is induced in response to bacterial (Escherichia coli and Bacillus thuringiensis) or fungal (Beauveria bassiana) injection and that recombinant AcNPC2a binds directly to the cell walls of these microorganisms, but it does not show antimicrobial activity, which suggests that AcNPC2a may function as a factor in innate immune reactions.

Materials and methods

cDNA cloning and sequence analysis

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A clone encoding AcNPC2a was selected from the set of expressed sequence tags

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(ESTs) that were generated from a cDNA library that was constructed using whole

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bodies of A. cerana (Kim et al., 2013a, b). Plasmid DNA was extracted using a Wizard

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Mini-Preparation kit (Promega, Madison, WI, USA), and the generated cDNA sequence

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was analyzed using an ABI310 automated DNA sequencer (Perkin-Elmer Applied Biosystems, Foster City, CA, USA). The sequenced cDNA was compared using the

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predicted

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sequence

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DNASIS and BLAST databanks (http://www.ncbi.nlm.nih.gov/BLAST). The signal using

the

SignalP

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software

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(http://www.cbs.dtu.dk/services/SignalP), and MacVector (ver. 6.5, Oxford Molecular Ltd., Oxford, UK) was used to align the predicted amino acid sequences of the NPC2 genes.

Protein expression and purification

A baculovirus expression system (Je et al., 2001) using Autographa californica nucleopolyhedrovirus (AcNPV) and the Spodoptera frugiperda (Sf9) insect cell line was employed to construct a recombinant virus expressing recombinant AcNPC2a. The

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AcNPC2a cDNA was PCR-amplified from pBluescript-AcNPC2a using the forward

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primer (20-37) 5’-CTCGAGATGGCAATCCTCACCTAT-3’ and the reverse primer

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(446-466)

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GAATTCTTAATGATGATGATGATGATGGTCGACAACTTTAGCTGG-3’;

5’the

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reverse primer was designed to include a His-tag sequence. PCR cycling conditions

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were as follows: 94 °C for 3 min, 30 cycles of amplification (94 °C for 30 sec, 55 °C for 30 sec, and 72 °C for 1 min), and 72 °C for 5 min. The PCR products were sequenced

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using the BigDye Terminator Cycle Sequencing Kit and an automated DNA sequencer

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(Perkin-Elmer Applied Biosystems). The isolated AcNPC2a fragment was inserted into

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the pBacPAK8 vector (Clontech, Palo Alto, CA, USA) to generate an expression vector under the control of the AcNPV polyhedrin promoter. For expression experiments, 500 ng of the construct (pBacPAK8-AcNPC2a) and 100 ng of AcNPV viral DNA (Je et al., 2001) were co-transfected into 1.0–1.5 × 106 Sf9 cells for 5 h using the Lipofectin transfection reagent (Gibco BRL, Gaithersburg, MD, USA). The transfected cells were cultured in TC100 medium (Gibco BRL) supplemented with 10% fetal bovine serum (Gibco BRL) at 27 °C for 5 days. Recombinant baculoviruses were propagated in Sf9 cells cultured in TC100 medium at 27 °C, and the recombinant proteins were then purified using the MagneHisTM Protein Purification System (Promega). The

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recombinant proteins were identified using 12% sodium dodecyl sulfate-polyacrylamide

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gel electrophoresis (SDS-PAGE) and Western blot analysis. Western blots were

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performed using an enhanced chemiluminescence Western blotting system (Amersham

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Biosciences) with anti-His antibody and horseradish peroxidase-conjugated anti-mouse

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IgG diluted 1:5,000 (v/v). The protein concentrations were determined using a Bio-Rad

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Protein Assay Kit (Bio-Rad, Hercules, CA, USA).

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Microbial binding assay

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The microbial binding assay was performed as previously described (You et al., 2010; Kim et al., 2013b). Bacillus thuringiensis 656-3 (Choi et al., 2004; Choo et al., 2010b; You et al., 2010; Kim et al., 2013b) and Escherichia coli DH5α (Choo et al., 2010b; You et al., 2010; Kim et al., 2013b) were grown in Luria-Bertani medium, and Beauveria bassiana SFB-205 (Kim and Je, 2010; You et al., 2010; Kim et al., 2013b) was grown in potato dextrose broth. When the cultures reached an OD600 of 0.4, 4 ml of each bacterial or fungal culture was harvested, washed in PBS, and resuspended in 40 µl of the recombinant AcNPC2a (0.8 µg). Following incubation at room temperature for 10 min, the suspensions were centrifuged, and the pellets were washed and resuspended

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in 40 µl of phosphate-buffered saline (PBS; 140 mM NaCl, 27 mM KCl, 8 mM

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Na2HPO4, and 1.5 mM KH2PO4, pH 7.4). Samples of the pellets and supernatants were

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subjected to 12% SDS-PAGE and Western blot analysis using an antiserum probe

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against the 6x His tag, as described above.

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Immunofluorescence staining

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As described in the microbial binding assay, B. thuringiensis, E. coli, and B. bassiana

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were harvested, washed three times with PBS, and resuspended in 40 µl of recombinant

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AcNPC2a (0.8 µg). After a 10-min incubation at room temperature, the bacteria and fungi were fixed in methanol (-20 °C) for 3 min and air-dried. The bacteria and fungi were washed three times in PBS and then pre-incubated in PBS containing 2% bovine serum albumin (BSA) at room temperature for 20 min. After being washed once with PBS, the bacteria and fungi were incubated for 1 h with anti-6×His tag mouse polyclonal antiserum diluted 1:500 (v/v) in PBS containing 1% BSA. The bacteria and fungi were washed two times with PBS for 10 min and then incubated for 1 h with fluorescein-conjugated goat anti-mouse antibody (Santa Cruz Biotech., Inc., Santa Cruz, CA, USA) diluted 1:400 (v/v) in PBS containing 1% BSA. After five successive washes

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in PBS for 25 min, the bacteria and fungi were wet-mounted. AcNPC2a was then

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visualized and localized within the bacterial and fungal cells using laser scanning

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confocal microscopy (Carl Zeiss LSM 510, Zeiss), as previously described (You et al.,

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2010; Kim et al., 2013b).

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Antimicrobial assay

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The antimicrobial activity of recombinant AcNPC2a was assayed as previously

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described (Choo et al., 2010b; You et al., 2010; Kim et al., 2013b). The antimicrobial

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activity of purified recombinant AcNPC2a was tested against the Gram-positive bacterium B. thuringiensis and the Gram-negative bacterium E. coli using a liquid growth inhibition assay. Two hundred microliters of inoculum (105 cfu/ml) was added to each well of a 96-well plate containing serial dilutions (1 µg, 2.5 µg, or 5 µg) of recombinant AcNPC2a; the same volume of PBS was used as a control. The 96-well plates were incubated at 37 °C for 24 h with shaking at 220 rpm, and bacterial growth inhibition was determined by measuring the absorbance at 595 nm. The growth inhibition results are expressed as the mean values from three independent replicates. The minimal inhibitory concentration (MIC) for the antibacterial assay was the lowest

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concentration that caused 50% inhibition of bacterial growth (Choo et al., 2010b).

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Recombinant AcNPC2a was also tested for antifungal activity against the fungus B.

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bassiana using a liquid growth inhibition assay. Two hundred microliters of inoculum

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(2 × 104 conidia/ml) was added to each well of a 96-well plate containing serial

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dilutions of AcNPC2a, and the same volume of PBS was used as a control. The 96-well

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plates were incubated at 22 °C for 48 h with shaking at 220 rpm, and fungal growth inhibition was determined by measuring the absorbance at 595 nm. The growth

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inhibition results are expressed as the mean values of three independent replicates. The

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IC50 values for antifungal activity are expressed as the concentration of the AcNPC2a

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that was required to inhibit 50% of fungal growth (Choo et al., 2010b; Kim et al.,

RNA extraction and Northern blot analysis

The Asian honeybee A. cerana (Hymenoptera: Apidae) that was used in this study was supplied by the Department of Agricultural Biology, National Academy of Agricultural Science, Republic of Korea (Kim et al., 2013a, b). A. cerana worker bees were dissected on ice using a stereomicroscope (Zeiss, Jena, Germany). Tissue samples

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(epidermis, fat body, gut, muscle, and venom gland) were collected and washed with

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PBS. Total RNA was isolated from the epidermis, fat body, gut, muscle, and venom

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gland of A. cerana using a Total RNA Extraction Kit (Promega). Total RNA (5 µg/lane)

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was separated using a 1.0% formaldehyde agarose gel, transferred onto a nylon blotting

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membrane (Schleicher & Schuell, Dassel, Germany), and hybridized at 42 °C with the

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appropriate probe diluted in hybridization buffer containing 5× SSC (0.75 M sodium chloride and 0.75 M sodium citrate), 5× Denhardt’s solution (0.1% each of BSA, Ficoll,

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and polyvinylpyrrolidone), 0.5% SDS, and 100 mg/ml denatured salmon sperm DNA.

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AcNPC2a cDNA was labeled with [α-32P] dCTP (Amersham Biosciences, Piscataway,

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NJ, USA) using the Prime-It II Random Primer Labeling kit (Stratagene, La Jolla, CA, USA), and the labeled cDNA was used as a probe for hybridization. After hybridization, the membrane filter was washed three times for 30 min each in 0.1% SDS and 0.2× SSC at 65 °C and then exposed to autoradiography film. Images of Northern blots were analyzed using a computerized image analysis system (Alpha Innotech Co., San Leandro, CA, USA). Alpha Imager 1220 (ver. 5.5) was used to aid the analyses. The integrated density value was used to determine the area of each band. Each analysis was performed with a total of three biological replicates.

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Microorganism injection

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A. cerana worker bees at random periods after adult emergence were injected with sample solutions between the first and second abdominal segments using a sterile

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needle (Yoon et al., 2009; You et al., 2010). For PBS injection or fungal challenge, A.

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cerana worker bees were injected with 5-µl samples after anesthesia by chilling (Yoon et al., 2009; You et al., 2010). Control A. cerana worker bees were injected with 5 µl of

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PBS. Experimental A. cerana worker bees were inoculated with bacteria (E. coli or B.

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thuringiensis) or fungi (B. bassiana). The bacterial cells were isolated through centrifugation of 10-ml overnight cultures, washed with PBS, and resuspended in PBS. A. cerana worker bees were injected with a 5-µl solution containing 5.5 × 103 bacterial cells. Fungal spores collected from a culture plate were resuspended in PBS, and A. cerana worker bees were injected with a 5-µl solution containing 5.0 × 103 spores. A. cerana worker bee tissues were collected at various times (1, 3, or 5 h post-injection) and washed twice with PBS. Total RNA extraction and Northern blot analysis were performed as described above.

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Results

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AcNPC2a is a bee NPC2a protein

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To characterize the bee-derived NPC2 protein, we identified an EST for a gene

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encoding an NPC2a protein from an A. cerana cDNA library. An AcNPC2a cDNA sequence that included the full-length NPC2a protein-coding sequence was identified

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by searching the set of A. cerana ESTs (GenBank accession number KJ633823).

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Database searches using the predicted AcNPC2a protein sequence confirmed that

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AcNPC2a consisted of 148 amino acids that included six cysteine residues (Fig. 1A and B). Analysis of the predicted AcNPC2a protein sequence revealed similarities to other members of the NPC2 family. Additionally, the predicted AcNPC2a protein sequence revealed the highest protein sequence identity to NPC2a (27% protein sequence identity) among the D. melanogaster NPC2 proteins (Fig. 1A). Thus, we named A. cerana NPC2a (AcNPC2a) based on the protein sequence identity and the numbers of certain amino acids, including the six cysteine residues, compared with D. melanogaster NPC2a. In addition, sequence analysis of the predicted AcNPC2a protein showed that it

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shared high identity with the bee NPC2 proteins (Fig. 1B), which also had six conserved

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cysteine residues, similar to D. melanogaster NPC2 proteins. The AcNPC2a protein

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sequence exhibited high identity to the NPC2 proteins of the honeybees, A. mellifera

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(94% protein sequence identity) and A. florea (93% protein sequence identity), while

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AcNPC2a showed relatively low identity to the NPC2 proteins of the bumblebees,

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Bombus terrestris (75% protein sequence identity) and B. impatiens (72% protein sequence identity). Furthermore, the honeybee NPC2 proteins consisted of 148 amino

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acids, while the bumblebee NPC2 proteins consisted of 149 amino acids.

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AcNPC2a expression is induced in the fat body after microbial challenge

Because NPC2 is involved in innate immune reactions (Dong et al., 2006; Ao et al., 2008; Horáčková et al., 2010; Shi et al., 2012), we hypothesized that AcNPC2a functions in innate immunity against microorganisms, such as B. thuringiensis, E. coli, and B. bassiana. To test this hypothesis, we examined the expression profile of AcNPC2a in A. cerana worker bees. First, the expression pattern of AcNPC2a in A. cerana worker bees was examined to confirm that it is an A. cerana-derived NPC2a protein. Northern blot analysis was performed using total RNA prepared from the

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epidermis, fat body, gut, muscle, and venom gland of A. cerana. Northern blotting

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showed that the AcNPC2a gene was constitutively expressed in the epidermis and fat

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body (Fig. 2A).

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Next, we assessed whether AcNPC2a is induced in response to microbial challenge.

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To determine the transcriptional expression profile of AcNPC2a in the fat body and

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epidermis following microbial challenge, A. cerana worker bees were injected with Gram-negative bacteria (E. coli), Gram-positive bacteria (B. thuringiensis), or

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entomopathogenic fungi (B. bassiana), and AcNPC2a transcription was characterized

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using Northern blot analysis. AcNPC2a transcription was significantly induced in the fat

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body of A. cerana worker bees injected with E. coli, B. thuringiensis, or B. bassiana, but not in the epidermis (Fig. 2B). The expression of AcNPC2a was acutely increased after microbial challenge (Fig. 2C).

AcNPC2a binds to bacterial and fungal cell wall components

To assess the function of AcNPC2a, recombinant AcNPC2a was expressed in baculovirus-infected insect cells. Purified recombinant AcNPC2a, which contained an additional six His residues compared to the natively encoded protein, was 16.6 kDa (Fig.

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3A).

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Using recombinant AcNPC2a, we investigated whether AcNPC2a can bind to

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bacterial and fungal cell wall components. To test this issue, we examined the microbial

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binding activity of AcNPC2a. We used Western blotting and immunofluorescence

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staining to assay the ability of AcNPC2a to bind directly to live microorganisms, such

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as E. coli, B. thuringiensis, and B. bassiana. Based on the Western blot analysis, AcNPC2a bound to E. coli, B. thuringiensis, and B. bassiana (Fig. 3B). Consistent with

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these data, immunofluorescence staining revealed that AcNPC2a was localized to the

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cell walls of bacteria and fungi (Fig. 3C).

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To assess whether the binding of AcNPC2a to live bacterial and fungal cells was correlated with cell death, we determined the antimicrobial activity of AcNPC2a against E. coli, B. thuringiensis, and B. bassiana. However, unlike the binding assay results, recombinant AcNPC2a did not exhibit antimicrobial activity against bacteria and fungi.

Discussion

NPC proteins are involved in lipid metabolism and innate immunity (Inohara and

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Nuňez, 2002; Huang et al., 2005, 2007; Dong et al., 2006; Ao et al., 2008; Horáčková et

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al., 2010; Liao et al., 2011; Shi et al., 2012; Adachi et al., 2014). Although NPC gene

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sequences from bees are found in databases, no NPC proteins from bees have been

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functionally characterized. In this study, we identified the first bee (A. cerana)-derived

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NPC2a protein (AcNPC2a) that may function in innate immune reactions. We

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hypothesized that AcNPC2a is similar to other NPC proteins because it possesses six conserved cysteine residues. Recently, Drosophila NPC2 genes were divided into three

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subgroups based on their 6 (NPC2a, -2b, -2c, and -2f), 7 (NPC2d and -2e), or 8 cysteine

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residues (NPC2g and -2h) (Shi et al., 2012). We named the protein AcNPC2a because it

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shares the highest sequence similarity to NPC2a (27% protein sequence identity) of the Drosophila NPC2 proteins and because it consists of 148 amino acids, including six cysteine residues, like Drosophila NPC2a. Additionally, sequence analysis showed that AcNPC2a shares high protein sequence identity with bee NPC proteins, as well as Drosophila NPC proteins. AcNPC2a and honeybee NPC proteins with 148 amino acids cluster together and share high sequence similarity each other (93-94% protein sequence identity), while two bumblebee NPC proteins with 149 amino acids show relatively low protein sequence identity (72-75%). Collectively, these data suggest that AcNPC2a is a member of the NPC protein family.

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Insect ML genes, such as NPC2- or MD-2-like genes, have been reported in An.

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gambiae (Dong et al., 2006), M. sexta (Ao et al., 2008), and I. ricinus (Horáčková et al.,

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2010), suggesting that they may have functions in innate immune reactions. D.

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melanogaster NPC2 proteins bind bacterial cell wall components, including LPS, lipid

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A, peptidoglycan (PG), and lipoteichoic acid (LTA), and NPC2e and NPC2a may play a

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role in the immune deficiency (Imd) pathway (Shi et al., 2012). Therefore, we first determined whether AcNPC2a is induced in response to microbial challenge. A

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transcriptional expression profile indicated that AcNPC2a is significantly induced in the

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fat body of A. cerana worker bees exposed to live E. coli, B. thuringiensis, or B.

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bassiana, which is partially consistent with the previous report that D. melanogaster NPC2a is not induced by Gram-negative E. coli or Gram-positive Staphylococcus aureus, while NPC2e is significantly induced by S. aureus and E. coli (Shi et al., 2012). The finding that AcNPC2a is acutely induced in response to Gram-negative E. coli, Gram-positive B. thuringiensis, and entomopathogenic fungus B. bassiana injection suggests that AcNPC2a is stimulated by LPS, PG, and β-1,3-glucans, which might be involved in innate immune reactions. Given these observations and the function of NPC2 in innate immune reactions (Dong et al., 2006; Ao et al., 2008; Horáčková et al., 2010; Shi et al., 2012), our results show that AcNPC2a is induced in response to

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invading bacterial and fungal microorganisms, suggesting that AcNPC2a may be

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directly involved in the innate immune response.

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Given the binding ability of the NPC2 proteins to bacterial cell wall components

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(Ao et al., 2008; Shi et al., 2012), we also tested AcNPC2a for its ability to bind to

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Gram-positive and Gram-negative bacteria, as well as fungi. Our results show that

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AcNPC2a binds directly to E. coli, B. thuringiensis, and B. bassiana and that the binding ability of AcNPC2a is correlated with the transcriptional expression profile of

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AcNPC2a in A. cerana worker bees. These findings clearly show that AcNPC2a binds

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to bacterial and fungal cell wall components. Although D. melanogaster NPC2 proteins

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bind to LPS and PG (Shi et al., 2012), no report about the binding of NPC2 to β-1,3glucans has been published. Our results suggest that AcNPC2a bound to β-1,3-glucans, as well as PG and LPS. Taken together, our findings show that AcNPC2a binds directly to live bacteria and fungi and may function in innate immune reactions. However, further work will be required to better understand the mechanism by which AcNPC2a directly binds to live bacteria and fungi. We next tested whether the binding ability of AcNPC2a is correlated with a reduction in bacterial and fungal viability. AcNPC2a did not show antimicrobial activity against bacteria and fungi. AgMDL1, MsML-1, and some Drosophila NPC2 proteins

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may function as pattern recognition receptors (PRRs) or co-receptors for different

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bacterial ligands to modulate innate immune signal pathways (Dong et al., 2006; Ao et

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al., 2008; Shi et al., 2012). Given these observations and the functions of Drosophila

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NPC2 proteins in immune signal pathways (Shi et al., 2012), our results suggest that,

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although AcNPC2a does not function as an antimicrobial peptide, it may be a factor in

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the innate immune response following bacterial and fungal infection. In this study, we provide the first evidence that AcNPC2a may function as a factor in

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innate immune reactions. Taken together, our findings suggest a role for AcNPC2a in

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innate immune reactions of A. cerana worker bees: one in which AcNPC2a is induced

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in response to bacterial and fungal infection, and another in which AcNPC2a binds directly to live bacteria and fungi. Our findings that AcNPC2a has a role in innate immune reactions could serve as the molecular basis for future studies on bee NPC2 proteins.

Acknowledgments

This work was supported by a grant from the Rural Development Administration (Next-Generation Biogreen 21 Program), Republic of Korea. 21

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Figure legends

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Fig. 1. Alignment of the amino acid sequences for AcNPC2a and known NPC2 proteins.

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(A) Alignment of AcNPC2a and D. melanogaster NPC2 proteins. The six conserved

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cysteine residues are indicated by solid circles, and the predicted signal peptides are

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underlined. The sources of the aligned sequences are AcNPC2a (this study, GenBank accession no. KJ633823), D. melanogaster NPC2a (DmNPC2a, GenBank accession no.

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NM_134793), D. melanogaster NPC2b (DmNPC2b, GenBank accession no.

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NM_169568), D. melanogaster NPC2c (DmNPC2c, GenBank accession no.

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NM_141719), D. melanogaster NPC2d (DmNPC2d, GenBank accession no. NM_141718), D. melanogaster NPC2e (DmNPC2e, GenBank accession no. NM_169323), D. melanogaster NPC2f (DmNPC2f, GenBank accession no. NM_142962), D. melanogaster NPC2g (DmNPC2g, GenBank accession no. NM_143566), D. melanogaster NPC2h {A} (DmNPC2h {A}, GenBank accession no. NM_143567), and D. melanogaster NPC2h {B} (DmNPC2h {B}, GenBank accession no. NM_170521). The AcNPC2a sequence was used as the reference for the identity/similarity (Id/Si) values. (B) Alignment of AcNPC2a and the bee NPC2 proteins. The six conserved cysteine residues are indicated by solid circles. The sources

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of the aligned sequences are AcNPC2a (this study, GenBank accession no. KJ633823),

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A. mellifera NPC2 (GenBank accession no. XM_001120220), A. florea NPC2

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(GenBank accession no. XM_003696195), Bombus terrestris NPC2 (GenBank

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accession no. XM_003395399), and B. impatiens NPC2 (GenBank accession no.

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XM_003488747). The AcNPC2a sequence was used as the reference for the

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identity/similarity (Id/Si) values.

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Fig. 2. Transcriptional expression profiles of the AcNPC2a gene. (A) Expression of

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AcNPC2a in A. cerana worker bees. Total RNA was isolated from the epidermis, fat body, gut, muscle, and venom gland of A. cerana worker bees. RNA was separated by 1.2% formaldehyde agarose gel electrophoresis, transferred onto a nylon membrane, and hybridized with radiolabeled AcNPC2a cDNA (lower panel). AcNPC2a transcripts are indicated with an arrow. The ethidium bromide-stained RNA gel shows uniform loading (upper panel). (B) Transcriptional expression profiles of AcNPC2a gene in the epidermis and fat body of A. cerana worker bees by E. coli, B. thuringiensis, or B. bassiana injection. A. cerana worker bees injected with PBS were used as injection controls. Total RNA was isolated from the epidermis and fat body of worker bees at different time points, as indicated above the corresponding lanes. AcNPC2a transcripts

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are indicated with an arrow. (C) The levels of AcNPC2a mRNA are the means of three

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injection (shown as 100%). Bars represent the means ± SD.

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measurements that were calculated relative to the levels at 0 h before PBS or microbial

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Fig. 3. Microbial binding of recombinant AcNPC2a. (A) SDS-PAGE of purified

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recombinant AcNPC2a expressed in baculovirus-infected Sf9 insect cells. (B) Western blot analysis of AcNPC2a microbial binding activity. Live E. coli, B. thuringiensis, or B.

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bassiana was incubated with AcNPC2a for 10 min. Bound AcNPC2a (P) was separated

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from free AcNPC2a in the supernatant (S) using centrifugation, and the samples were

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analyzed using Western blotting with an anti-His antibody. (C) Immunofluorescence staining was performed to visualize the binding of AcNPC2a to live bacterial and fungal cells. E. coli, B. thuringiensis, or B. bassiana was treated with AcNPC2a for 10 min. AcNPC2a (green) bound to the cell walls of E. coli, B. thuringiensis, and B. bassiana. Merged confocal images are shown in the third column. The scale bar corresponds to 10 µm.

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Figure 1A

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Figure 1B

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Figure 2A

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Figure 2BC

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Figure 3

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Graphical abstract

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Highlights

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► The cDNA of bee (Apis cerana) Niemann-Pick disease type C2 (NPC2) protein

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(AcNPC2a) was cloned

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► AcNPC2a is induced in response to bacterial and fungal injection

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► AcNPC2a binds directly to the cell walls of bacteria and fungi ► AcNPC2a does not show antimicrobial activity

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► AcNPC2a may function in innate immune reactions

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