Identification of Proteasome Subunit Beta Type 6 (PSMB6) Associated with Deltamethrin Resistance in Mosquitoes by Proteomic and Bioassay Analyses

Deltamethrin (DM) insecticides are currently being promoted worldwide for mosquito control, because of the high efficacy, low mammalian toxicity and less environmental impact. Widespread and improper use of insecticides induced resistance, which has become a major obstacle for the insect-borne disease management. Resistance development is a complex and dynamic process involving many genes. To better understand the possible molecular mechanisms involved in DM resistance, a proteomic approach was employed for screening of differentially expressed proteins in DM-susceptible and -resistant mosquito cells. Twenty-seven differentially expressed proteins were identified by two-dimensional electrophoresis (2-DE) and mass spectrometry (MS). Four members of the ubiquitin-proteasome system were significantly elevated in DM-resistant cells, suggesting that the ubiquitin-proteasome pathway may play an important role in DM resistance. Proteasome subunit beta type 6 (PSMB6) is a member of 20S proteasomal subunit family, which forms the proteolytic core of 26S proteasome. We used pharmaceutical inhibitor and molecular approaches to study the contributions of PSMB6 in DM resistance: the proteasome inhibitor MG-132 and bortezomib were used to suppress the proteasomal activity and siRNA was designed to block the function of PSMB6. The results revealed that both MG-132 and bortezomib increased the susceptibility in DM-resistant cells and resistance larvae. Moreover, PSMB6 knockdown decreased cellular viability under DM treatment. Taken together, our study indicated that PSMB6 is associated with DM resistance in mosquitoes and that proteasome inhibitors such as MG-132 or bortezomib are suitable for use as a DM synergist for vector control.


Introduction
Mosquito-borne diseases, such as malaria, dengue fever, yellow fever, filariasis and encephalitis, cause severe mortality and morbidity around the world, and pose significant threats to public health [1][2][3][4]. For a long period of time, insecticides have been the primary method for managing mosquito-borne diseases [5,6]. Pyrethroid, one of the most prevalent insecticides, interacts with ion channels, which disrupt the transmembrane potentials, and damage the insect nervous system [7]. Deltamethrin (DM), a representative synthetic pyrethroid insecticide, is widely used for bed net impregnation and residual spraying for mosquito control [8,9]. Widespread and improper use of insecticides has induced the development of insecticide resistance [10,11], which has become the main obstacle for the mosquito-borne disease management [12][13][14].
Insecticide resistance is polygenic inheritance phenomenon, which suggests that multiple genes are associated with resistance [15]. Large-scale transcriptional gene expression profiling based on suppression subtractive hybridization (SSH) and cDNA microarray studies had been carried out to identify DM resistance-associated genes in Culex pipiens pallens [16,17]. Although novel genes associated with DM resistance have been identified, the mechanisms underlying DM resistance are still not fully understood. Proteomics research, a strategy focusing on protein expression profiling, has great advantages in the study of complicated biological events [18]. Therefore, characterization and comparison of the protein profiles of susceptible and resistant strains will provide valuable information regarding the resistance mechanisms in mosquitoes.
The proteasome 20S core is a multi-subunit protein complex comprised of two a-rings and two b-rings, which have regulatory activity and proteolytic activity, respectively [28,29]. The 20S proteasome is responsible for the breakdown of shortlived proteins involved in cellular apoptosis, DNA repair, endocytosis, cell cycle regulation and for the rapid removal of misfolded proteins [30]. PSMB6 belongs to the 20S proteasomal subunit family, which participates in catalyzing ubiquitin-protein degradation [31].
This study was designed to isolate differentially expressed proteins identified by protein profiling of DM-susceptible andresistant mosquito cells, and to study the role of PSMB6 in mosquito deltamethrin resistance. A DM-susceptible strain of Cx. pipiens pallen (strain 01, the LC50 was 0.008 mg/L) which had never been exposed to insecticides, was obtained from the Shanghai Insect Institute of the Chinese Academy of Sciences (Shanghai, China). The DM (Sigma; St. Louis, USA) concentration used for selection was determined by LC50, which was calculated by larval bioassays. Two DMresistant strains (strain 03 and 07) were used in this study. Strain 03 were selected by 0.05 mg/L DM for every generation and strain 07 were selected with increasing dose of DM for generations. For MG-132-treatment (Santa Cruz; Santa Cruz, USA) experiment, mosquitoes were subjected to DM selection for more than 10 generations, the LC50 for strain 03 and strain 07 were 0.08 mg/L and 0.56 mg/L, respectively. For bortezomib-treatment (LC Laboratories; Woburn, USA) experiment, mosquitoes were subjected to DM selection for more than 40 generations, the LC50 for strain 03 and strain 07 were 0.74 mg/L and 3.8 mg/L, respectively. All strains were maintained with approximately 14 h:10 h light/dark cycle at 28uC.

In-Gel Tryptic Digestion and MALDI-TOF/TOF
Silver-stained protein spots were excised, dehydrated in acetonitrile, and dried at room temperature. Proteins were reduced with 10 mM DTT and 25 mM ammonium bicarbonate (NH 4 HCO 3 ) at 56uC for 1 h and 55 mM iodoacetamide and 25 mM NH 4 HCO 3 at RT for 45 min in the dark. Then gel pieces were completely washed with 25 mM NH 4 HCO 3 , 50% acetonitrile and 100% acetonitrile in succession and were thoroughly dried using Speedvac (Concentrator 5301, Eppendorf, Hamburg, Germany). The dried gel pieces were rehydrated with 2 ml trypsin (Promega, Madison, WI, USA) solution (10 mg/L trypsin in 25 mM NH4HCO3) and incubated at 4uC for 40 min. The excess liquid was discarded, and the gel plugs were incubated at 37uC for 12 h before the reaction was stopped by the addition of trifluoroacetic acid (TFA) (Sigma; St. Louis, USA) at a final concentration of 0.1%.
The extracted peptide mixture was then analyzed by MALDI-TOF mass spectrometry and tandem TOF/TOF mass spectrometry which was carried out on a time-of-flight Ultraflex II mass spectrometer (Biflex; Bruker Daltonics, Germany). Peptide mass maps were acquired in positive ion mode using a SmartBeam solid laser (averaging 800 laser shots per MALDI-TOF spectrum and 800 laser shots per TOF/TOF spectrum). Resolution was 15,000 to 20,000. The spectrum was calibrated by bruker calibration mixtures to a mass tolerance within 0.1 Da [33].

Database Queries and Protein Identifications
The m/z and resolution for mass spectra were ranging from 700 to 4,000 and 10,000 to 20,000, respectively. Results were analyzed using the FlexAnalysis software (version 2.4, Bruker Daltonik GmbH) with the following parameters: peak detection algorithm, Sort Neaten Assign and Place (SNAP); S/N threshold, 3.0; quality factor threshold, 50. The tryptic autodigestion peptides (842.51 and 1,045.56 Da) were used as internal standards. The matrix or auto-proteolytic trypsin fragments and known contaminants (e.g., keratins) were removed. The search conditions used were as described [36]. The detailed principle is available online (http:// www.matrixscience.com/pdf/2003WKSHP2.pdf). Search parameters for MS data: 100 ppm for the precursor ion and 0.3 Da for the fragment ions. Covalent modifications and cleavage specificity were recognized to be the same as those described for peptide mass fingerprint (PMF) analysis. Confidence intervals exceeding 95% were considered significant. All significant MS results identified results by Mascot were manually validated for spectral quality, and y and b ion series matches.

Larvicidal Activity Assay
Three strains of Cx. pipiens pallen (strain 01, 03 and 07) were used for these experiments. In each strain, 180 early fourth instar larvae, randomly divided into three groups, were subjected to different treatments. Larvae were pre-treated with 1 mM MG-132 or 0.125 mM bortezomib for 4 h, followed by DM treatment. The LC50 of each strain was used as the test concentration. The larvae were exposed to these solutions at 28uC for a14 h:10 h light/dark cycle and the percentage of survival was recorded. MG-132 or bortezomib treatment alone and DM treatment alone were applied as control and each assay was repeated three times.

RNA extraction and cDNA synthesis
Total RNA of mosquito cells was extracted by TriZol Reagent (Invitrogen; Carlsbad, USA) and the cDNA was reverse transcribed from 1 mg of total RNA by the SuperScriptH VILO TM cDNA Synthesis kit (Invitrogen; Carlsbad, USA), according to the manufacturer's instructions. The PCR reactions were carried out using an AdvantageH2 PCR Kit (Clontech; Mountain View, USA) following the manufacturer's instructions. PCR products were separated by 1% agarose gel electrophoresis and purified using a QIA quick Gel extraction kit (QIAGEN; GmbH, Germany). The purified products were sequenced by the Shanghai Invitrogen Biotechnology Company (Shanghai, China). Finally, all sequences were assembled to generate the putative full-length cDNA of PSMB6.

Sequence Alignment and Phylogenetic Analysis
The standard protein/protein BLAST sequence comparison programs (http://beta.uniprot.org/?tab = blast) were used to search sequences with similarities to the translated sequences of PSMB6 in the SWISS-PROT databases. Deduced amino acid sequences were aligned by the ClustalW2 computer program (http://www.ebi.ac.uk/Tools/clustalw2/index.html). The MEGA 5.0 program was used to construct the phylogenic tree.

Quantitative PCR analysis
Quantitative PCR assays were performed with the ABI PRISM 7300 equipment (Applied Biosystems, Foster City, USA). The primers used are all listed in Table S1. According to the manufacturer's instruction, each reaction was performed in a total volume of 20 ml containing cDNA, specific forward and reverse primers and LightCycler FastStart DNA Master SYBR Green I (Roche; Rockford, USA). A melting curve was generated immediately after the reaction to check the specificity and the data were analyzed with 7300 System SDS Software v1.2.1 (Applied Biosystems). The parameters for PCR were set as: 95uC for 30 s, followed by 40 cycles of 94uC for 30 s, 55uC for 30 s, and 72uC for 30 s, and the dissociation curve was inspected for quality control purposes. b-actin was used as the internal control. The relative gene expression level was calculated from the threshold cycle (C t ) value of each reaction through Delta-delta Ct method [37].

Cell Viability Analysis
Resistant and susceptible cells were seeded (2610 4 /well) in 100 ml per well of complete media in four 96-well plates and incubated for 24 h. Cells were pre-treated with 1 mM MG-132 or 0.1 mM bortezomib. 1% (v/v) Dimethyl sulfoxide (DMSO) (Sigma; St. Louis, USA) alone was used as a negative control. Cells (resistance and susceptible) were then treated with 100 ml MG-132 or bortezomib for 4 h. Other batches of cell were transfected with PSMB6-siRNA for 7 h. siRNA (6 ml) and X-tremeGENE siRNA Transfection Reagent (6 ml) (Roche; Rockford, USA) were mixed in 1.2 ml DMEM in a RNase-free tube for 15 min at room temperature, then 4.8 ml DMEM was added to the transfection mixture, mixed again and added to cells (100 ml per well). Cells transfected with scrambled siRNA were used as negative controls. Cells were treated with 10 0.5 , 10 1 , 10 1.5 , 10 2 , 10 2.5 mg/L of DM for 68 h. According to the manufacturer's instructions, CCK-8 reagents (Dojindo; Gaithersburg, USA) was added to the medium and incubated for a further 4 h at 28uC. A microplate reader (Biotek Instruments; Winooski, USA) was used to measure the absorbance at 450 nm. Cell viability was calculated as a percentage based upon control cell viability.

Statistical analysis
All statistical analysis was performed with GraphPad 5.0 (GraphPad Software). Statistically significant differences were evaluated with the Student's t-test (*P,0.05, **P,0.01). All experiments were performed in triplicates on at least three separate occasions.

Characterization of DM-resistant Mosquito Cells
DM-resistance mosquito cells were selected with increasing doses of DM for hundreds of generations to generate the DMresistance cells and cell viability was analyzed using a modified MTT method (CCK-8). As shown in Figure 1, after DM selection, the LC 50 for the resistant cells was 251 mg/L, much higher than 17 mg/L for the susceptible cells ( Figure 1A). DM-selection in resistance cells significantly improved cell viability and proliferation compared with susceptible cells ( Figure 1B).

Identification and quantification protein spots on 2-DE gels
A representative 2-DE gel image for protein expression of DMresistant and -susceptible mosquito cell is presented in Figure S1. Thirty-six proteins were identified to be significantly differently expressed between two groups (P,0.05 with average spot intensity greater than 1.2-fold). All spots were subjected to tryptic digestion and MALDI-TOF/MS analysis. Using PMF analysis, twenty-seven proteins were identified (summary of these proteins, including accession numbers, protein names, scores, sequence coverage, Mr, and pI, are listed in Table 1 and the raw data of each protein are shown in Table S2). The other nine proteins were unidentified because of incomplete polypeptide fragments or low abundance. A further magnified 2-DE gel image of four DMresistant upregulated proteins: proteasome subunit beta type 6 (PSMB6), 26S proteasome non-ATPase regulatory subunit 14 (PSMD14/POH1), ubiquitin-conjugating enzyme (E2) and ubiquitin-specific protease, putative (USP) was shown in Figure 2. These proteins are components of the ubiquitin-proteasome system. Quantitative PCR confirmed that gene expression of PSMB6, POH1, E2 and USP was 1.59, 1.46, 1.80 and 2.93-fold increased in DM-resistant mosquito strain (strain 07, LC50 was 3.8 mg/L) compared with those of DM-susceptible strain (strain 01) ( Figure 2C).

Proteasome inhibitor increases the sensitivity of DMresistant cells to DM treatment
The proteasome inhibitor MG-132 and bortezomib were used to elucidate the involvement of the ubiquitin-proteasome system in DM resistance. A dose-dependent viability experiment was

Molecular cloning and sequence analysis of PSMB6
PSMB6 is a catalytic subunit of proteasome b-rings [25,26]. The full-length cDNA of PSMB6 from Cx. pipiens pallen were cloned and submitted to GenBank (GenBank Accession NO: JQ037858) ( Figure S3A). The deduced peptide is composed of 227 amino acids. Homology analysis of the Cx. pipiens pallen PSMB6 sequence revealed 99% and 91% and 90% identity with PSMB6 of Cx. Quinquefasciatus, Ae. aegypti and An. Gambiae, respectively ( Figure  S3B). Phylogenetic analysis showed that Cx. pipiens pallen, Cx. quinquefasciatus, Ae. albopictus, Ae. aegypti and An. gambiae share the most recent common ancestry ( Figure S3C).

PSMB6 is essential for DM resistance in mosquito cells
The expression of PSMB6 in DM-resistant cell was shown to be 2.5-fold higher than that in DM-susceptible cell assessed by quantitative PCR ( Figure 5A). Combined with the 2-DE results, these results indicated that PSMB6 expression is upregulated at both the transcriptional and translational levels in DM-resistant cells. Subsequent investigation of the role of PSMB6 in DM resistance was conducted using PSMB6 siRNA. The knockdown efficiency of PSMB6 was confirmed by quantitative PCR ( Figure 5B). Cell viability over a wide range of DM concentrations (10 0.5 , 10 1 , 10 1.5 , 10 2 , 10 2.5 mg/L) of DM was measured using the CCK-8 method. As shown in Figure 5C, transient knock down of PSMB6 expression in DM-resistant cells resulted in decreased cell viability, indicating that cell lack of PSMB6 became more sensitized to DM treatment. No significant difference in cell viability was observed between control and PSMB6 siRNA-treated groups of DM-susceptible cells.

Discussion
In the present study, a global comparative proteomic analysis was performed between DM-susceptible and -resistant mosquito cells. Twenty-seven DM resistance-associated candidate proteins, involved in metabolism, energy generation, translation and signalling transduction, were identified. Four proteins (PSMB6, POH1, E2 enzymes and USP) involved in different steps of ubiquitin-proteasome degradation process were found to be upregulated. Together with the upregulated transcription of these genes, our results suggested that ubiquitin-proteasome system may play critical roles in DM-resistance.
E2 is involved in the formation of ubiquitin chains during protein ubiquitination. Ubiquitin specific proteinase (USP) is an important regulator that mediates the removal, and recycling of ubiquitin to maintain adequate free ubiquitin levels [38][39][40]. POH1 is a component of the proteasomal complex [41]. PSMB6 is a member of the proteasome b-type family, which participate in the formation of proteolytic centers of proteasome machinery [42]. The upregulated expression of these ubiquitin-proteasome proteins involved in each stage of the degradation process suggests that this ubiquitin-proteasome system is functionally enhanced and may contribute to DM resistance in mosquitoes.
Enhanced proteasomal activity has been demonstrated as a mediator of resistance to chemotherapy [20]. Overexpression of members of the ubiquitin-proteasome pathway has been implicated in cancer chemotherapy resistance. Inhibiting E2 enzyme activity with CDC34 could enhance the anti-cancer activity of bortezomib, dexamethasone and 2-methoxyestradiol [43].  reported that the over-expression of the PSMB1 proteasomal subunit is associated with resistance to cisplatin in cancer cell lines [44]. Furthermore, PSMB7 has been proved to be associated with anthracycline-resistance in breast cancer [45]. Overexpression of the POH1 subunit confers resistance to vinblastine, cisplatin, doxorubicin and paclitaxel in mammalian cells [43]. In this study, we identified PSMB6 as a DM-resistance mediator in mosquitoes for the first time.
MG-132 and Bortezomib are both reversible and cell-permeable proteasome inhibitor [46][47][48]. Bortezomib is reported to be more specific with no significant inhibitory activity towards other enzymes or receptors [48]. As we anticipated, pre-treatment with MG-132 or bortezomib was associated with significantly decreased viability of DM-resistant cells or mosquito larvae indicating that proteasome do involved in DM-resistant. PSMB6 was highly at transcriptional level in DM-resistant cells, which was consistent with the protein profile, indicating that transcriptional upregulation of PSMB6 leads to translational upregulation of the protein.
PSMB6 silencing resulted in significantly decreased cell viability under DM stress. Inhibition of proteasome activity using pharmaceutical inhibitor or knocking down the expression of PSMB6 through molecular methods resulted in sensitization of mosquito cells to DM-treatment, which strongly suggests that ubiquitin-proteasome system maybe involved in the DM resistance. Taken together, it is possible to manage DM resistance by regulating the ubiquitin-proteasome activity.
Under continuous selective pressure from insecticides, mosquitoes have attained stable inheritance of DM-resistance through over-expression of ubiquitin-proteasome proteins. It can be speculated that hyperactivation of the proteasome degradation pathway is associated with pyrethroid resistance, although further studies are required to elucidate the underlying mechanism.
This study provides compelling evidences that PSMB6 is associated with DM resistance, which indicated the potential of proteasome inhibitors as synergistic agent for insecticides.