Salmonella manipulates macrophage migration via SteC-mediated myosin light chain activation to penetrate the gut-vascular barrier.

The intestinal pathogen Salmonella enterica rapidly enters the bloodstream after the invasion of intestinal epithelial cells, but how Salmonella breaks through the gut-vascular barrier is largely unknown. Here, we report that Salmonella enters the bloodstream through intestinal CX3CR1+ macrophages during early infection. Mechanistically, Salmonella induces the migration/invasion properties of macrophages in a manner dependent on host cell actin and on the pathogen effector SteC. SteC recruits host myosin light chain protein Myl12a and phosphorylates its Ser19 and Thr20 residues. Myl12a phosphorylation results in actin rearrangement, and enhanced migration and invasion of macrophages. SteC is able to utilize a wide range of NTPs other than ATP to phosphorylate Myl12a. We further solved the crystal structure of SteC, which suggests an atypical dimerization-mediated catalytic mechanism. Finally, in vivo data show that SteC-mediated cytoskeleton manipulation is crucial for Salmonella breaching the gut vascular barrier and spreading to target organs.

Force-Activated Isomerization of a Single Molecule.

Understanding and controlling isomerization at the single molecular level should provide new insight into the molecular dynamics and design guidelines of functional devices. Scanning tunneling microscopy (STM) has been demonstrated to be a powerful tool to study isomerization of single molecules on a substrate, by either electric field or inelastic electron tunneling mechanisms. A similar molecular isomerization process can in principle be induced by mechanical force; however, relevant study has remained elusive. Here, we demonstrate that isomerization of a N,N-dimethylamino-dianthryl-benzene molecule on Ag(100) can be mechanically driven by the STM tip. The existence of an out-of-plane dimethylamino group in the molecule is found to play a pivotal role in the isomerization process by providing a steric hindrance effect for asymmetric interaction between the STM tip and the molecule. This underlying mechanism is further confirmed by performing molecular dynamics simulations, which show agreement with experimental results. Our work opens the opportunity to manipulate the molecular configuration on the basis of mechanical force.

GhMAP3K65, a Cotton Raf-Like MAP3K Gene, Enhances Susceptibility to Pathogen Infection and Heat Stress by Negatively Modulating Growth and Development in Transgenic Nicotiana benthamiana.

Mitogen-activated protein kinase kinase kinases (MAP3Ks), the top components of MAPK cascades, modulate many biological processes, such as growth, development and various environmental stresses. Nevertheless, the roles of MAP3Ks remain poorly understood in cotton. In this study, GhMAP3K65 was identified in cotton, and its transcription was inducible by pathogen infection, heat stress, and multiple signalling molecules. Silencing of GhMAP3K65 enhanced resistance to pathogen infection and heat stress in cotton. In contrast, overexpression of GhMAP3K65 enhanced susceptibility to pathogen infection and heat stress in transgenic Nicotiana benthamiana. The expression of defence-associated genes was activated in transgenic N. benthamiana plants after pathogen infection and heat stress, indicating that GhMAP3K65 positively regulates plant defence responses. Nevertheless, transgenic N. benthamiana plants impaired lignin biosynthesis and stomatal immunity in their leaves and repressed vitality of their root systems. In addition, the expression of lignin biosynthesis genes and lignin content were inhibited after pathogen infection and heat stress. Collectively, these results demonstrate that GhMAP3K65 enhances susceptibility to pathogen infection and heat stress by negatively modulating growth and development in transgenic N. benthamiana plants.

A glutathione S-transferase gene associated with antioxidant properties isolated from Apis cerana cerana.

Glutathione S-transferases (GSTs) are an important family of multifunctional enzymes in aerobic organisms. They play a crucial role in the detoxification of exogenous compounds, especially insecticides, and protection against oxidative stress. Most previous studies of GSTs in insects have largely focused on their role in insecticide resistance. Here, we isolated a theta class GST gene designated AccGSTT1 from Apis cerana cerana and aimed to explore its antioxidant and antibacterial attributes. Analyses of homology and phylogenetic relationships suggested that the predicted amino acid sequence of AccGSTT1 shares a high level of identity with the other hymenopteran GSTs and that it was conserved during evolution. Quantitative real-time PCR showed that AccGSTT1 is most highly expressed in adult stages and that the expression profile of this gene is significantly altered in response to various abiotic stresses. These results were confirmed using western blot analysis. Additionally, a disc diffusion assay showed that a recombinant AccGSTT1 protein may be roughly capable of inhibiting bacterial growth and that it reduces the resistance of Escherichia coli cells to multiple adverse stresses. Taken together, these data indicate that AccGSTT1 may play an important role in antioxidant processes under adverse stress conditions.

The cotton WRKY transcription factor GhWRKY17 functions in drought and salt stress in transgenic Nicotiana benthamiana through ABA signaling and the modulation of reactive oxygen species production.

Drought and high salinity are two major environmental factors that significantly limit the productivity of agricultural crops worldwide. WRKY transcription factors play essential roles in the adaptation of plants to abiotic stresses. However, WRKY genes involved in drought and salt tolerance in cotton (Gossypium hirsutum) are largely unknown. Here, a group IId WRKY gene, GhWRKY17, was isolated and characterized. GhWRKY17 was found to be induced after exposure to drought, salt, H2O2 and ABA. The constitutive expression of GhWRKY17 in Nicotiana benthamiana remarkably reduced plant tolerance to drought and salt stress, as determined through physiological analyses of the germination rate, root growth, survival rate, leaf water loss and Chl content. GhWRKY17 transgenic plants were observed to be more sensitive to ABA-mediated seed germination and root growth. However, overexpressing GhWRKY17 in N. benthamiana impaired ABA-induced stomatal closure. Furthermore, we found that GhWRKY17 modulated the increased sensitivity of plants to drought by reducing the level of ABA, and transcript levels of ABA-inducible genes, including AREB, DREB, NCED, ERD and LEA, were clearly repressed under drought and salt stress conditions. Consistent with the accumulation of reactive oxygen species (ROS), reduced proline contents and enzyme activities, elevated electrolyte leakage and malondialdehyde, and lower expression of ROS-scavenging genes, including APX, CAT and SOD, the GhWRKY17 transgenic plants exhibited reduced tolerance to oxidative stress compared with wild-type plants. These results therefore indicate that GhWRKY17 responds to drought and salt stress through ABA signaling and the regulation of cellular ROS production in plants.

A c-di-GMP binding effector STM0435 modulates flagellar motility and pathogenicity in Salmonella.

Flagella play a crucial role in the invasion process of Salmonella and function as a significant antigen that triggers host pyroptosis. Regulation of flagellar biogenesis is essential for both pathogenicity and immune escape of Salmonella. We identified the conserved and unknown function protein STM0435 as a new flagellar regulator. The ∆stm0435 strain exhibited higher pathogenicity in both cellular and animal infection experiments than the wild-type Salmonella. Proteomic and transcriptomic analyses demonstrated dramatic increases in almost all flagellar genes in the ∆stm0435 strain compared to wild-type Salmonella. In a surface plasmon resonance assay, purified STM0435 protein-bound c-di-GMP had an affinity of ~8.383 µM. The crystal structures of apo-STM0435 and STM0435&c-di-GMP complex were determined. Structural analysis revealed that R33, R137, and D138 of STM0435 were essential for c-di-GMP binding. A Salmonella with STM1987 (GGDEF protein) or STM4264 (EAL protein) overexpression exhibits completely different motility behaviours, indicating that the binding of c-di-GMP to STM0435 promotes its inhibitory effect on Salmonella flagellar biogenesis.

Identification and characterization of an Apis cerana cerana Delta class glutathione S-transferase gene (AccGSTD) in response to thermal stress.

Glutathione S-transferases (GSTs) are members of a multifunctional enzyme super family that plays a pivotal role in both insecticide resistance and protection against oxidative stress. In this study, we identified a single-copy gene, AccGSTD, as being a Delta class GST in the Chinese honey bee (Apis cerana cerana). A predicted antioxidant response element, CREB, was found in the 1,492-bp 5'-flanking region, suggesting that AccGSTD may be involved in oxidative stress response pathways. Real-time PCR and immunolocalization studies demonstrated that AccGSTD exhibited both developmental- and tissue-specific expression patterns. During development, AccGSTD transcript was increased in adults. The AccGSTD expression level was the highest in the honey bee brain. Thermal stress experiments demonstrated that AccGSTD could be significantly upregulated by temperature changes in a time-dependent manner. It is hypothesized that high expression levels might be due to the increased levels of oxidative stress caused by the temperature challenges. Additionally, functional assays of the recombinant AccGSTD protein revealed that AccGSTD has the capability to protect DNA from oxidative damage. Taken together, these data suggest that AccGSTD may be responsible for antioxidant defense in adult honey bees.

Capping Layer Determined Self-assembly of Au-Ag Bimetallic Janus Nanoparticles at An Oil/Water Interface by Molecular Dynamics Simulations.

Bimetallic Janus nanoparticles (BJNPs) have gained more attention due to their unique catalytic and optical properties. The self-assembly of BJNPs is expected as an effective way to fabricate metamaterials suitable for different potential applications. However, the self-assembly dynamic process of BJNPs, which is key to achieving a controllable synthesis, is limited in both experimental and theoretical investigations. Herein, all-atom molecular dynamics (MD) simulations were employed to investigate the self-assembly process of 1-dodecanethiol (DDT)-decorated Au-Ag BJNPs at an oil-water interface. We demonstrate that DDT's van der Waals (vdW) interaction dominates the self-assembly process. BJNPs form close-packed structures at both fast and slow evaporation rates. Au-Ag BJNPs exhibit relatively larger rotations at a low evaporation rate than those at a high evaporation rate, suggesting that the evaporation rate influences the orientation of the Au-Ag BJNPs. BJNPs tend to orient their electric dipole moments toward the external electric field, according to the ab initio MD simulation results. Based on the energy comparison and model analysis, it is found that the parallel array is more stable than the antiparallel one for the Au-Ag BJNP arrays. The dipole-dipole interaction difference between the parallel and antiparallel BJNP arrays obtained according to dipole moment obtained from ab initio calculation is qualitatively consistent with that obtained from MD simulations, indicating that the dipole plays a decisive role in determining the orientation of the BJNP array. This work uncovers the self-assembly dynamic process of BJNPs, paving the way for future applications.

Ligand-Ligand-Interaction-Dominated Self-Assembly of Gold Nanoparticles at the Oil/Water Interface: An Atomic-Scale Simulation.

The self-assembly of nanoparticles (NPs) into ordered superlattices is a powerful strategy to fabricate functional nanomaterials. Subtle variations in the interactions between NPs will influence the self-assembled superlattices. Using all-atom molecular dynamics simulations, we explore the self-assembly of 16 gold NPs, 4 nm in diameter, capped with ligands at the oil-water interface, and quantify the interactions between NPs at the atomic scale. We demonstrate that the interaction between capping ligands rather than that between NPs is dominant during the assembly process. For dodecanethiol (DDT)-capped Au NPs, the assembled superlattice is highly ordered in a close-packed configuration at a slow evaporation rate, while it is disordered at a fast evaporation rate. When replacing the capping ligands with stronger polarization than DDT molecules, the NPs form a robust ordered configuration at different evaporation rates due to the stronger electrostatic attraction between capping ligands from different NPs. Moreover, Au-Ag binary clusters exhibit similar assembly behavior with Au NPs. Our work uncovers the nonequilibrium nature of NP assembly at the atomic scale and would be helpful in rationally controlling NPs superlattice by changing passivating ligands, solvent evaporation rate, or both.

Cooperative Regulation of Flagellar Synthesis by Two EAL-Like Proteins upon Salmonella Entry into Host Cells.

When Salmonella enters host cells, the synthesis of flagella is quickly turned off to escape the host immune system. In this study, we investigated the cooperative regulatory mechanism of flagellar synthesis by two EAL-like proteins, STM1344 and STM1697, in Salmonella. We found that Salmonella upregulated the expression of both STM1344 and STM1697 to various degrees upon invading host cells. Importantly, deletion of STM1697 or STM1344 led to failure of Salmonella flagellar control within host cells, suggesting that the two factors are not redundant but indispensable. STM1697 was shown to modulate Salmonella flagellar biogenesis by preventing the flagellar master protein FlhDC from recruiting RNA polymerase. However, STM1344 was identified as a bifunctional factor that inhibits RNA polymerase recruitment of FlhDC at low molar concentrations and the DNA binding activity of FlhDC at high molar concentrations. Structural analysis demonstrated that STM1344-FlhD binds more tightly than STM1697-FlhD, and size exclusion chromatography (SEC) experiments showed that STM1344 could replace STM1697 in a STM1697-FlhDC complex. Our data suggest that STM1697 might be a temporary flagellar control factor upon Salmonella entry into the host cell, while STM1344 plays a more critical role in persistent flagellar control when Salmonella organisms survive and colonize host cells for a long period of time. Our study provides a more comprehensive understanding of the complex flagellar regulatory mechanism of Salmonella based on regulation at the protein level of FlhDC. IMPORTANCE Salmonella infection kills more than 300,000 people every year. After infection, Salmonella mainly parasitizes host cells, as it prevents host cell pyroptosis by turning off the synthesis of flagellar antigen. Previous studies have determined that there are two EAL-like proteins, STM1344 and STM1697, encoded in the Salmonella genome, both of which inhibit flagellar synthesis by interacting with the flagellar master protein FlhDC. However, the expression order and simultaneous mechanism of STM1344 and STM1697 are not clear. In this study, we determined the expression profiles of the two proteins after Salmonella infection and demonstrated the cooperative mechanism of STM1344 and STM1697 interaction with FlhDC. We found that STM1344 might play a more lasting regulatory role than STM1697. Our results reveal a comprehensive flagellar control process after Salmonella entry into host cells.

Switching off Bacterial Flagellar Biogenesis by YdiU-Mediated UMPylation of FlhDC.

Bacterial flagellin activates the host immune system and triggers pyroptosis. Salmonella reduces flagellin expression when it survives within host cells. Here, we found that the UMPylator YdiU significantly altered the Salmonella flagellar biogenesis process upon host cell entry. The expression levels of class II and class III flagellar genes, but not the class I flagellar genes flhDC, were dramatically increased in a ΔydiU strain compared to wild-type (WT) Salmonella in a host-simulating environment. A direct interaction between YdiU and FlhDC was detected by bacterial two-hybrid assay. Furthermore, YdiU efficiently catalyzed the UMPylation of FlhC but not FlhD, FliA, or FliC. UMPylation of FlhC completely eliminated its DNA-binding activity. In vivo experiments showed that YdiU was required and sufficient for Salmonella flagellar control within host cells. Mice infected with the ΔydiU strain died much earlier than WT strain-infected mice and developed much more severe inflammation and injury in organs and much higher levels of cytokines in blood, demonstrating that early host death induced by the ΔydiU strain is probably due to excessive inflammation. Our results indicate that YdiU acts as an essential factor of Salmonella to mediate host immune escape. IMPORTANCE Salmonella is an important facultative pathogen of foodborne illness and typhoid fever in humans. Flagella allow bacterial motility and are required for Salmonella to successfully invade the host cells. In parallel, flagellin triggers the host immune system. Salmonella reduces flagellar biogenesis to avoid detection within host cells by a largely unknown mechanism. Here, we report that the UMPylator YdiU inhibits flagellin expression in response to host signals in an UMPylation-dependent manner. The target of YdiU is the major flagellar transcription factor FlhDC. YdiU UMPylates the FlhC subunit on its Ser31 residue and prevents FlhDC from binding to flagellar genes, thus switching off flagellar biogenesis. Our results reveal a novel mechanism by which Salmonella adopts posttranslational modification to shut down flagellar synthesis as a strategy to achieve immune escape.

Salmonella Facilitates Iron Acquisition through UMPylation of Ferric Uptake Regulator.

Iron limitation is a universal strategy of host immunity during bacterial infection. However, the mechanisms by which pathogens antagonize host nutritional immunity have not been fully elucidated. Here, we identified a requirement for the UMPylator YdiU for this process in Salmonella. The expression of YdiU was dramatically induced by the metal starvation signal. The intracellular iron content was much lower in the ΔydiU strain than in wild-type Salmonella, and the ΔydiU strain exhibited severe growth defect under metal deficiency environments. Genome-wide expression analyses revealed significantly decreased expression of iron uptake genes in ΔydiU strain compared with the wild-type strain. Interestingly, YdiU did not affect the expression level of the major iron uptake regulator Fur but directly UMPylated Fur on its H118 residue in vivo and in vitro. UMPylation destroyed the Fur dimer, promoted Fur aggregation, and eliminated the DNA-binding activity of Fur, thus abolishing the ability of Fur to inhibit iron uptake. Restricting Fur to the deUMPylated state dramatically eliminates Salmonella iron uptake in iron deficiency environments. In parallel, YdiU facilitates Salmonella survival within host cells by regulating the iron uptake pathway. IMPORTANCE Salmonella is the major pathogen causing bacterial enteric illness in both humans and animals. Iron availability is strictly controlled upon Salmonella entry into host cells. The mechanisms by which Salmonella balances the acquisition of sufficient iron while preventing a toxic overload has not been fully understood. Here, we reveal a novel regulation process of iron acquisition mediated by the UMPylator YdiU. Fur acts as the central regulator of bacterial iron homeostasis. YdiU UMPylates Fur on H118 and prevents Fur from binding to target DNA, thus activating the expression of iron uptake genes under iron-deficient conditions. We describe the first posttranslational modification-based regulation of Fur and highlight a potential mechanism by which Salmonella can adapt to eliminate host nutritional immunity.

Host acid signal controls Salmonella flagella biogenesis through CadC-YdiV axis.

Upon entering host cells, Salmonella quickly turns off flagella biogenesis to avoid recognition by the host immune system. However, it is not clear which host signal(s) Salmonella senses to initiate flagellum control. Here, we demonstrate that the acid signal can suppress flagella synthesis and motility of Salmonella, and this occurs after the transcription of master flagellar gene flhDC and depends on the anti-FlhDC factor YdiV. YdiV expression is activated after acid treatment. A global screen with ydiV promoter DNA and total protein from acid-treated Salmonella revealed a novel regulator of YdiV, the acid-related transcription factor CadC. Further studies showed that CadCC, the DNA binding domain of CadC, directly binds to a 33 nt region of the ydiV promoter with a 0.2 µM KD affinity. Furthermore, CadC could separate H-NS-ydiV promoter DNA complex to form CadC-DNA complex at a low concentration. Structural simulation and mutagenesis assays revealed that H43 and W106 of CadC are essential for ydiV promoter binding. No acid-induced flagellum control phenotype was observed in cadC mutant or ydiV mutant strains, suggesting that flagellum control during acid adaption is dependent on CadC and YdiV. The intracellular survival ability of cadC mutant strain decreased significantly compared with WT strain while the flagellin expression could not be effectively controlled in the cadC mutant strain when surviving within host cells. Together, our results demonstrated that acid stress acts as an important host signal to trigger Salmonella flagellum control through the CadC-YdiV-FlhDC axis, allowing Salmonella to sense a hostile environment and regulate flagellar synthesis during infection.

On-surface synthesis of size- and shape-controlled two-dimensional Aun nanoclusters using a flexible fullerene molecular template.

Synthesizing nano-clusters with a well-defined size, shape, and composition is an important and challenging goal in nanotechnology. Here we report the application of a single layer C60 molecule as an effective molecular template for the synthesis of size- and shape-selected two-dimensional gold clusters (Aun) on a graphite substrate. This molecular template facilitates the preferential formation of Au19 clusters with a selectivity as high as 90%. Density-functional-theory (DFT) calculations found an energy minimum associated with C60-stabilized two-dimensional Au19 clusters.

The YdiU Domain Modulates Bacterial Stress Signaling through Mn2+-Dependent UMPylation.

Sensing stressful conditions and adjusting the cellular metabolism to adapt to the environment are essential activities for bacteria to survive in variable situations. Here, we describe a stress-related protein, YdiU, and characterize YdiU as an enzyme that catalyzes the covalent attachment of uridine-5'-monophosphate to a protein tyrosine/histidine residue, an unusual modification defined as UMPylation. Mn2+ serves as an essential co-factor for YdiU-mediated UMPylation. UTP and Mn2+ binding converts YdiU to an aggregate-prone state facilitating the recruitment of chaperones. The UMPylation of chaperones prevents them from binding co-factors or clients, thereby impairing their function. Consistent with the recent finding that YdiU acts as an AMPylator, we further demonstrate that the self-AMPylation of YdiU padlocks its chaperone-UMPylation activity. A detailed mechanism is proposed based on the crystal structures of Apo-YdiU and YdiU-AMPNPP-Mn2+ and on molecular dynamics simulation models of YdiU-UTP-Mn2+ and YdiU-UTP-peptide. In vivo data demonstrate that YdiU effectively protects Salmonella from stress-induced ATP depletion through UMPylation.

Correction: GhWRKY68 Reduces Resistance to Salt and Drought in Transgenic Nicotiana benthamiana.

[This corrects the article DOI: 10.1371/journal.pone.0120646.].

Identification of an Apis cerana cerana MAP kinase phosphatase 3 gene (AccMKP3) in response to environmental stress.

MAP kinase phosphatase 3 (MKP3), a member of the dual-specificity protein phosphatase (DUSP) superfamily, has been widely studied for its role in development, cancer, and environmental stress in many organisms. However, the functions of MKP3 in various insects have not been well studied, including honeybees. In this study, we isolated an MKP3 gene from Apis cerana cerana and explored the role of this gene in the resistance to oxidation. We found that AccMKP3 is highly conserved in different species and shares the closest evolutionary relationship with AmMKP3. We determined the expression patterns of AccMKP3 under various stresses. qRT-PCR results showed that AccMKP3 was highly expressed during the pupal stages and in adult muscles. We further found that AccMKP3 was induced in all the stress treatments. Moreover, we discovered that the enzymatic activities of peroxidase, superoxide dismutase, and catalase increased and that the expression levels of several antioxidant genes were affected after AccMKP3 was knocked down. Collectively, these results suggest that AccMKP3 may be associated with antioxidant processes involved in response to various environmental stresses.

Characteristics of AccSTIP1 in Apis cerana cerana and its role during oxidative stress responses.

Various environmental stresses, such as heat shock, heavy metals, ultraviolet (UV) radiation and different pesticides, induce a cellular oxidative stress response. The cellular oxidative stress response is usually regulated by heat shock proteins (Hsps) acting as molecular chaperones. Stress-induced phosphoprotein 1 (STIP1), one of the most widely studied co-chaperones, functions as an adaptor that directs Hsp90 to Hsp70-client protein complexes. However, the biological functions of STIP1 remain poorly understood in honeybee (Apis cerana cerana). In this study, AccSTIP1 was identified in Apis cerana cerana. AccSTIP1 transcription was found to be induced by heat (42 °C), HgCl2, H2O2 and different pesticides (emamectin benzoate, thiamethoxam, hexythiazox and paraquat) and inhibited by CdCl2, UV and kresoxim-methyl. Moreover, western blot analysis indicated that the expression profiles of AccSTIP1 were consistent with its transcriptional expression levels. The disc diffusion assay showed that chemically competent transetta (DE3) bacteria expressing a recombinant AccSTIP1 protein displayed the smaller death zones than did control bacteria after exposure to paraquat and HgCl2. The DNA nicking assay suggested that recombinant purified AccSTIP1 protected supercoiled pUC19 plasmid DNA from damage caused by a thiol-dependent mixed-function oxidation (MFO) system. After knocking down AccSTIP1 gene expression via RNA interference (RNAi), the transcript levels of antioxidation-related genes were obviously lower in dsAccSTIP1 honeybees compared with those in the uninjected honeybees. Collectively, these results demonstrated that AccSTIP1 plays an important role in counteracting oxidative stress. This study lays a foundation for revealing the mechanism of AccSTIP1 in the Apis cerana cerana antioxidant system.

Isolation of carboxylesterase (esterase FE4) from Apis cerana cerana and its role in oxidative resistance during adverse environmental stress.

Carboxylesterases (CarEs) play vital roles in metabolising different physiologically important endogenous compounds and in detoxifying various harmful exogenous compounds in insects. Multiple studies of CarEs have focused on pesticide metabolism in insects, while few studies have aimed to identify CarE functions in oxidative resistance, particularly in Apis cerana cerana. In this study, we isolated a carboxylesterase gene, esterase FE4, from Apis cerana cerana and designated it towards an exploration of its roles as an antioxidant and in detoxification. We investigated AcceFE4 expression patterns in response to various stressors. A quantitative real-time PCR analysis revealed that AcceFE4 was up-regulated by H2O2, imidacloprid, and paraquat, and was down-regulated by 4 °C, UV radiation, CdCl2, and HgCl2. Additionally, the protein expression of this gene was down-regulated at 4 °C and up-regulated by H2O2. Disc diffusion assays showed that the AcceFE4 recombinant protein-expressing bacteria had a smaller killing zone than the control group with the paraquat, HgCl2 and cumyl hydroperoxide treatments. Moreover, when the gene was knocked down by RNA interference, we observed that multiple oxidant genes (i.e., AccSOD, AccGST, AccTrx, AccMsrA, and others) were down-regulated in the knockdown samples. Superoxide dismutase (SOD), peroxidase (POD) and catalase (CAT) activity levels were reduced in the knockdown samples relative to the control group. Finally, we measured the enzyme activity of carboxylesterase and found that the enzyme activity was also reduced in the silent samples. Together, these data suggest that AcceFE4 may be involved in the oxidative resistance response during adverse stress.

Roles of a mitochondrial AccSCO2 gene from Apis cerana cerana in oxidative stress responses.

In eukaryotes, cytochrome c oxidase (COX) is a multimeric protein complex that is the last enzyme in the respiratory electron transport chain of mitochondria. Syntheses of cytochrome c oxidase (SCO) proteins are copper-donor chaperones involved in metalation of the CuA redox center of COX. However, its other precise actions are not yet understood. Here, we report the characterization of AccSCO2 from Apis cerana cerana (Acc). Our data showed that AccSCO2 expression was induced by cold (4°C), CdCl2, HgCl2, ultraviolet (UV) light, and H2O2 and was inhibited by different pesticide treatments. In addition, a disc diffusion assay of recombinant AccSCO2, AccSCO2-R1, and AccSCO2-R2 proteins showed that they played a functional role in protecting cells from oxidative stress involved in copper-dependent manner. Further, following knockdown of AccSCO2 in A. cerana cerana using RNA interference (RNAi), the expression levels of most antioxidant genes (AccGSTD, AccGSTO1, AccGSTS4, AccSOD1, AccSOD2, etc.) were significantly decreased in the AccSCO2-silenced bees compared with the control bees. Moreover, the antioxidant enzymatic activities of superoxide dismutase (SOD), peroxidase (POD) and catalase (CAT) were all lower in the silenced bees than in the control bees. Finally, the in vivo activity of COX was measured after AccSCO2 knockdown, which revealed a strong reduction in COX activity in the silenced bees. Thus, we hypothesize that AccSCO2 plays important roles in cellular stress responses and anti-oxidative processes, which help to regulate the production of mitochondrial reactive oxygen species and/or the impairment of mitochondrial activity under oxidative stress.