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Abstract Background Deformed wing virus (DWV) is a major honey bee pathogen that is actively transmitted by the parasitic mite Varroa destructor and plays a primary role in Apis mellifera winter colony losses. Despite intense investigation on this pollinator, which has a unique environmental and economic importance, the mechanisms underlying the molecular interactions between DWV and honey bees are still poorly understood. Here, we report on a group of honey bee proteins, identified by mass spectrometry, that specifically co-immunoprecipitate with DWV virus particles. Results Most of the proteins identified are involved in fundamental metabolic pathways. Among the co-immunoprecipitated proteins, one of the most interesting was arginine kinase (ArgK), a conserved protein playing multiple roles both in physiological and pathological processes and stress response in general. Here, we investigated in more detail the relationship between DWV and this protein. We found that argK RNA level positively correlates with DWV load in field-collected honey bee larvae and adults and significantly increases in adults upon DWV injection in controlled laboratory conditions, indicating that the argK gene was upregulated by DWV infection. Silencing argK gene expression in vitro, using RNAi, resulted in reduced DWV viral load, thus confirming that argK upregulation facilitates DWV infection, likely through interfering with the delicate balance between metabolism and immunity. Conclusions In summary, these data indicate that DWV modulates the host ArgK through transcriptional regulation and cooptation to enhance its fitness in honey bees. Our findings open novel perspectives on possible new therapies for DWV control by targeting specific host proteins.

Self-labelling protein tags (SLPs) are resourceful tools that revolutionized sensor imaging, having the versatile ability of being genetically fused with any protein of interest and undergoing activation with alternative probes specifically designed for each variant (namely, SNAP-tag, CLIP-tag and Halo-tag). Commercially available SLPs are highly useful in studying molecular aspects of mesophilic organisms, while they fail in characterizing model organisms that thrive in harsh conditions. By applying an integrated computational and structural approach, we designed a engineered variant of the alkylguanine-DNA-alkyl-transferase (OGT) from the hyper-thermophilic archaeon Saccharolobus solfataricus (SsOGT), with no DNA-binding activity, able to covalently react with O6-benzyl-cytosine (BC-) derivatives, obtaining the first thermostable CLIP-tag, named SsOGT-MC8.The presented construct is able to recognize and to covalently bind BC- substrates with a marked specificity, displaying a very low activity on orthogonal benzyl-guanine (BG-) substrate and showing a remarkable thermal stability that broadens the applicability of SLPs. The rational mutagenesis that, starting from SsOGT, led to the production of SsOGT-MC8 was first evaluated by structural predictions to precisely design the chimeric construct, by mutating specific residues involved in protein stability and substrate recognition. The final construct was further validated by biochemical characterization and X-ray crystallography, allowing us to present here the first structural model of a CLIP-tag establishing the molecular determinants of its activity, as well as proposing a general approach for the rational engineering of any O6-alkylguanine-DNA-alkyl-transferase turning it into a SNAP- and a CLIP-tag variant.

SNAP-tag® is a powerful technology for the labelling of protein/enzymes by using benzyl-guanine (BG) derivatives as substrates. Although commercially available or ad hoc produced, their synthesis and purification are necessary, increasing time and costs. To address this limitation, here we suggest a revision of this methodology, by performing a chemo-enzymatic approach, by using a BG-substrate containing an azide group appropriately distanced by a spacer from the benzyl ring. The SNAP-tag® and its relative thermostable version (SsOGT-H5) proved to be very active on this substrate. The stability of these tags upon enzymatic reaction makes possible the exposition to the solvent of the azide-moiety linked to the catalytic cysteine, compatible for the subsequent conjugation with DBCO-derivatives by azide-alkyne Huisgen cycloaddition. Our studies propose a strengthening and an improvement in terms of biotechnological applications for this self-labelling protein-tag.

Carbonic anhydrases (CAs, EC 4.2.1.1) are a superfamily of ubiquitous metalloenzymes present in all living organisms on the planet. They are classified into seven genetically distinct families and catalyse the hydration reaction of carbon dioxide to bicarbonate and protons, as well as the opposite reaction. CAs were proposed to be used for biotechnological applications, such as the post-combustion carbon capture processes. In this context, there is a great interest in searching CAs with robust chemical and physical properties. Here, we describe the enhancement of thermostability of the α-CA from Sulfurihydrogenibium yellowstonense (SspCA) by using the anchoring-and-self-labelling-protein-tag system (ASLtag). The anchored chimeric H5-SspCA was active for the CO2 hydration reaction and its thermostability increased when the cells were heated for a prolonged period at high temperatures (e.g. 70 °C). The ASLtag can be considered as a useful method for enhancing the thermostability of a protein useful for biotechnological applications, which often need harsh operating conditions.

The use of natural systems, such as outer membrane protein A (OmpA), phosphoporin E (PhoE), ice nucleation protein (INP), etc., has been proved very useful for the surface exposure of proteins on the outer membrane of Gram-negative bacteria. These strategies have the clear advantage of unifying in a one-step the production, the purification and the in vivo immobilisation of proteins/biocatalysts onto a specific biological support. Here, we introduce the novel Anchoring-and-Self-Labelling-protein-tag (ASLtag), which allows the in vivo immobilisation of enzymes on E. coli surface and the labelling of the neosynthesised proteins with the engineered alkylguanine-DNA-alkyl-transferase (H5) from Sulfolobus solfataricus. Our results demonstrated that this tag enhanced the overexpression of thermostable enzymes, such as the carbonic anhydrase (SspCA) from Sulfurihydrogenibium yellowstonense and the β-glycoside hydrolase (SsβGly) from S. solfataricus, without affecting their folding and catalytic activity, proposing a new tool for the improvement in the utilisation of biocatalysts of biotechnological interest.

Abstract Background The Bacillus subtilis spore has long been used to display antigens and enzymes. Spore display can be accomplished by a recombinant and a non-recombinant approach, with the latter proved more efficient than the recombinant one. We used the non-recombinant approach to independently adsorb two thermophilic enzymes, GH10-XA, an endo-1,4-β-xylanase (EC 3.2.1.8) from Alicyclobacillus acidocaldarius, and GH3-XT, a β-xylosidase (EC 3.2.1.37) from Thermotoga thermarum. These enzymes catalyze, respectively, the endohydrolysis of (1-4)-β-d-xylosidic linkages of xylans and the hydrolysis of (1-4)-β-d-xylans to remove successive d-xylose residues from the non-reducing termini. Results We report that both purified enzymes were independently adsorbed on purified spores of B. subtilis. The adsorption was tight and both enzymes retained part of their specific activity. When spores displaying either GH10-XA or GH3-XT were mixed together, xylan was hydrolysed more efficiently than by a mixture of the two free, not spore-adsorbed, enzymes. The high total activity of the spore-bound enzymes is most likely due to a stabilization of the enzymes that, upon adsorption on the spore, remained active at the reaction conditions for longer than the free enzymes. Spore-adsorbed enzymes, collected after the two-step reaction and incubated with fresh substrate, were still active and able to continue xylan degradation. The recycling of the mixed spore-bound enzymes allowed a strong increase of xylan degradation. Conclusion Our results indicate that the two-step degradation of xylans can be accomplished by mixing spores displaying either one of two required enzymes. The two-step process occurs more efficiently than with the two un-adsorbed, free enzymes and adsorbed spores can be reused for at least one other reaction round. The efficiency of the process, the reusability of the adsorbed enzymes, and the well documented robustness of spores of B. subtilis indicate the spore as a suitable platform to display enzymes for single as well as multi-step reactions.