A Vitellogenin Antibody in Honey Bees (Apis mellifera): Characterization and Application as Potential Biomarker for Insecticide Exposure.

The insect yolk precursor vitellogenin is a lipoglycoprotein synthesized and stored in the fat body and secreted into the hemolymph. In honey bees, vitellogenin displays crucial functions in hormone signaling, behavioral transition of nurse bees to foragers, stress resistance, and longevity in workers. Plant protection products such as neonicotinoids, pyrethroids, and organophosphates alter the transcriptional expression of vitellogenin. To assess plant protection product-induced alterations on the protein level, we developed a rabbit polyclonal vitellogenin antibody. After characterization, we assessed its specificity and vitellogenin levels in different tissues of worker bees. The vitellogenin antibody recognized full-length 180-kDa vitellogenin and the lighter fragment of 150 kDa in fat body, hemolymph, and brain. In hemolymph, a band of approximately 75 kDa was detected. Subsequent mass spectrometric analysis (liquid chromatography-mass spectrometry) confirmed the 180- and 150-kDa bands as vitellogenin. Subsequently, we evaluated vitellogenin expression in brain, fat body, and hemolymph on 24-h exposure of bees to 3 ng/bee to the neonicotinoid clothianidin. Full-length vitellogenin was upregulated 3-fold in the fat body, and the 150-kDa fragment was upregulated in the brain of exposed honey bees, whereas no alteration occurred in the hemolymph. Upregulation of the vitellogenin protein by the neonicotinoid clothianidin is in line with the previously shown induction of its transcript. We conclude that vitellogenin might serve as a potential biomarker for neonicotinoid and other pesticide exposure in bees. Environ Toxicol Chem 2019;00:1-10. © 2019 SETAC.

Characterization of the housekeeping sortase from the human pathogen Propionibacterium acnes: first investigation of a class F sortase.

Sortase enzymes play an important role in Gram-positive bacteria. They are responsible for the covalent attachment of proteins to the surface of the bacteria and perform this task via a highly sequence-specific transpeptidation reaction. Since these immobilized proteins are often involved in pathogenicity of Gram-positive bacteria, characterization of this type of enzyme is also of medical relevance. Different classes of sortases (A-F) have been found, which recognize characteristic recognition sequences present in substrate proteins. Up to date, sortase A from Staphylococcus aureus, a housekeeping class A sortase, is the most thoroughly studied representative of the sortase family of enzymes. Here we report the in-depth characterization of the class F sortase from Propionibacterium acnes, a class of sortases that has not been investigated before. As Sortase F is the only transpeptidase found in the P. acnes genome, it is the housekeeping sortase of this organism. Sortase F from P. acnes shows a behavior similar to sortases from class A in terms of pH dependence, recognition sequence and catalytic activity; furthermore, its activity is independent of bivalent ions, which contrasts to sortase A from S. aureus We demonstrate that sortase F is useful for protein engineering applications, by producing a site-specifically conjugated homogenous antibody-drug conjugate with a potency similar to that of a conjugate prepared with sortase A. Thus, the detailed characterization presented here will not only enable the development of anti-virulence agents targeting P. acnes but also provides a powerful alternative to sortase A for protein engineering applications.

Transcriptional and physiological effects of the pyrethroid deltamethrin and the organophosphate dimethoate in the brain of honey bees (Apis mellifera).

The pyrethroid deltamethrin and the organophosphate insecticide dimethoate are widely used in agriculture and in urban areas. Both plant protection products (PPPs) unintendedly result in adverse effects in pollinators. Currently, the sublethal effects of both compounds are poorly known, particularly on the molecular and biochemical level. Here we analysed effects of deltamethrin and dimethoate at environmental and sublethal concentrations in honey bee workers by focusing on transcriptional changes of target genes in the brain. In addition, expression of vitellogenin protein and activity of acetylcholinesterase were assessed upon dimethoate exposure to assess physiological effects. Deltamethrin resulted in induction of the cyp9q2 transcript at 0.53 ng/bee, while dimethoate led to induction of vitellogenin on the mRNA and protein level at 2 ng/bee. Transcripts of additional cytochrome P450-dependent monooxygenases (cyps) and genes related to immune system regulation were not differentially expressed upon PPP exposure. Dimethoate but not deltamethrin led to a strong and concentration-related inhibition of the acetylcholinesterase at 2 and 20 ng/bee. Our data demonstrate that deltamethrin and dimethoate exhibit transcriptional effects at environmental concentrations in the brain of honey bees. Dimethoate also strongly affected physiological traits, which may translate to adverse effects in forager bees.

Development of hepatitis B virus capsids into a whole-chain protein antigen display platform: new particulate Lyme disease vaccines.

The immunogenicity of peptides and small protein fragments can be considerably enhanced by their presentation on particulate carriers such as capsid-like particles (CLPs) from hepatitis B virus (HBV). HBV CLPs are icosahedral nanoparticles formed by 90 or 120 core protein dimers. Insertions into the immunodominant c/e1 B cell epitope, a surface-exposed loop on the HBV capsid protein, are especially immunogenic. Here we investigated whether the HBV core protein can be exploited as a vaccine carrier for whole-chain protein antigens, using two clinically relevant proteins derived from a bacterial human pathogen, the Lyme disease agent Borrelia burgdorferi. For this purpose we analyzed CLP formation by core fusions with the entire 255-amino-acid ectodomain of outer surface lipoprotein A (OspA), and with two distinct, 189 amino acid long variants of the dimeric OspC (OspC(a), OspC(b)) of B. burgdorferi. OspA appropriately inserted into the HBV core protein yielded a multimerization-competent fusion protein, termed coreOspA. Although only partially assembling into regular CLPs, coreOspA induced antibodies to OspA, including the Ig isotype profile and specificity for the protective epitope "LA-2", with an efficiency similar to that of recombinant lipidated OspA, the first generation vaccine against Lyme disease. Moreover, coreOspA actively and passively protected mice against subsequent challenge with B. burgdorferi. Fusions with the two OspC variants were found to efficiently form regular CLPs, most probably by OspC dimerization across different core protein dimers. In mice, both coreOspC preparations induced high-titered antibody responses to the homologous but also to the heterologous OspC variant, which conferred protection against challenge with B. burgdorferi. The data demonstrate the principal applicability of HBV CLPs to act as potent immunomodulator even for structurally complex full-length polypeptide chains, and thus open new avenues for novel vaccine designs.

High plasticity of the hepatitis B virus capsid revealed by conformational stress.

Hepatitis B virus (HBV) replicates through reverse transcription inside its icosahedral nucleocapsid. The internal genome status is signaled to the capsid surface, predicting regulated conformational changes in the capsid structure. To probe their nature and extent, we imposed local conformational stress on the outer surface of HBV capsid-like particles, and monitored its consequences by electron cryomicroscopy and image reconstruction. The capsid structure had an enormous flexibility and robustness as a whole, as well as within the subunits, whose spikes were able to rotate by as much as 40 degrees against the distal interdimer contact sites. The likely hinge for the swiveling movement was the conserved Gly111 residue at the inner surface of the capsid. The stress imposed from the outside also affected the internal capsid organization, implying a specific route for the flow of conformational information between capsid interior and exterior as required for signaling of the genome status.

In vitro assembly of mosaic hepatitis B virus capsid-like particles (CLPs): rescue into CLPs of assembly-deficient core protein fusions and FRET-suited CLPs.

Hepatitis B virus core protein self-assembles into icosahedral, highly immunogenic capsid-like particles (CLPs) that can serve as molecular platforms for heterologous proteins. Insertion into the centrally located c/e1 epitope leads to surface display, fusion to the C terminus to internal disposition of the foreign domains. However, symmetry-defined space restrictions on the surface and particularly inside the CLPs limit the size of usable heterologous fusion partners. Further, CLPs carrying differing foreign domains are desirable for applications such as multivalent vaccines, and for structure probing by distance sensitive interactions like fluorescence resonance energy transfer (FRET). Here, we report an in vitro co-assembly system for such mosaic-CLPs allowing successful CLP formation with a per se assembly-deficient fusion protein, and of CLPs from two different fluoroprotein-carrying fusions that exert FRET in an assembly-status dependent way.

Quaternary structure is critical for protein display on capsid-like particles (CLPs): efficient generation of hepatitis B virus CLPs presenting monomeric but not dimeric and tetrameric fluorescent proteins.

Self-organizing assemblies such as viral capsids may be used as symmetrical molecular platforms for the display of heterologous sequences, with applications ranging from vaccines to structural studies. The 183-amino-acid hepatitis B virus (HBV) core protein assembles spontaneously into icosahedral capsid-like particles (CLPs). The most exposed, and most immunogenic, substructure on the CLPs is a small loop that connects two long antiparallel alpha-helices which act as dimerization interface. Ninety (90) or 120 dimers multimerize into the capsid; the four-helix bundles formed by the dimers protrude as spikes from the surface. We recently demonstrated that the entire enhanced green fluorescent protein (eGFP) can be inserted into this loop, yielding CLPs that natively displayed eGFP on their surface. The central location of the insertion site requires that any insert be fixed to the carrier via both termini, with corresponding restrictions regarding insert size and structure. eGFP obviously satisfied these criteria but, surprisingly, all attempts to produce CLPs with the isostructural red fluorescent proteins DsRed1, DsRed2, and HcRed failed. Suspecting their oligomerization tendency to be responsible, we generated fusions containing instead monomeric yellow, cyan, and red fluorescent proteins (mYFP, mCFP and mRFP1). This strongly increased the yields of YFP and CFP-CLPs, and it allowed for the first time to efficiently generate red fluorescent CLPs. Hence insert quaternary structure is a highly critical factor for CLP assembly. These data have important implications for the rational design of self-assembling fusion proteins.

dNTP versus NTP discrimination by phenylalanine 451 in duck hepatitis B virus P protein indicates a common structure of the dNTP-binding pocket with other reverse transcriptases.

Hepatitis B viruses, or hepadnaviruses, are small DNA-containing viruses that replicate through reverse transcription. Their prototype, HBV, causes severe liver disease in humans. The hepadnaviral P protein is an unusual reverse transcriptase (RT) that initiates DNA synthesis by host-factor-dependent protein priming on a specific RNA stem-loop template, epsilon, yielding a short DNA oligonucleotide covalently attached to the RT. This priming reaction can be reconstituted with in vitro-translated duck hepatitis B virus (DHBV) P protein. No direct structural data are available for any P protein. However, P proteins share a number of conserved motifs with other polymerases. Box A contains an invariant bulky residue recently shown to be crucial for dNTP versus NTP discrimination in RTs and some DNA polymerases; its equivalent in DHBV P protein would be phenylalanine 451 (F451). Four mutants, containing glycine (F451G), alanine (F451A), valine (F451V) and aspartate (F451D), were therefore analyzed for their ability to utilize dNTPs and NTPs in in vitro priming. Priming efficiencies with dNTPs decreased with decreasing side chain size but GTP utilization increased; the wild-type enzyme was inactive with GTP. In the context of complete DHBV genomes, all mutant proteins were competent for RNA encapsidation, indicating the absence of global structural alterations. Because the function of the discriminatory residue depends on its specific spatial disposition this strongly suggests a similar architecture for the P protein dNTP-binding pocket as in other RTs.