Diatom adhesive trail proteins acquired by horizontal gene transfer from bacteria serve as primers for marine biofilm formation.

Biofilm-forming benthic diatoms are key primary producers in coastal habitats, where they frequently dominate sunlit intertidal substrata. The development of gliding motility in raphid diatoms was a key molecular adaptation that contributed to their evolutionary success. However, the structure-function correlation between diatom adhesives utilized for gliding and their relationship to the extracellular matrix that constitutes the diatom biofilm is unknown. Here, we have used proteomics, immunolocalization, comparative genomics, phylogenetics and structural homology analysis to investigate the evolutionary history and function of diatom adhesive proteins. Our study identified eight proteins from the adhesive trails of Craspedostauros australis, of which four form a new protein family called Trailins that contain an enigmatic Choice-of-Anchor A (CAA) domain, which was acquired through horizontal gene transfer from bacteria. Notably, the CAA-domain shares a striking structural similarity with one of the most widespread domains found in ice-binding proteins (IPR021884). Our work offers new insights into the molecular basis for diatom biofilm formation, shedding light on the function and evolution of diatom adhesive proteins. This discovery suggests that there is a transition in the composition of biomolecules required for initial surface colonization and those utilized for 3D biofilm matrix formation.

The molecular basis for pore pattern morphogenesis in diatom silica.

Biomineral-forming organisms produce inorganic materials with complex, genetically encoded morphologies that are unmatched by current synthetic chemistry. It is poorly understood which genes are involved in biomineral morphogenesis and how the encoded proteins guide this process. We addressed these questions using diatoms, which are paradigms for the self-assembly of hierarchically meso- and macroporous silica under mild reaction conditions. Proteomics analysis of the intracellular organelle for silica biosynthesis led to the identification of new biomineralization proteins. Three of these, coined dAnk1-3, contain a common protein-protein interaction domain (ankyrin repeats), indicating a role in coordinating assembly of the silica biomineralization machinery. Knocking out individual dank genes led to aberrations in silica biogenesis that are consistent with liquid-liquid phase separation as underlying mechanism for pore pattern morphogenesis. Our work provides an unprecedented path for the synthesis of tailored mesoporous silica materials using synthetic biology.

The Protein Corona Does Not Influence Receptor-Mediated Targeting of Virus-like Particles.

Protein corona formation has been regarded as an obstacle to developing diagnostic and therapeutic nanoparticles for in vivo applications. Serum proteins that assemble around nanoparticles can hinder their targeting efficiency. Virus-based nanoparticles should be naturally predisposed to evade such barriers in host organisms. Here, we demonstrate that virus-like particles derived from mouse polyomavirus do not form a rich protein corona. These particles can be efficiently targeted to cells that overproduce transferrin receptors, e.g., cancer cells, by conjugating transferrin to the particle surface. In this study, we provide evidence that the interaction of virus-like particles with their newly assigned target receptor is not obstructed by serum proteins. The particles enter target cells via a clathrin-dependent endocytic pathway that is not naturally used by the virus. Our results support the notion that the natural properties of virus-like particles make them well-suited for development of nanosized theranostic tools resistant to detargeting by protein coronas.

Influence of cell-penetrating peptides on the activity and stability of virus-based nanoparticles.

Viral nanoparticles represent potential natural versatile platforms for targeted gene and drug delivery. Improving the efficiency of gene transfer mediated by viral vectors could not only enhance their therapeutic potential, but also contribute to understanding the limitations in interactions of nanoparticles with cells and the development of new therapeutic approaches. In this study, four cell-penetrating peptides (CPPs), cationic octaarginine (R8), histidine-rich peptides (LAH4 and KH27K) and fusogenic peptide (FUSO), are investigated for their effect on infection by mouse polyomavirus (MPyV) or on transduction of reporter genes delivered by MPyV or related viral vectors. Peptides noncovalently associated with viral particles enhance gene transfer (with the exception of FUSO). Removal of cellular heparan sulfates by the heparinase does not significantly change the enhancing potential of CPPs. Instead, CPPs influences the physical state of viral particles: R8 slightly destabilizes the intact virus, KH27K induces its aggregation and LAH4 promotes disassembly and aggregation of the particles that massively and rapidly associate with cells. The findings indicate that peptides acting as transduction-enhancing agents of polyomavirus-based nanoparticles modulate their physical state, which can be an important prerequisite for sensitization of cells and determination of the further fate of viral particles inside cells.

Prevalence of antibodies against BKPyV subtype I and IV in kidney transplant recipients and in the general Czech population.

Active infection with BK polyomavirus (BKPyV) may cause serious complications in transplantation settings. Recently, the level of BKPyV IgG seroreactivity in graft donors has been shown to predict viremia and BKPyV-associated nephropathy in kidney transplant (KTx) recipients. Pretransplantation testing of the donor and recipient BKPyV serostatus could, therefore, identify patients at high risk. For the development of serological immunoassays, antibody response to the predominant BKPyV subtypes (BKPyV-I and BKPyV-IV) was studied using virus-like particle (VLP)-based enzyme-linked immunosorbent assay (ELISA). VLPs made from the capsid protein, VP1, derived from BKPyV-I and BKPyV-IV subtypes were produced using a baculovirus expression system and used as antigens. The tests were used for IgG antibody determination in 50 KTx recipients and 111 healthy blood donors. While 87% of samples reacted with mixed BKPyV-I and BKPyV-IV antigens, only 49% of samples were reactive in both ELISA tests when using BKPyV-I or BKPyV-IV antigens separately. Twenty-seven percent of healthy blood donors and 26% of KTx recipients were reactive only with BKPyV-I, while 9% and 20% were reactive only with BKPyV-IV, respectively. To determine the specificities of the antigens, selected seropositive samples were retested after preadsorption with soluble BKPyV-I, BKPyV-IV, or JC polyomavirus antigens. The experiments confirmed that recombinant VP1 VLP-based ELISAs predominantly detected BKPyV type-specific antibodies. The results imply that anti-BKPyV antibody ELISA tests should contain a mixture of subtype-specific VLP-based antigens instead of antigen derived from the most prevalent BKPyV-I subtype. The tests can be used for serological surveys of BKPyV infection and improved KTx patient management.

Inhibitor-GCPII Interaction: Selective and Robust System for Targeting Cancer Cells with Structurally Diverse Nanoparticles.

Glutamate carboxypeptidase II (GCPII) is a membrane protease overexpressed by prostate cancer cells and detected in the neovasculature of most solid tumors. Targeting GCPII with inhibitor-bearing nanoparticles can enable recognition, imaging, and delivery of treatments to cancer cells. Compared to methods based on antibodies and other large biomolecules, inhibitor-mediated targeting benefits from the low molecular weight of the inhibitor molecules, which are typically stable, easy-to-handle, and able to bind the enzyme with very high affinity. Although GCPII is established as a molecular target, comparing previously reported results is difficult due to the different methodological approaches used. In this work, we investigate the robustness and limitations of GCPII targeting with a diverse range of inhibitor-bearing nanoparticles (various structures, sizes, bionanointerfaces, conjugation chemistry, and surface densities of attached inhibitors). Polymer-coated nanodiamonds, virus-like particles based on bacteriophage Qß and mouse polyomavirus, and polymeric poly(HPMA) nanoparticles with inhibitors attached by different means were synthesized and characterized. We evaluated their ability to bind GCPII and interact with cancer cells using surface plasmon resonance, inhibition assay, flow cytometry, and confocal microscopy. Regardless of the diversity of the investigated nanosystems, they all strongly interact with GCPII (most with low picomolar Ki values) and effectively target GCPII-expressing cells. The robustness of this approach was limited only by the quality of the nanoparticle bionanointerface, which must be properly designed by adding a sufficient density of hydrophilic protective polymers. We conclude that the targeting of cancer cells overexpressing GCPII is a viable approach transferable to a broad diversity of nanosystems.

Retargeting Polyomavirus-Like Particles to Cancer Cells by Chemical Modification of Capsid Surface.

Virus-like particles based on polyomaviruses (PVLPs) are promising delivery devices for various cargoes, including nucleic acids, imaging probes, and therapeutic agents. In biological environments, the major coat protein VP1 interacts with ubiquitously distributed sialic acid residues, and therefore PVLPs show a broad tropism. For selective targeting, appropriate engineering of the PVLP surface is needed. Here, we describe a chemical approach to retarget PVLPs to cancer cells displaying abnormally high levels of transferrin receptor. We created an array of transferrin molecules on the surface of PVLPs by combining a high-yielding bioconjugation approach with specific point modification of transferrin. This artificial surface protein architecture enables (i) suppression of natural VP1-specific interactions by blocking the surface conformational epitope on the VP1 protein, (ii) unusually high cellular uptake efficiency, and (iii) selective retargeting of PVLPs to osteosarcoma (U2OS) and lymphoblastoid leukemia (CCRF-CEM) cells.

The encapsidation of polyomavirus is not defined by a sequence-specific encapsidation signal.

Mouse polyomavirus (MPyV) is considered a potential tool for the application of gene therapy; however, the current knowledge of the encapsulation of DNA into virions is vague. We used a series of assays based on the encapsidation of a reporter vector into MPyV pseudovirions to identify putative cis-acting elements that are involved in DNA encapsidation. None of the sequences that were derived from MPyV have been shown to solely enhance the encapsidation of a reporter vector in the assay. The frequency of encapsidation strongly correlated with the total intracellular amount of the vector after transfection. The encapsidation of target DNA into the pseudovirions was shown to be non-specific, and the packaging of non-replicated DNA was observed. We propose that the actual concentration of target DNA at the sites of virion formation is the primary factor that determines its selection for encapsidation.

The Cre/loxP recombination system for production of infectious mouse polyomavirus.

Murine polyomavirus mutants are frequently produced for experimental as well as therapy purposes. Commonly used methods for preparation of mutant viral genomes from recombinant vectors are laborious and give variable yields and quality. We describe an efficient and reproducible Cre/loxP-mediated recombination system that generates polyomavirus genomes from recombinant plasmid in vivo. We designed and constructed two variants of recombinant vectors containing the wild-type polyomavirus genome flanked by loxP homologous sites. The loxP sites were introduced either into the intronic region of early genes or between the two poly(A) signal sites of convergent transcriptional units. After cotransfection of the recombinant plasmids with the Cre-expressing vector into mouse 3T6 cells, we obtained infectious virus from the genome variant containing loxP site in the intronic region, but we failed to isolate any infectious virus from the viral genome containing loxP site between poly(A) signals. We show that the Cre/loxP-based method of polyomavirus production is simple, expedient, and reproducible and works with satisfactory efficiency.