Whole-bacterium ribosome display selection for isolation of highly specific anti-Staphyloccocus aureus Affitins for detection- and capture-based biomedical applications.

Detection and capture methods using antibodies have been developed to ensure identification of pathogens in biological samples. Though antibodies have many attractive properties, they also have limitations and there are needs to expand the panel of available affinity proteins with different properties. Affitins, that we developed from the Sul7d proteins, are a solid class of affinity proteins, which can be used as substitutes to antibodies or to complement them. We report the generation and characterization of antibacterial Affitins with high specificity for Staphylococcus aureus. For the first time, ribosome display selections were carried out using whole-living-cell and naïve combinatorial libraries, which avoid production of protein targets and immunization of animals. We showed that Affitin C5 exclusively recognizes S. aureus among dozens of strains, including clinical ones. C5 binds staphylococcal Protein A (SpA) with a K D of 108 ± 2 nM and has a high thermostability (T m = 77.0°C). Anti-S. aureus C5 binds SpA or bacteria in various detection and capture applications, including ELISA, western blot analysis, bead-fishing, and fluorescence imaging. Thus, novel anti-bacteria Affitins which are cost-effective, stable, and small can be rapidly and fully designed in vitro with high affinity and specificity for a surface-exposed marker. This class of reagents can be useful in diagnostic and biomedical applications.

Prosthetic groups for radioiodination and astatination of peptides and proteins: A comparative study of five potential bioorthogonal labeling strategies.

125I- and 211At-labeled azide and tetrazine based prosthetic groups for bioorthogonal conjugation were designed and tested in a comparative study of five bioorthogonal systems. All five bioconjugation reactions conducted on a model clickable peptide led to quantitative yields within less than a minute to several hours depending on the system used. Transferability to the labeling of an IgG was demonstrated with one of the bioorthogonal system. This study provides several new alternatives to the conventional and suboptimal approach currently in use for radioiodination and astatination of biomolecules and should accelerate the development of new probes with these radionuclides for applications in nuclear imaging and targeted alpha-therapy.

Novel Tn916-like elements confer aminoglycoside/macrolide co-resistance in clinical isolates of Streptococcus gallolyticus ssp. gallolyticus.

Background: Streptococcus gallolyticus ssp. gallolyticus (Sgg) is a commensal bacterium and an opportunistic pathogen. In humans it has been clinically associated with the incidence of colorectal cancer (CRC) and epidemiologically recognized as an emerging cause of infective endocarditis (IE). The standard therapy of Sgg includes the administration of a penicillin in combination with an aminoglycoside. Even though penicillin-resistant isolates have still not been reported, epidemiological studies have shown that this microbe is a reservoir of multiple acquired genes, conferring resistance to tetracyclines, aminoglycosides, macrolides and glycopeptides. However, the underlying antibiotic resistance mobilome of Sgg remains poorly understood. Objectives: To investigate the mobile genetic basis of antibiotic resistance in multiresistant clinical Sgg. Methods: Isolate NTS31106099 was recovered from a patient with IE and CRC at Nantes University Hospital, France and studied by Illumina WGS and comparative genomics. Molecular epidemiology of the identified mobile element(s) was performed using antibiotic susceptibility testing (AST), PCR, PFGE and WGS. Mobility was investigated by PCR and filter mating. Results: Two novel conjugative transposons, Tn6263 and Tn6331, confer aminoglycoside/macrolide co-resistance in clinical Sgg. They display classical family Tn916/Tn1545 modular architecture and harbour an aph(3')-III→sat4→ant(6)-Ia→erm(B) multiresistance gene cluster, related to pRE25 of Enterococcus faecium. These and/or closely related elements are highly prevalent among genetically heterogeneous clinical isolates of Sgg. Conclusions: Previously unknown Tn916-like mobile genetic elements conferring aminoglycoside/macrolide co-resistance make Sgg, collectively with other gut Firmicutes such as enterococci and eubacteria, a potential laterally active reservoir of these antibiotic resistance determinants among the mammalian gastrointestinal microbiota.

A novel, smaller scaffold for Affitins: Showcase with binders specific for EpCAM.

Affitins are highly stable engineered affinity proteins, originally derived from Sac7d and Sso7d, two 7 kDa DNA-binding polypeptides from Sulfolobus genera. Their efficiency as reagents for intracellular targeting, enzyme inhibition, affinity purification, immunolocalization, and various other applications has been demonstrated. Recently, we have characterized the 7 kDa DNA-binding family, and Aho7c originating from Acidianus hospitalis was shown to be its smallest member with thermostability comparable to those of Sac7d and Sso7d. Here, after four rounds of selection by ribosome display against the human recombinant Epithelial Cell Adhesion Molecule (hrEpCAM), we obtained novel Aho7c-based Affitins. The binders were expressed in soluble form in Escherichia coli, displayed high stability (up to 74°C; pH 0-12) and were shown to be specific for the hrEpCAM extracellular domain with picomolar affinities (KD = 110 pM). Thus, we propose Aho7c as a good candidate for the creation of Affitins with a 10% smaller size than the Sac7d-based ones (60 vs. 66 amino acids).

In vitro emergence of fluoroquinolone resistance in Cutibacterium (formerly Propionibacterium) acnes and molecular characterization of mutations in the gyrA gene.

In vitro occurrence of levofloxacin (LVX) resistance in C. acnes and characterization of its molecular background were investigated. The mutation frequency was determined by inoculation of 108 cfu of C. acnes ATCC 11827 (LVX MIC = 0.25 mg/L) on LVX-containing agar plates. The progressive emergence of resistance was studied by a second exposure to increasing LVX concentrations. For mutants, the QRDR regions including the gyrA and parC genes were sequenced and compared to both C. acnes ATCC 11827 and C. acnes KPA171202 reference sequences (NC006085). The importance of the efflux pump system in resistance was investigated by using inhibitors on selected resistant mutants with no mutation in the QRDR. C. acnes growth was observed on LVX-containing plates with mutation frequencies of 3. 8 cfu × 10-8 (8 × MIC) and 1.6 cfu × 10-7 (4 × MIC). LVX resistance emerged progressively after one-step or two-step assays. In LVX-resistant isolates, the MIC ranged from 0.75 to >32 mg/L. Mutations were detected exclusively in the gyrA gene. Ten genotypes were identified: G99 C, G99 D, D100N, D100 H, D100 G, S101L, S101W, A102 P, D105 H and A105 G. Mutants S101L and S101W were always associated with a high level of resistance. Mutants with no mutation in the QRDR were more susceptible when incubated with an efflux pump inhibitor (phenyl-arginine ß-naphthylamide) only, suggesting, for the first time, the expression of such a system in C. acnes LVX-resistant mutants.

Construction of Synthetic VHH Libraries in Ribosome Display Format.

Single-domain antibodies, or VHH, represent an attractive molecular basis to design affinity proteins with favorable properties. Beyond high affinity and specificity for their cognate target, they usually show high stability and high production yields in bacteria, yeast, or mammalian cells. In addition to these favorable properties, their ease of engineering makes them useful for many applications. Until the past few years, the generation of VHH involved the immunization of a Camelidae with the target antigen, followed by a phage display selection using phage libraries encoding the VHH repertoire of the animal blood sample. However, this approach is constrained by the accessibility to the animals, and the output relies on the animal's immune system.Recently, synthetic VHH libraries have been designed to avoid the use of animals. Here, we describe the construction of VHH combinatorial libraries and their use for the selection of binders by ribosome display, a fully in vitro selection technique.

Engineering of a phosphorylatable tag for specific protein binding on zirconium phosphonate based microarrays.

A phosphorylatable tag was designed and fused at the C-terminal end of proteins, which allowed efficient and oriented immobilization of capture proteins on glass substrates coated with a zirconium phosphonate monolayer. The concept is demonstrated using Nanofitin directed against lysozyme. This peptide tag (DSDSSSEDE) contains four serines in an acidic environment, which favored its in vitro phosphorylation by casein kinase II. The resulting phosphate cluster at the C-terminal end of the protein provided a specific, irreversible, and multipoint attachment to the zirconium surface. In a microarray format, the high surface coverage led to high fluorescence signal after incubation with Alexa Fluor 647 labeled lysozyme. The detection sensitivity of the microarray for the labeled target was below 50 pM, owing to the exceptionally low background staining, which resulted in high fluorescence signal to noise ratios. The performance of this new anchoring strategy using a zirconium phosphonate modified surface compares favorably with that of other types of microarray substrates, such as nitrocellulose-based or epoxide slides, which bind proteins in a nonoriented way.

Application of Affitins for Affinity Purification of Proteins.

Affinity chromatography is a powerful purification technique, as it allows proteins of interest to be obtained at a high degree of purity in a single step. This technique can be applied on a research laboratory scale as well as on an industrial scale. The interaction involved in affinity separation most often involves a natural ligand or an antibody specific for the protein of interest, or the recognition of a peptide tag artificially added to the recombinant protein. Unfortunately, natural ligands are not always available and it may be undesirable or impossible to add a purification tag, especially for the production of therapeutic proteins. We have developed Affitins as a new class of artificial affinity proteins that can be generated against virtually any protein of interest. Due to their very high selectivity, their remarkable robustness against extreme acid or alkaline conditions and their low production cost, Affitins are particularly suited to this technique. We describe here the production of Affitins and their immobilization on resin beads to prepare affinity chromatography columns. The protocol also describes the use of these columns.

A novel and efficient approach to high-throughput production of HLA-E/peptide monomer for T-cell epitope screening.

Over the past two decades, there has been a great interest in the study of HLA-E-restricted αß T cells during bacterial and viral infections, including recently SARS-CoV-2 infection. Phenotyping of these specific HLA-E-restricted T cells requires new tools such as tetramers for rapid cell staining or sorting, as well as for the identification of new peptides capable to bind to the HLA-E pocket. To this aim, we have developed an optimal photosensitive peptide to generate stable HLA-E/pUV complexes allowing high-throughput production of new HLA-E/peptide complexes by peptide exchange. We characterized the UV exchange by ELISA and improved the peptide exchange readout using size exclusion chromatography. This novel approach for complex quantification is indeed very important to perform tetramerization of MHC/peptide complexes with the high quality required for detection of specific T cells. Our approach allows the rapid screening of peptides capable of binding to the non-classical human HLA-E allele, paving the way for the development of new therapeutic approaches based on the detection of HLA-E-restricted T cells.

Remodeling a DNA-binding protein as a specific in vivo inhibitor of bacterial secretin PulD.

We engineered a class of proteins that binds selected polypeptides with high specificity and affinity. Use of the protein scaffold of Sac7d, belonging to a protein family that binds various ligands, overcomes limitations inherent in the use of antibodies as intracellular inhibitors: it lacks disulfide bridges, is small and stable, and can be produced in large amounts. An in vitro combinatorial/selection approach generated specific, high-affinity (up to 140 pM) binders against bacterial outer membrane secretin PulD. When exported to the Escherichia coli periplasm, they inhibited PulD oligomerization, thereby blocking the type II secretion pathway of which PulD is part. Thus, high-affinity inhibitors of protein function can be derived from Sac7d and can be exported to, and function in, a cell compartment other than that in which they are produced.

Characterization of Affitin proteolytic digestion in biorelevant media and improvement of their stabilities via protein engineering.

Affitins are a novel class of small 7 kDa artificial proteins which can be used as antibody substitutes in therapeutic, diagnostic and biotechnological applications. One challenge for this type of protein agent is their behaviour in the context of oral administration. The digestive system is central, and biorelevant media have fast emerged as relevant and reliable tools for evaluating the bioavailability of drugs. This study describes, for the first time, the stability of Affitins under simulated gastric and intestinal digestion conditions. Affitins appear to be degraded into stable fragments in in vitro gastric medium. We identified cleavage sites generated by pepsin that were silenced by site-directed mutagenesis. This protein engineering allowed us to enhance Affitin properties. We showed that a mutant M1 containing a double mutation of amino acid residues 6 and 7 in H4 and C3 Affitins acquired a resistance against proteolytic digestion. In addition, these mutations were beneficial for target affinity, as well as for production yield. Finally, we found that the mutated residues kept or increased the important pH and temperature stabilities of Affitins. These improvements are particularly sought after in the development of engineered binding proteins for research tools, preclinical studies and clinical applications.

Affitins: Ribosome Display for Selection of Aho7c-Based Affinity Proteins.

Engineered protein scaffolds have made a tremendous contribution to the panel of affinity tools owing to their favorable biophysical properties that make them useful for many applications. In 2007, our group paved the way for using archaeal Sul7d proteins for the design of artificial affinity ligands, so-called Affitins. For many years, Sac7d and Sso7d have been used as molecular basis to obtain binders for various targets. Recently, we characterized their old gifted protein family and identified Aho7c, originating from Acidianus hospitalis, as the shortest member (60 amino-acids) with impressive stability (96.5 °C, pH 0-12). Here, we describe the construction of Aho7c combinatorial libraries and their use for selection of binders by ribosome display.

Type II secretion system secretin PulD localizes in clusters in the Escherichia coli outer membrane.

The cellular localization of a chimera formed by fusing a monomeric red fluorescent protein to the C terminus of the Klebsiella oxytoca type II secretion system outer membrane secretin PulD (PulD-mCherry) in Escherichia coli was determined in vivo by fluorescence microscopy. Like PulD, PulD-mCherry formed sodium dodecyl sulfate- and heat-resistant multimers and was functional in pullulanase secretion. Chromosome-encoded PulD-mCherry formed fluorescent foci on the periphery of the cell in the presence of high (plasmid-encoded) levels of its cognate chaperone, the pilotin PulS. Subcellular fractionation demonstrated that the chimera was located exclusively in the outer membrane under these circumstances. A similar localization pattern was observed by fluorescence microscopy of fixed cells treated with green fluorescent protein-tagged affitin, which binds with high affinity to an epitope in the N-terminal region of PulD. At lower levels of (chromosome-encoded) PulS, PulD-mCherry was less stable, was located mainly in the inner membrane, from which it could not be solubilized with urea, and did not induce the phage shock response, unlike PulD in the absence of PulS. The fluorescence pattern of PulD-mCherry under these conditions was similar to that observed when PulS levels were high. The complete absence of PulS caused the appearance of bright and almost exclusively polar fluorescent foci.

Bisphosphonate adaptors for specific protein binding on zirconium phosphonate-based microarrays.

Two bisphosphonate adaptors were designed to immobilize histidine-tagged proteins onto glass substrates coated with a zirconium phosphonate monolayer, allowing efficient and oriented immobilization of capture proteins, affitins directed to lysozyme, on a microarray format. These bifunctional adaptors contain two phosphonic acid anchors at one extremity and either one nitrilotriacetic acid (NTA) or two NTA groups at the other. The phosphonate groups provide a stable bond to the zirconium interface by multipoint attachment and allow high density of surface coverage of the linkers as revealed by X-ray photoelectron spectroscopy (XPS). Reversible high-density capture of histidine-tagged proteins is shown by real-time surface plasmon resonance enhanced ellipsometry and in a microarray format using fluorescence detection of AlexaFluor 647-labeled target protein. The detection sensitivity of the microarray for the target protein was below 1 nM, despite the monolayer arrangement of the probes, due to very low background staining, which allows high fluorescent signal-to-noise ratio. The performance of these Ni-NTA-modified zirconium phosphonate coated slides compared favorably to other types of microarray substrates, including slides with a nitrocellulose-based matrix, epoxide slides, and epoxide slides functionalized with Ni-NTA groups. This immobilization strategy has a large potential to fix any histidine-tagged proteins on zirconium or titanium ion surfaces.

Llama VHH antibody fragments against GFAP: better diffusion in fixed tissues than classical monoclonal antibodies.

Camelids produce antibodies made of homodimeric heavy chains, and the antigen-binding region being composed of a single domain called VHH. These VHHs are much smaller than complete IgG. They are also more thermostable and more soluble in water; they should, therefore, diffuse more readily in the tissues. VHHs, expressed in bacteria, are easier to produce than conventional monoclonal antibodies. Because of these special characteristics, these antibody fragments could have interesting developments in immunohistochemistry and in the development of biomarkers. To test the possibility of their use in immunohistochemistry (IHC), we selected the glial fibrillary acidic protein (GFAP), a well-known marker of astrocytes. One alpaca (Lama pacos) was immunized against GFAP. Lymphocytes were isolated; the DNA was extracted; the VHH-coding sequences were selectively amplified. Three VHHs with a high affinity for GFAP and their corresponding mRNA were selected by ribosome display. Large quantities of the recombinant VHHs coupled with different tags were harvested from transfected bacteria. One of them was shown to immunolabel strongly and specifically to GFAP of human astrocytes in tissue sections. The quality of the IHC was comparable or, in some aspects, superior to the quality obtained with conventional IgG. The VHH was shown to diffuse on a longer distance than conventional monoclonal antibodies in fixed cortical tissue: a property that may be useful in immunolabeling of thick sections.

Multivalent Affidendrons with High Affinity and Specificity toward Staphylococcus aureus as Versatile Tools for Modulating Multicellular Behaviors.

Multivalency is a widely occurring natural phenomenon often exploited in nanotechnology to enhance biorecognition. We report the preparation and characterization of versatile, multivalent Affitin-dendrimer conjugates (Affidendrons) showcased by a set targeting Staphylococcus aureus ( S. aureus), an opportunistic pathogen causing numerous hospital- and community-acquired infections. Affitins are small affinity proteins characterized by higher stability and lower cost-effective production than antibodies. The strategy presented provides a platform for the rational design of multivalent nanodevices that, retaining the ability of Affitins to recognize their target with high specificity, achieve a largely enhanced affinity. Affidendrons with precisely designed size and valency have been exploited to modulate complex multicellular behaviors of S. aureus, such as agglutination and biofilm formation. Agglutination assays showed that Affidendrons rapidly cross-link S. aureus strains with high bacterial cell selectivity. Moreover, remarkably low concentrations of Affidendrons were able to effectively prevent biofilm formation. Overall, Affidendrons represent a promising platform for the rapid and selective pathogen identification, infection imaging, and theranostic applications.

Model Affitin and PEG modifications onto siRNA lipid nanocapsules: cell uptake and in vivo biodistribution improvements.

Malignant melanoma is an aggressive tumor, associated with the presence of local and/or distant metastases. The development of gene therapy by the use of small interfering RNA (siRNA) represents a promising new treatment. However, the protection of this biomolecule is necessary in order for it to be intravenously administrated, for example via its incorporation into nanomedicines. In parallel to the passive targeting usually obtained by pegylation, various studies have aimed at developing "smart" nanomedicines to efficiently deliver the drug to tumor sites. In this work, siRNA loaded lipid nanocapsules (LNCs) were modified with DSPE-polyethylene glycol (DSPE-PEG), tetraether-PEG (TE-PEG) and/or with an Affitin model, to assay multiple targeting strategies. The uptake of fluorescently labelled LNCs, nanocarrier integrity and siRNA release into human SK-Mel28 melanoma cells were studied by flow cytometry, conventional confocal microscopy and by confocal spectral imaging in a Förster Resonance Energy Transfer (FRET) mode. Surface modified siRNA LNCs were followed after human plasma incubation and after intravenous injection, in order to compare the stealth properties. Finally, the biodistribution of the different siRNA LNCs in healthy and melanoma tumor bearing mice models was assessed by in vivo biofluorescence imaging (BFI), to evaluate the potential tumor targeting ability. The post-insertion of DSPE-PEG induced a strong decrease of the internalization into melanoma cells compared to TE-PEG modification. Both PEG polymer decorations induced a great plasma protection of siRNA but only DSPE-PEG led to stealth properties, even at low concentration (5 mM). The Affitin grafting by thiolation of DSPE-PEG was validated on siRNA LNCs. DSPE-PEG-Affitin LNCs were not detected in this melanoma tumor model but did not show unspecific accumulation in organs. DSPE-PEG and TE-PEG LNCs induced a significant intratumoral accumulation of modified LNCs.

A designed ankyrin repeat protein evolved to picomolar affinity to Her2.

Designed ankyrin repeat proteins (DARPins) are a novel class of binding molecules, which can be selected to recognize specifically a wide variety of target proteins. DARPins were previously selected against human epidermal growth factor receptor 2 (Her2) with low nanomolar affinities. We describe here their affinity maturation by error-prone PCR and ribosome display yielding clones with zero to seven (average 2.5) amino acid substitutions in framework positions. The DARPin with highest affinity (90 pM) carried four mutations at framework positions, leading to a 3000-fold affinity increase compared to the consensus framework variant, mainly coming from a 500-fold increase of the on-rate. This DARPin was found to be highly sensitive in detecting Her2 in human carcinoma extracts. We have determined the crystal structure of this DARPin at 1.7 A, and found that a His to Tyr mutation at the framework position 52 alters the inter-repeat H-bonding pattern and causes a significant conformational change in the relative disposition of the repeat subdomains. These changes are thought to be the reason for the enhanced on-rate of the mutated DARPin. The DARPin not bearing the residue 52 mutation has an unusually slow on-rate, suggesting that binding occurred via conformational selection of a relatively rare state, which was stabilized by this His52Tyr mutation, increasing the on-rate again to typical values. An analysis of the structural location of the framework mutations suggests that randomization of some framework residues either by error-prone PCR or by design in a future library could increase affinities and the target binding spectrum.

Structures of in vitro evolved binding sites on neocarzinostatin scaffold reveal unanticipated evolutionary pathways.

We have recently applied in vitro evolution methods to create in Neocarzinostatin a new binding site for a target molecule unrelated to its natural ligand. The main objective of this work was to solve the structure of some of the selected binders in complex with the target molecule: testosterone. Three proteins (1a.15, 3.24 and 4.1) were chosen as representative members of sequence families that came out of the selection process within different randomization schemes. In order to evaluate ligand-induced conformational adaptation, we also determined the structure of one of the proteins (3.24) in the free and complexed forms. Surprisingly, all these mutants bind not one but two molecules of testosterone in two very different ways. The 3.24 structure revealed that the protein spontaneously evolved in the system to bind two ligand molecules in one single binding crevice. These two binding sites are formed by substituted as well as by non-variable side-chains. The comparison with the free structure shows that only limited structural changes are observed upon ligand binding. The X-ray structures of the complex formed by 1a.15 and 4.1 Neocarzinostatin mutants revealed that the two variants form very similar dimers. These dimers were observed neither for the uncomplexed variants nor for wild-type Neocarzinostatin but were shown here to be induced by ligand binding. Comparison of the three complexed forms clearly suggests that these unanticipated structural responses resulted from the molecular arrangement used for the selection experiments.