bacterial-inner-membrane-display-for-screening-library-antibody

Bacterial Inner-membrane Display for Screening a Library of Antibody Fragments

Antibodies engineered for intracellular function must not only have affinity for their target antigen, but must also be soluble and correctly folded in ...

quantifying-antifungal-activity-peptides-against-candida

Quantifying the Antifungal Activity of Peptides Against <em>Candida albicans</em>

Traditional methods for performing antifungal susceptibility testing for Candida albicans are time-consuming and lack quantitative results. For example, a ...

University of Maryland

Antibody fragments, such as the single-chain variable fragment (scFv), have much potential in research and diagnostics because of their antigen-binding ability similar to a full-sized antibody and their ease of production in microorganisms. Some applications of antibody fragments require immobilization on a surface, and we have established a simple immobilization method that is based on the biotin-streptavidin interaction and does not require a separate purification step. We genetically fused two biotinylation tags-the biotin carboxyl carrier protein (BCCP) or the AviTag minimal sequence-to six different scFvs (scFv13R4, scFvD10, scFv26-10, scFv3, scFv5, and scFv12) for site-specific biotinylation in vivo by endogenous biotin ligases produced by Escherichia coli. The biotinylated scFvs were immobilized onto streptavidin-coated plates directly from cell lysates, and immobilization was detected through enzyme-linked immunosorbent assays. All scFvs fusions were successfully immobilized, and scFvs biotinylated via the BCCP tag tended to immobilize better than those biotinylated via the AviTag, even when biotinylation efficiency was improved with the biotin ligase BirA. The ability of immobilized scFvs to bind antigens was confirmed using scFv13R4 and scFvD10 with their respective targets β-galactosidase and bacteriophage lambda head protein D (gpD). The immobilized scFv13R4 bound to β-galactosidase at the same level for both biotinylation tags when the surface was saturated with the scFv, and immobilized scFvs retained their functionality for at least 100days after immobilization. The simplicity and robustness of our method make it a promising approach for future applications that require antibody fragment immobilization.

A simple and robust approach to immobilization of antibody fragments.

Candida albicans is an opportunistic fungal pathogen and a commensal organism that commonly colonizes mucosal surfaces, including those inside the human mouth. To help control C. albicans, human saliva contains the antifungal peptide histatin 5 (Hst-5), which has strong antifungal activity against C. albicans. However, the pathogen produces secreted aspartic proteases (Saps) that cleave Hst-5 at lysine residues and eliminate its antifungal properties. We designed variants of Hst-5 with its lysine residues substituted with arginine or leucine to evaluate the effect on proteolysis by Saps. We found site-, residue-, and Sap-dependent effects from single amino acid substitutions. The K17R and K17L modifications led to dramatic results, with over 77% and 100% intact peptide remaining after incubation with Sap9 and Sap2, respectively, compared to 47% and 61% of Hst-5. This decrease in proteolysis was accompanied by a reduction in cleavage on the C-terminal side of K17, suggesting the Saps prefer lysine at K17 for cleavage. Incubation with C. albicans cells and culture supernatant corroborated the results with purified Saps and highlighted their biological relevance. The modifications to Hst-5 do not diminish the antifungal activity of Hst-5, and, in fact, the K17R, K17L, and K11R peptides retained significantly more antifungal activity after treatment with Saps than Hst-5. Our results indicate that single amino acid modifications drastically impact both proteolysis at the modification sites and the overall level of proteolysis of the peptide, demonstrating the potential of designing peptides for resistance to proteolysis as a means for improving therapeutic efficacy.

Engineering improved variants of the antifungal peptide histatin 5 with reduced susceptibility to Candida albicans secreted aspartic proteases and enhanced antimicrobial potency.

Cell-penetrating peptides (CPPs) are peptides that cross cell membranes, either alone or while carrying molecular cargo. Although their interactions with mammalian cells have been widely studied, much less is known about their interactions with fungal cells, particularly at the biophysical level. We analyzed the interactions of seven CPPs (penetratin, Pep-1, MPG, pVEC, TP-10, MAP, and cecropin B) with the fungal pathogen Candida albicans using experiments and molecular simulations. Circular dichroism (CD) of the peptides revealed a structural transition from a random coil or weak helix to an α-helix occurs for all peptides when the solvent is changed from aqueous to hydrophobic. However, CD performed in the presence of C. albicans cells showed that proximity to the cell membrane is not necessarily sufficient to induce this structural transition, as penetratin, Pep-1, and MPG did not display a structural shift in the presence of cells. Monte Carlo simulations were performed to further probe the molecular-level interaction with the cell membrane, and these simulations suggested that pVEC, TP-10, MAP, and cecropin B strongly penetrate into the hydrophobic domain of the membrane lipid bilayer, inducing a transition to an α-helical conformation. In contrast, penetratin, Pep-1 and MPG remained in the hydrophilic region without a shift in conformation. The experimental data and MC simulations combine to explain how peptide structure affects their interaction with cells and their mechanism of translocation into cells (direct translocation vs. endocytosis). Our work also highlights the utility of combining biophysical experiments, biological experiments, and molecular modeling to understand biological phenomena.

Secondary structure of cell-penetrating peptides during interaction with fungal cells.

Single-chain variable fragment antibodies (scFvs) are attractive for use in applications that require high specificity and binding to a target, such as biosensors. Previously, we demonstrated that a variety of scFvs can be immobilized onto a streptavidin surface through in vivo biotinylation of the biotin carboxyl carrier protein (BCCP) or smaller AviTag fused to the scFvs. However, the BCCP constructs showed better immobilization than the AviTag constructs. In this work, we investigated whether the discrepancy between the biotinylation tags could be alleviated by incorporating a flexible (G S) linker of varying lengths or a rigid (EA K) linker between the biotinylation tags and the scFvs scFv13R4 and scFv5. Fusion of the (G S) linker or the (G S) linker to the AviTag construct of scFv13R4 or scFv5, respectively, and fusion of the (EA K) linkers to the AviTag constructs of both scFvs enhanced immobilization. Meanwhile, the robust immobilization of the BCCP construct of the scFv constructs remained unaffected. The positive to neutral effects of the linkers, with no adverse effects, make them beneficial tools to incorporate into fusion proteins that show poor immobilization without a linker.

Effect of linkers on immobilization of scFvs with biotin-streptavidin interaction.

Histatin 5 (Hst-5) is an antimicrobial peptide with strong antifungal activity against Candida albicans, an opportunistic pathogen that is a common cause of oral thrush. The peptide is natively secreted by human salivary glands and shows promise as an alternative therapeutic against infections caused by C. albicans. However, Hst-5 can be cleaved and inactivated by a family of secreted aspartic proteases (Saps) produced by C. albicans. Single-residue substitutions can significantly affect the proteolytic resistance of Hst-5 to Saps and its antifungal activity; the K17R substitution increases resistance to proteolysis, while the K11R substitution enhances antifungal activity. In this work, we showed that the positive effects of these two single-residue modifications can be combined in a single peptide, K11R-K17R, with improved proteolytic resistance and antifungal activity. We also investigated the effect of additional single-residue substitutions, with a focus on the effect of addition or removal of negatively charged residues, and found Sap-dependent effects on degradation. Both single- and double-substitutions affected the kinetics of proteolytic degradation of the intact peptide and of the fragments formed during degradation. Our results demonstrate the importance of considering proteolytic stability and not just antimicrobial activity when designing peptides for potential therapeutic applications.

Effects of histatin 5 modifications on antifungal activity and kinetics of proteolysis.

Histatin-5 (Hst-5) is a human salivary peptide with antibacterial and antifungal activities. Thorough characterization and reliable quantification of Hst-5 and its degradation products are essential for understanding the Hst-5 degradation pathway. Due to the highly basic and strong cationic nature of the Hst-5 peptide, the quantitative analysis of Hst-5 and its degradation forms by online mass spectrometry remains challenging. Here, we adopt a recently developed electrokinetically pumped sheath liquid capillary electrophoresis - mass spectrometry (CE-MS) coupling technology, and successfully apply it for the analysis of Hst-5 and its degradation products. Our CE-MS method is demonstrated to be robust and quantitative. This novel analytical platform is reproducible and free of sample carryover. The efficacy of this method is demonstrated with a kinetic study of Hst-5 degradation by Sap9, a secreted aspartic peptidase. Our work demonstrates the potential of online CE-MS as a powerful approach for characterizing highly basic peptides.

Svetlana P. Ikonomova

Department of Chemical and Biomolecular Engineering

University of Maryland

Bacterial Inner-membrane Display for Screening a Library of Antibody Fragments

Quantifying the Antifungal Activity of Peptides Against Candida albicans

Svetlana P. Ikonomova

.css-o250i3{font-size:18px;font-family:Open Sans,sans-serif;line-height:21.6px;}Department of Chemical and Biomolecular Engineering

University of Maryland

Bacterial Inner-membrane Display for Screening a Library of Antibody Fragments

Quantifying the Antifungal Activity of Peptides Against Candida albicans

Department of Chemical and Biomolecular Engineering