PROTEIN ENGINEERING APPROACHES TO IMPROVING DIAGNOSIS AND TREATMENT OF CANDIDA ALBICANS INFECTIONS

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dc.contributor.advisorKarlsson, Amy Jen_US
dc.contributor.authorIkonomova, Svetlana Pavlovaen_US
dc.contributor.departmentChemical Engineeringen_US
dc.contributor.publisherDigital Repository at the University of Marylanden_US
dc.contributor.publisherUniversity of Maryland (College Park, Md.)en_US
dc.date.accessioned2017-10-06T05:30:36Z
dc.date.available2017-10-06T05:30:36Z
dc.date.issued2017en_US
dc.description.abstractCandida albicans is an opportunistic pathogen that can cause infections that range from mild rashes to life-threatening bloodstream infections. The antifungal drug resistance of some Candida strains and the deficiencies in the current methods used to identify infection-causing species call for improvements in treatment and diagnosis of Candida infections. Towards improving therapeutics, the antimicrobial peptide histatin 5 (Hst-5) was studied. Hst-5 is a salivary peptide with strong antifungal activity against C. albicans. However, the pathogen produces a family of secreted aspartic proteases (Saps), some of which can degrade and inactivate Hst-5. Of the family, Sap2 and Sap9 have been previously shown to degrade Hst-5. Variants of Hst-5 with one or two amino acid substitutions were designed and tested to evaluate the effect on the proteolysis of the peptide by the Saps. The results showed that even a single amino acid substitution results in a significant reduction or enhancement of proteolysis by Saps. The K17R and K17L modifications led to significantly enhanced resistance to proteolysis to both Saps. The substitutions also had site-, residue-, and Sap-dependent effects and the proteases have preferences for certain amino acids at the substitution sites, such as leucine at the 13th residue by Sap9. The substitutions affect not only the cleavage at the substitution sites but also the degradation of the peptide as a whole. Some of the modifications (K11R, E16R, E16L) also led to enhanced antifungal activity. Furthermore, combination of two modifications (K11R-K17R) resulted in a peptide with both enhanced proteolytic resistance to Saps and enhanced antifungal activity, demonstrating its potential as an alternative therapeutic. Towards improving diagnostics, a purification-free method to immobilize single-chain variable fragment (scFv) antibodies was developed. Two biotinylation tags—the biotin carboxyl carrier protein or the AviTag minimal sequence—were genetically fused to scFvs to allow for in vivo, site-specific biotinylation by endogenous Escherichia coli biotin ligases during protein expression. scFvs with different frameworks and antigens were all successfully immobilized directly from cell lysates onto streptavidin-coated plates, and the immobilized scFvs maintained their functionality for over 100 days at 4 °C. The simplicity and robustness of this method make it a suitable approach to create diagnostic arrays and could be used to capture and identify Candida infections.en_US
dc.identifierhttps://doi.org/10.13016/M2H12V85R
dc.identifier.urihttp://hdl.handle.net/1903/20154
dc.language.isoenen_US
dc.subject.pqcontrolledChemical engineeringen_US
dc.subject.pquncontrolledbiotinen_US
dc.subject.pquncontrolledCandida albicansen_US
dc.subject.pquncontrolledhistatin 5en_US
dc.subject.pquncontrolledimmobilizationen_US
dc.subject.pquncontrolledscFven_US
dc.subject.pquncontrolledsecreted aspartic proteasesen_US
dc.titlePROTEIN ENGINEERING APPROACHES TO IMPROVING DIAGNOSIS AND TREATMENT OF CANDIDA ALBICANS INFECTIONSen_US
dc.typeDissertationen_US

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