The Study Of The ScFv-Phage For Candida Albicanstargeting Therapy

Candida albicans(C.albicans)is the most prevalent fungal pathogen of the human micobiota,which can cause infections that range from superficial infections of the skin to life-threatening systemic infections,leading to mortality rates as high as 81% because of poor treatment options.Currently,even after treatment with classical antifungal drugs,mortality caused by Candida infections remains high,in part because of the rise of drug-resistant fungi,adverse drug reactions,side effects,and the high cost of treatment.Thus,new approaches to control this pathogen are imperative.Filamentous bacteriophage(phage)is an artificially modifiable supramacromolecule,which is surrounded by a cylinder of coat proteins.Compared with conventional medical drugs,phage is suitable for not only advancing novel nanostructures and biomaterials but also developing important fields in biomedicine.To date,phage display technology is a powerful method for selection and generating monoclonal antibodies.In the phage format all gene? molecules are expressed as antibody-fusions and 3-5 copies are displayed on the tip of bacteriophage,which allow the selection of binders with high affinities.In this study,we use two methods to treat against C.albicans.1.Antibody therapyRecently,some evidence suggested that antibodies could be regarded as therapeutic agents against various fungal infections including C.albicans.Specifically targeting virulence factors,such as the secreted proteases and surface antigens specific to dimorphic forms,have been considered as attractive strategies.Secreted aspartyl proteinases(Saps)are a major virulence trait of C.albicans,and among these proteases Sap2 has the highest expression levels.It is possible that antibodies against Sap2 could provide an antifungal effect.In this study,two phages displaying anti-r Sap2 single chain variable fragments(sc Fvs)were screened from human single fold sc Fv libraries,and their potential therapeutic roles were evaluated using a murine model infected by C.albicans.The in vivo efficacies were assessed by mortality rates,fungal burden and histological examination.Overall survival rates were significantly increased while the colony counts and infectious foci were significantly decreased after treatment with the sc Fv-phages relative to the control groups.In order to investigate the immune response provoked by sc Fv-phages,three kinds of cytokines(Th1,Th2 and Th17 types)were measured and a clear immmue response was observed.Th1 and Th17 cytokines were significantly increased in mice treated with anti-r Sap2 sc Fv-phage,which meant the cellular immunoresponse may represent one of the therapeutic mechanisms in vivo.2.Photodynamic therapyNowadays,photodynamic therapy(PDT)is an emerging alternative to treat infections based on the usage of visible light and photosensitisers(PSs),in which pheophorbide a(PPa)is a chlorophyll-based photosensitizer that could induce cell death after light irradiation.In this study,a sc Fv phage with high affinity to MP65 was screened from the human single fold sc Fv libraries and acted as a binding target for C.albicans cells,and then it was integrated into PPa to generate a combined nano-scale material.After PDT,the growth of C.albicans was inhibited by PPa-phage as well as the induction of apoptosis.Scanning electron microscopy(SEM)analysis revealed the structure of C.albicans was shrank and ruptured.Furthermore,the mechanism of the antifungal activity was delineated.Depolarization of mitochondrial membrane potential was decreased and intracellular reactive oxygen species(ROS)levels were elevated significantly.In addition,PPa-phage could also lead to S phase arrest.Finally,metacaspase activation resulting from mitochondrial dysfunction was further confirmed to be involved in the C.albicans apoptosis.In conclusion,PPa-phage induced C.albicans apoptosis through a caspase-dependent pathway and the results herein will shed lights on the potential application of phtototheraputic nanostructures in fungal therapy.Therefore,these novel therapeutic materials combine the advantages of bacteriophage and single chain variable fragments,to provide a potential candidate for the therapy of systemic infection caused by C.albicans.Otherwise,it can be concluded that the combination of photosensitizers-PPa and targeting agents,phage,could improve the anti-fungal capabilities of the newly synthesized difunctional anti-fungi drugs,which will broaden the applicability of nanophototheraputics.