Investigation of the antimycobacterial activity of fungal endophytes and their magnetic dendrimer stabilized nanoparticles against mycobacteria

Date
2024-03
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Stellenbosch : Stellenbosch University
Abstract
ENGLISH ABSTRACT: Tuberculosis (TB), is a disease caused by a single infectious agent Mtb, remains a global health threat that claimed the lives of 1.4 million people worldwide between 2020- 2021. Despite current anti tuberculosis treatment, TB remains one of the leading causes of infection highlighting the imminent need for drug discovery of alternative and novel treatments with distinct modes of action against Mycobacteria. In this context, the present study investigated the antimycobacterial activities of fungal endophyte metabolites functionalized on magnetic dendrimer stabilized nanoparticles (DSNPs) against mycobacterial models. To achieve the aim, fungal endophytes were isolated from four Fynbos families (Asteraceae, Lamiaceae, Ericaceae, and Droseraceae), sequenced and identified by phylogeny. Methanolic crude extracts obtained from cultivated fungi were tested for antimycobacterial activity against Mycobacterium tuberculosis H37Rv (Mtb H37Rv) and Mycobacterium smegmatis mc2155 (Msmeg mc2155). Metabolites within the bioactive fungal extracts were identified by untargeted liquid chromatography-mass spectrometry (LC-QTOF-MS/MS). The infection of THP-1 macrophage cells with Msmeg mc2155 was performed to determine the intracellular antimycobacterial activity of fungal crude extracts. The DSNPs were synthesized from modified magnetic iron oxide nanoparticles (MIONs) and G3-dendrimer micelles. Microscopic and spectral techniques (SEM, FTIR, UV-Vis, and ICP-AES) were used to characterize nanoparticles, followed by antimycobacterial activity assays. Twenty unique fungal isolates were identified with Penicillium being the abundant genera. Penicillium thomii, Diaporthe leucospermi, Penicillium rubens, Cadophora sp. and Penicillium sp. possess antimycobacterial activity. Cadophora sp. demonstrated the most significant inhibition of Msmeg mc2155 and the bioactivity was enhanced in the host-directed approach against intracellular Msmeg mc2155. The metabolites N-benzyl-1-tetradecanamine, N-benzyl-1-hexadecanamine, asperthecin, meleagrin, roquerfortine F, chlamydocin and neoxaline, (-)5-methylmellein, and 9-octadecenamide were identified with the latter two possessing antimycobacterial activity. The nanoparticles (G3- dendrimer micelles, MIONs and DSNPs) were successfully synthesized. G3-dendrimer micelles and DNSPs inhibited Msmeg mc2155 and the former Mtb H37Rv. MIONs possessed enhanced bioactivities against intracellular Msmeg mc2155. Functionalization of DSNPs with bioactive metabolites (nano metabolites) demonstrated no antimycobacterial activity against Msmeg mc2155 directly but antimycobacterial activity was observed intracellularly. Enhanced activity of nano-metabolites was observed directly against Mtb H37RV. Fungal extracts, nanoparticles and functionalized nanoparticles have distinct mechanisms of antimycobacterial activities against the strains of mycobacteria (Msmeg mc2155 and Mtb H37Rv). This study highlighted that the properties of nanoparticles greatly influence their antimycobacterial activities i.e., the ferrous content of a naked nanoparticle opposed to a modified nanoparticle. Functionalization of a nanoparticle contributes to its modification which has an impact on the antimycobacterial activity. Hence, this necessitates further investigations about nanoparticles, their properties and modifications against mycobacterial models. In addition, the nanoparticles have the potential to be modified to accommodate or scaffold existing anti-Tb drugs and to potentiate antimycobacterial effects through pathogen- and host directed approaches.
AFRIKAANSE OPSOMMING: Tuberkulose (TB), 'n siekte wat deur 'n enkele aansteeklike middel Mycobacterium tuberculosis (Mtb) veroorsaak word, bly 'n wêreldwye gesondheidsbedreiging wat die lewens van 1,4 miljoen mense wêreldwyd tussen 2020 en 2021 geëis het. Ten spyte van huidige behandeling teen TB, bly dit een van die hoofoorsake van infeksie wat dus die dreigende behoefte aan geneesmiddelontdekking van alternatiewe en nuwe behandelings met duidelike maniere van werking teen Mycobacteria beklemtoon. In hierdie konteks het die studie die patogeen- en gasheergerigte antimikobakteriese aktiwiteite van swam-endofietmetaboliete wat op magnetiese dendrimeer-gestabiliseerde nanopartikels (DSNPs) gefunksionaliseer is, ondersoek. Om hierdie doel te bereik, is swam-endofiete uit vier Fynbos-families (Asteraceae, Lamiaceae, Ericaceae en Droseraceae) geïsoleer en volgens filogenie opeengevolg en geïdentifiseer. Metanoliese ru-ekstrakte wat van gekweekte swamme verkry is, is getoets vir antimikobakteriese aktiwiteit teen Mtb H37Rv (Mtb H37Rv) en Mycobacterium smegmatis mc2155 (Msmeg mc2155). Metaboliete binne die bioaktiewe swamekstrakte is geïdentifiseer deur onbedoelde vloeistofchromatografie-massaspektrometrie (LC-QTOF-MS/MS). Die infeksie van THP-1 makrofaag selle met Msmeg mc2155 is uitgevoer om die intrasellulêre antimikobakteriese aktiwiteit van swam ru ekstrakte te bepaal. Die DSNPs is gesintetiseer uit gemodifiseerde magnetiese ysteroksied nanopartikels (MIONs) en G3-dendrimeer-miselle. Mikroskopiese en spektrale tegnieke (SEM, FTIR, UV-Vis en ICP-AES) is gebruik om nanopartikels te karakteriseer, gevolg deur patogeen- en gasheergerigte antimikobakteriële aktiwiteittoetse. Twintig unieke swam-isolate is geïdentifiseer met Penicillium as die volop genera. Penicillium thomii, Diaporthe leucospermi, Penicillium rubens, Cadophora sp. en Penicillium sp. beskik oor antimikobakteriese aktiwiteit. Cadophora sp. het die mees betekenisvolle inhibisie van Msmeg mc2155 getoon en die bioaktiwiteit is verbeter in die gasheergerigte benadering teen intrasellulêre Msmeg mc2155. Die metaboliete N-bensiel-1-tetradekanamien, N-bensiel-1- heksadekanamien, asperthecin, meleagrien, roquerfortine F, chlamydocin en neoxaline, (-)5- methylmelleïn, en 9-oktadeceenamied is geïdentifiseer met laasgenoemde twee wat antimikobakteriële aktiwiteit besit. Die nanopartikels (G3-dendrimeer-miselle, MION's en DSNP's) is suksesvol gesintetiseer. G3-dendrimeer-miselle en DNSP's het Msmeg mc2155 en die voormalige Mtb H37Rv geïnhibeer. MIONs beskik oor verbeterde bioaktiwiteite binne die gasheergerigte benadering, dit wil sê teen intrasellulêre Msmeg mc2155. Funksionalisering van DSNPs met bioaktiewe metaboliete (nano metaboliete) het geen antimikobakteriële aktiwiteit direk teen Msmeg mc2155 getoon nie, maar antimikobakteriële aktiwiteit is intrasellulêr waargeneem. Verhoogde aktiwiteit van nano-metaboliete is direk teen Mtb H37RV waargeneem. Swamekstrakte, nanopartikels en gefunksionaliseerde nanopartikels het duidelike meganismes van antimikobakteriese aktiwiteite teen die stamme van mikobakterieë (Msmeg mc2155 en Mtb H37Rv) (patogeengerigte benadering). Swamekstrakte, nanopartikels en nano-metaboliete beskik oor duidelike antimikobakteriese aktiwiteite teen die intrasellulêre patogeen (gasheergerigte benadering). Hierdie studie het uitgelig dat die eienskappe van nanopartikels hul antimikobakteriese aktiwiteite grootliks beïnvloed, dit wil sê die ysterhoudende inhoud van 'n naakte nanopartikel in teenstelling met 'n gemodifiseerde nanopartikel. Funksionalisering van 'n nanopartikel dra by tot die modifikasie daarvan wat 'n impak op die antimikobakteriese aktiwiteit het. Dit noodsaak dus verdere ondersoeke oor nanopartikels, hul eienskappe en modifikasies teen mikobakteriese modelle. Daarbenewens het die nanopartikels die potensiaal om aangepas te word om bestaande anti-Tb-middels te akkommodeer of te steier en om antimikobakteriese effekte deur patogeen- en gasheergerigte benaderings te versterk.
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Thesis (PhD)--Stellenbosch University, 2024.
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