Molecular manipulation and new antimicrobial identification in Acanthamoeba spp

  • Alemao Carpinteyro Sanchez

Student thesis: Doctoral Thesis

Abstract

Acanthamoeba spp. are causative agents of a painful and severe sight-threatening corneal infection that can lead to blindness known as Acanthamoeba keratitis and a subacute disease in the brain which is usually fatal known as granulomatous amoebic encephalitis. Over the last few years, there has been a notorious increase in the number of infections due to Acanthamoeba spp. Poor diagnosis, problems of side effects, toxicity of the current drug treatment and the lack of gene editing tools as potential future therapy contribute to a high mortality rate. Strathclyde Minor Groove Binders (S-MGBs), compounds that bind to the minor groove of the DNA that designed and synthesised at University of Strathclyde were evaluated as potential alternative inhibitors against Acanthamoeba infections. Through cell viability microplate alamarBlue assays 42 S-MGBs were screened from which S-MGB 235 showed the most potent inhibitory effect with IC50 in the nanomolar range against five Acanthamoeba isolates after 24 h and 96 h incubation. Confocal microscopy of trophozoites labelled with fluorescent S-MGB 363 (analogue of S-MGB235) showed this compound in the nucleus, nucleolus and distributed over the granuloplasm causing cell lysis, supporting the potent effect observed in vitro by S-MGB 235. Furthermore, conditions were standardised to establish Galleria mellonella larvae as a new in vivo infection model for A. castellanii Neff infections to assess the efficacy and toxicity of voriconazole, miltefosine and S-MGB 235. Voriconazole and miltefosine did not protect larvae from trophozoite infection, however S-MGB 235 significantly protected larvae when compared with the negative control. It was attempted to establish a CRISPR-Cas9 system for gene editing in Acanthamoeba. The plasmids pBRFPT7NeoCas9 and pBRFPT7PhleoCas9 (containing genes for the red fluorescent protein, T7 RNA polymerase, Cas9 along with the neomycin and phleomycin resistance genes, respectively, were constructed and transfected into A. castellanii Neff trophozoites using Xfect. Expression of RFP was confirmed by fluorescence microscopy and fluorescence-activated cell sorting.
Date of Award13 Jan 2022
Original languageEnglish
Awarding Institution
  • University Of Strathclyde
SponsorsUniversity of Strathclyde
SupervisorCraig Roberts (Supervisor) & Martin Wiese (Supervisor)

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