Molecular manipulation and new antimicrobial identification in Acanthamoeba spp

Date on title page is date of submission (November 2021). Date of award is 2022.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.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

Characterization of Acanthamoeba castellanii recombinant calreticulin and evaluation as a potential immunodiagnostic

Acanthamoeba é uma ameba de vida livre, capaz de causar raras e graves infecções oportunistas em olhos, pele e sistema nervoso de humanos e animais. É um protozoário ubiquitário e frequentemente isolado do ambiente. Pode infectar humanos através do uso de lentes de contato, cortes ou feridas na pele assim como ser inalada e conduzida aos pulmões. Possui duas formas em seu ciclo de vida: trofozoíto móvel e infectiva capaz de causar ceratite assim como uma fatal encefalite e de cisto resistente ao ambiente e ao ataque do sistema imune, facilitando a ocorrência da infecção. Existem vários fatores que contribuem na patogênese de Acanthamoeba, sendo a proteína de união à Manose (MBP) a única proteína de superfície descrita como principal fator de patogenicidade. Não existem trabalhos sobre o papel funcional ou patogênico de outras proteínas de superfície que poderiam ser relevantes na invasão ou infecção do hospedeiro por esta ameba. O objetivo geral deste estudo é identificar e analisar uma proteína de superfície de Acanthamoeba castellanii e avaliar seu potencial para utilização em diagnóstico da ceratite amebiana ou encefalite granulomatosa. Predições in silico deste estudo demostram que a calreticulina é uma proteína de superfície. A clonagem da sequência codificadora da calreticulina por recombinação homóloga in vivo foi realizada no vetor pGEX4T1 para permitir a expressão em Escherichia coli BL21 pLysE da proteína recombinante em fusão com a Glutariona-STransferase nas condições de 14°C por 16 horas tendo um rendimento final de 3,64 mg/L de proteína purificada. O potencial imunodiagnóstico foi avaliado através de ELISA usando soros de pacientes com encefalite e soros de rato com ceratite e encefalite testando como antígeno a proteína recombinante. A proteína foi reconhecida pelos soros de pacientes infectados e saudáveis, diferentemente dos soros de ratos, em que só foi reconhecida pelos ratos infectados. Estes resultados preliminares apontam que a proteína calreticulina de A. castellanii poderia ser um candidato para imunodiagnóstico das doenças causadas pelo protozoário em ratos; no caso dos resultados em pacientes humanos ainda são necessárias maiores investigações.Acanthamoeba is a microscopic free-living amoeba able to cause rare and serious opportunistic infections in eyes, skin and nervous system in humans and animals. It’s one of the most common protist widely distributed and it has been isolated from the environment, including water and soil. The amoeba can infect contact-lenses wearers through skin lesions or via the nasal route. Acanthamoeba exists in two distinct forms: an active-infective trophozoite form during which Acanthamoeba reproduces being capable of cause Acanthamoeba keratitis and a fatal granulomatous amoebic encephalitis, and a dormant cyst form resistant to immune system defense making easier the recurrence of these infections. Several factors contribute with the pathogenesis of Acanthamoeba. The mannose bindingprotein (MBP) is the only surface protein as a main pathogenicity factor in Acanthamoeba; however, there are no evidence of any scientific work that describes the functional nor pathogenic role of another surface protein that could be relevant in the host-parasite invasion or infection by this amoeba. The general objective of this study is identify and analyze an Acanthamoeba castellanii surface protein and test its potential for use in diagnosis of amoebic keratitis or granulomatous encephalitis. In silico predictions showed that, A. castellanii calreticulin is a surface protein. In vivo homologous recombination cloning of the coding sequence was performed using the pGEX4T1 vector for expressing the GST fusion recombinant protein using an Escherichia coli BL21 pLysE strain at 14°C for 16 hours obtaining a final efficiency of 3,64 mg/L of purified protein. The immunodiagnostic potential was tested with specific antibody responses in serum from patients with encephalitis and infected rats with keratitis and encephalitis against recombinant calreticulin by ELISA. The protein was recognized by both infected and healthy patients serum, whereas the infected rat serum was the only recognized by the recombinant protein. These results would conclude that A. castellanii calreticulin probably could be an immunodiagnostic prospect of Acanthamoeba diseases in animals but in human further immunoassays are required

Characterization of Acanthamoeba castellanii recombinant calreticulin and evaluation as a potential immunodiagnostic

Acanthamoeba é uma ameba de vida livre, capaz de causar raras e graves infecções oportunistas em olhos, pele e sistema nervoso de humanos e animais. É um protozoário ubiquitário e frequentemente isolado do ambiente. Pode infectar humanos através do uso de lentes de contato, cortes ou feridas na pele assim como ser inalada e conduzida aos pulmões. Possui duas formas em seu ciclo de vida: trofozoíto móvel e infectiva capaz de causar ceratite assim como uma fatal encefalite e de cisto resistente ao ambiente e ao ataque do sistema imune, facilitando a ocorrência da infecção. Existem vários fatores que contribuem na patogênese de Acanthamoeba, sendo a proteína de união à Manose (MBP) a única proteína de superfície descrita como principal fator de patogenicidade. Não existem trabalhos sobre o papel funcional ou patogênico de outras proteínas de superfície que poderiam ser relevantes na invasão ou infecção do hospedeiro por esta ameba. O objetivo geral deste estudo é identificar e analisar uma proteína de superfície de Acanthamoeba castellanii e avaliar seu potencial para utilização em diagnóstico da ceratite amebiana ou encefalite granulomatosa. Predições in silico deste estudo demostram que a calreticulina é uma proteína de superfície. A clonagem da sequência codificadora da calreticulina por recombinação homóloga in vivo foi realizada no vetor pGEX4T1 para permitir a expressão em Escherichia coli BL21 pLysE da proteína recombinante em fusão com a Glutariona-STransferase nas condições de 14°C por 16 horas tendo um rendimento final de 3,64 mg/L de proteína purificada. O potencial imunodiagnóstico foi avaliado através de ELISA usando soros de pacientes com encefalite e soros de rato com ceratite e encefalite testando como antígeno a proteína recombinante. A proteína foi reconhecida pelos soros de pacientes infectados e saudáveis, diferentemente dos soros de ratos, em que só foi reconhecida pelos ratos infectados. Estes resultados preliminares apontam que a proteína calreticulina de A. castellanii poderia ser um candidato para imunodiagnóstico das doenças causadas pelo protozoário em ratos; no caso dos resultados em pacientes humanos ainda são necessárias maiores investigações.Acanthamoeba is a microscopic free-living amoeba able to cause rare and serious opportunistic infections in eyes, skin and nervous system in humans and animals. It’s one of the most common protist widely distributed and it has been isolated from the environment, including water and soil. The amoeba can infect contact-lenses wearers through skin lesions or via the nasal route. Acanthamoeba exists in two distinct forms: an active-infective trophozoite form during which Acanthamoeba reproduces being capable of cause Acanthamoeba keratitis and a fatal granulomatous amoebic encephalitis, and a dormant cyst form resistant to immune system defense making easier the recurrence of these infections. Several factors contribute with the pathogenesis of Acanthamoeba. The mannose bindingprotein (MBP) is the only surface protein as a main pathogenicity factor in Acanthamoeba; however, there are no evidence of any scientific work that describes the functional nor pathogenic role of another surface protein that could be relevant in the host-parasite invasion or infection by this amoeba. The general objective of this study is identify and analyze an Acanthamoeba castellanii surface protein and test its potential for use in diagnosis of amoebic keratitis or granulomatous encephalitis. In silico predictions showed that, A. castellanii calreticulin is a surface protein. In vivo homologous recombination cloning of the coding sequence was performed using the pGEX4T1 vector for expressing the GST fusion recombinant protein using an Escherichia coli BL21 pLysE strain at 14°C for 16 hours obtaining a final efficiency of 3,64 mg/L of purified protein. The immunodiagnostic potential was tested with specific antibody responses in serum from patients with encephalitis and infected rats with keratitis and encephalitis against recombinant calreticulin by ELISA. The protein was recognized by both infected and healthy patients serum, whereas the infected rat serum was the only recognized by the recombinant protein. These results would conclude that A. castellanii calreticulin probably could be an immunodiagnostic prospect of Acanthamoeba diseases in animals but in human further immunoassays are required