Abstract
Accurate and rapid diagnosis of Acanthamoeba keratitis (AK) is difficult. Although the diagnostic procedure for AK has improved, further development and effective diagnostic tool utilization for AK need to continue. Chorismate mutase is a key regulatory enzyme involved in the shikimate pathway, a metabolic pathway absent in mammals but central for amino acid biosynthesis in bacteria, fungi, algae, and plants. In this study, we describe the identification and production of a polyclonal peptide antibody targeting chorismate mutase secreted by A. castellanii, which could be used for AK diagnosis. Western blot was performed using the protein lysates and conditioned media of the human corneal epithelial (HCE) cells, non-pathogenic Acanthamoeba, pathogenic Acanthamoeba, clinical isolate of Acanthamoeba spp., and other causes of keratitis such as Fusarium solani, Pseudomonas aeruginosa, and Staphylococcus aureus. Polyclonal antibodies raised against A. castellanii chorismate mutase specifically interacted with lysates of Acanthamoeba origin and their culture media, while such interactions were not observed from other samples. Acanthamoeba-specificity of chorismate mutase was also confirmed using immunocytochemistry after co-culturing Acanthamoeba with HCE cells. Specific binding of the chorismate mutase antibody to Acanthamoeba was observed, which were absent in the case of HCE cells. These results indicate that the chorismate mutase antibody of Acanthamoeba may serve as a method for rapid and differential Acanthamoeba identification.
Highlights
Acanthamoeba keratitis (AK) is a painful and sight-threatening infection caused by several free-living amoebae belonging to the genus Acanthamoeba [1]
Secretory proteins isolated from both non-pathogenic (Fig 1A) and pathogenic (Fig 1B) A. castellanii were resolved by SDS-PAGE and compared for analysis
The fulllength open reading frame of chorismate mutase from pathogenic A. castellanii Castellani was identified by polymerase chain reaction (GenBank accession number MN630520)
Summary
Acanthamoeba keratitis (AK) is a painful and sight-threatening infection caused by several free-living amoebae belonging to the genus Acanthamoeba [1]. Early detection and diagnosis of AK could lead to successful treatment, accurately diagnosing AK remains difficult and this has frequently resulted in ocular Acanthamoeba infection being. Current diagnostic methods for AK primarily rely on microbiological culture and microscopic identification, while histochemical staining and PCR-based diagnosis are available [4,5,6,7]. These methods require corneal scrapings that inflict immense pain to the patients during the sample acquisition process and as such, a non-invasive diagnostic method encompassing a high degree of sensitivity and Acanthamoeba specificity is desired
Sign-in/Register to view highlights & summary Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a callDisclaimer: All third-party content on this website/platform is and will remain the property of their respective owners and is provided on "as is" basis without any warranties, express or implied. Use of third-party content does not indicate any affiliation, sponsorship with or endorsement by them. Any references to third-party content is to identify the corresponding services and shall be considered fair use under The CopyrightLaw.
