Specific Detection of Acanthamoeba species using Polyclonal Peptide Antibody Targeting the Periplasmic Binding Protein of A. castellanii

Abstract

Acanthamoeba keratitis (AK) is a rare ocular disease, but it is a painful and sight-threatening infectious disease. Early diagnosis and adequate treatment are necessary to prevent serious complications. While AK is frequently diagnosis via several PCR assays or Acanthamoeba-specific antibodies, a more specific and effective diagnostic method is required. This study described the production of a polyclonal peptide antibody against the periplasmic binding protein (PBP) of A. castellanii and investigated its diagnostic potential. Western blot analysis showed that the PBP antibody specifically reacted with the cell lysates of A. castellanii. However, the PBP antibody did not interact with human corneal epithelial (HCE) cells and the other 3 major causative agents of keratitis. Immunocytochemistry (ICC) results revealed the specific detection of A. castellanii trophozoites and cysts by PBP antibodies when A. castellanii were co-cultured with HCE cells. PBP antibody specificity was further confirmed by co-culture of A. castellanii trophozoites with F. solani, S. aureus, and P. aeruginosa via ICC. The PBP antibody specifically reacted with the trophozoites and cysts of A. polyphaga, A. hatchetti, A. culbertsoni, A. royreba, and A. healyi, thus demonstrated its genus-specific nature. These results showed that the PBP polyclonal peptide antibody of A. castellanii could specifically detect several species of Acanthamoeba, contributing to the development of an effective antibody-based AK diagnostics.

Keywords: Acanthamoeba keratitis; peptide antibody; periplasmic binding protein; species specificity.

Fig. 1

Analysis of amino acid sequence and 3-dimensional conformation of periplasmic binding protein (PBP). (A) Multiple amino acid sequence alignment was produced using the CLUSTAL_X version 2.1. Aligned PBP amino acid sequences of A. castellanii (Ac_PBP, QVH35977.1), F. albosuccineum (Fa_PBP, KAF4442825.1), S. aureus (Sa_PBP, BBA23260.1), and P. aeruginosa (Pa_PBP, KJJ10303.1) were compared. Conserved regions were denoted with black shading and the boxed sequence was used to raise the anti-PBP polyclonal peptide antibody. (B) The 3-dimensional structure of PBP was predicted by RoseTTAFold software.

Fig. 2

Specificity of the anti-periplasmic binding protein (PBP) of A. castellanii was determined by western blot analysis using cell lysates from different organisms. Lane 1; HCE cells, Lane 2; A. castellanii, Lane 3; F. solani, Lane 4; S. aureus, Lane 5; P. aeruginosa.

Fig. 3

Immunocytochemical staining using anti-PBP antibodies. Human corneal epithelial (HCE) cells and A. castellanii trophozoites (A) and cysts (B) were co-cultured. F. solani, S. aureus, and P. aeruginosa were inoculated into the cultures (A. castellanii trophozoites and HCE cells) and incubated for 1 h (C). The co-cultured cells were observed under a fluorescent microscope. Bright-field, DAPI staining (blue), PBP antibody combined with CFL488-conjugated secondary antibody (green), and merged images were acquired at 400× magnification.