Table 1.
Identification of cutinase amino acids as determined by BLASTP.
Fig 1.
Amino acid sequence alignments of cutinases from various organisms.
The aligned cutinase amino acid sequences of Fusarium solani (F.s_cut, AAA33334.1), Aspergillus fumigatus (A.f_cut, XP_755775.1), Pseudomonas aeruginosa (P.a_cut, AXL68988.1), and Staphylococcus aureus (S.a_cut, RZI07573.1) were compared. Multiple sequence alignment was performed using ClustalX software, with sequence conservation visualized through differential shading. A red boxed sequence from this alignment was employed in the production of the anti-cutinase polyclonal antibody.
Fig 2.
Antibody titration of F. solani-specific anti-cutinase polyclonal antibody.
The cutinase antibody was titrated through serial dilutions ranging from 1:5 to 1:5,000, using cell lysates (A-E) and conditioned media (F-J) from various organisms: A and F represent F. solani, B and G represent A. castellanii, C and H represent HCE cells, D and I represent P. aeruginosa, and E and J represent S. aureus. Positive groups (bold black line) consisted of immune mouse sera, while negative controls (dotted black line) consisted of naïve mouse sera. Statistical significance between the absorbance values of positive and negative sera at respective dilutions was indicated by asterisks (*** P < 0.001 and **** P < 0.0001).
Fig 3.
Assessing the sensitivity of F. solani anti-cutinase polyclonal antibody.
The sensitivity of the cutinase antibody was assessed using serial dilutions of cell lysates (A-E) and conditioned media (F-J), ranging from 100 to 0.001 μg/ml. Positive groups (bold black line) consisted of immune mouse sera, while negative controls (dotted black line) consisted of naïve mouse sera. Specifically, A and F correspond to F. solani, B and G to A. castellanii, C and H to HCE cells, D and I to P. aeruginosa, and E and J to S. aureus. Statistical significance between the absorbance values of positive and negative sera at respective dilutions was indicated by asterisks (*** P < 0.001 and **** P < 0.0001).
Fig 4.
Immunocytochemistry using the F. solani cutinase antibody.
HCE cells were co-cultured with F. solani, A. castellanii, P. aeruginosa, and S. aureus. The white arrow indicates A. castellanii, the yellow arrow indicates HCE cell, and the red arrows indicate F. solani. The co-cultured cells were subsequently incubated with a cutinase-specific antibody followed by a CFL488-labeled anti-mouse IgG secondary antibody (green). Acanthamoeba and HCE cell were counterstained with DAPI (blue) prior to fluorescence microscopy. Bright-field, DAPI, cutinase-antibody, and merged images were captured at 400X magnification. The black scale bar represents 10 μm.
Fig 5.
Detection of Fusarium antigens in FK mouse model samples using cutinase antibody.
Fungal keratitis was induced in BALB/c mice by exposing the scratched cornea of each mouse to contact lenses infected with F. solani for 10 days. Compared to naïve mice, typical inflammation and symptoms of keratitis were observed in Fusarium-infected mice (A). Fusarium antigens were detected by ELISA assay using cutinase-specific antibodies in both tear-wash samples (B) and sonicated eyeballs in PBS (C). Negative controls (□) correspond to naïve tear-wash samples, and positive groups (■) correspond to FK-mouse tear-wash samples. Similarly, negative controls (○) correspond to naïve eyeball lysates, and positive groups (●) correspond to FK-mouse eyeball lysates. Asterisks indicate statistical significance between the means (*** P < 0.001 and **** P < 0.0001).