Download - Supported by grant from Brazilian Scientific Foundations: FAPESP (08/53969-0), CAPES, CNPq
Fabio Carvalho, Linda C Carrijo-Carvalho, Ana Marisa Chudzinski-Tavassi, Annette Foronda, Denise de Freitas
Supported by grant from Brazilian Scientific Foundations: FAPESP (08/53969-0), CAPES, CNPq.
Corneal infections due to free-living amoebae have potentially devastating consequences. If not successfully treated, it can progress into the eye, causing corneal ulcer, loss of visual acuity and eventually blindness.
Proteases represent a class of enzymes which are related with pathogenicity and cytolysis of Acanthamoeba species and genotypes.
To evaluate the expression of extracellular proteins by Acanthamoeba trophozoites as predictive virulence factors in the corneal infection by the protozoa.
The research was conducted in accordance with the tenets of the Declaration of Helsinki Corneal scrapings
10 patients Contact-lens wearers
Acanthamoeba keratitis isolates (n=10)
Dialysis
CultivationNeff’s Broth Media
Acanthamoeba strain
CentrifugationSupernatant collected
SDS-PAGE Collagen2,0 µg of protein/ well
Protein DeterminationBradford Assay
Analysis of extracellular proteases
Secreted in Neff’s media
Dialysis
CultivationNeff’s Broth Media
Acanthamoeba strain
CentrifugationSupernatant collected
SDS-PAGE Collagen2,0 µg of protein/ well
Protein DeterminationBradford Assay
Analysis of extracellular proteases
Secreted in Neff’s media
Lane 1: Negative control
Lane 2: ATCC 30011 avirulent strain
Lanes 3 and 6: Single pattern
Lane 4: High diversity
KDa
109
95
46
36
Lane 8:
Single pattern
Lanes 11 and 12:
High diversity
36
55
76
96
117
141
KDa
Low molecular weight protein
AK: role in virulence.
Collagenases
Heterogeneous extracellular collagenases are produced by different Acanthamoeba isolates;
Avirulent Acanthamoeba strain could not produce collagenolytic enzymes;
Collagenases should be suggested as a virulence marker in the differentiation of Acanthamoeba species and genotypes.
The genotyping and phylogenetic inferences of clinical isolates are currently under investigation;
Collagenolytic enzymes are under isolation by chromatographic techniques.