Download - Supported by grant from Brazilian Scientific Foundations: FAPESP (08/53969-0), CAPES, CNPq
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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.
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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.
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Proteases represent a class of enzymes which are related with pathogenicity and cytolysis of Acanthamoeba species and genotypes.
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To evaluate the expression of extracellular proteins by Acanthamoeba trophozoites as predictive virulence factors in the corneal infection by the protozoa.
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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)
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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
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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
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Lane 8:
Single pattern
Lanes 11 and 12:
High diversity
36
55
76
96
117
141
KDa
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Low molecular weight protein
AK: role in virulence.
Collagenases
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Heterogeneous extracellular collagenases are produced by different Acanthamoeba isolates;
Avirulent Acanthamoeba strain could not produce collagenolytic enzymes;
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Collagenases should be suggested as a virulence marker in the differentiation of Acanthamoeba species and genotypes.
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The genotyping and phylogenetic inferences of clinical isolates are currently under investigation;
Collagenolytic enzymes are under isolation by chromatographic techniques.