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.