Acanthamoeba proteases in Pathogensis

Acanthamoeba Proteases & Pathogenesis

Page updated 16/8/02

Proteolytic activity of Acanthamoeba was first reported (I believe) in 1988 in the context of pathogenesis (Ferrante & Bates,1988), although protease activity had been identified prior to its involvement in pathogenesis was known. Collagenase activity was later discovered in culture supernatant in which Acanthamoeba had been cultured (He et al, 1990) in addition to amoeba, but not in cysts (Mitro et al, 1994). All of the Acanthamoeba spp that I have so far isolated have isolated and tested contain a protease active against collagen (see Amoeba methods) that is present in the growth medium presumably having been secreted by the amoebae. The proteases secreted from Acanthamoeba seem to be extremely variable in character with a range of molecular weights being reported as well as a range of physical characteristics (Table 1). Various proteases have described, even from the same strain including serine, cysteine and metalloproteinases (Mitro et al, 1994; Cao et al, 1998; Alfieri et al, 2000). It seems that serine type proteases are especially abundant in secretions from Acanthamoeba, and a subtilisin-like serine proteinase has been cloned from Acanthamoeba healyi (Hong, et al, 2000) .

The fact that all Acanthamoeba strain seem to secrete proteases whither or not they happen to be pathogenic, raises the question of why Acanthamoeba (and other amoeba) secrete proteases. It has been suggested that Acanthamoeba is just a "messy eater" spilling out hydrolytic enzymes from the phagolysosome in the course of membrane turnover (Homan & Bowers, 1984). What ever the purpose in protease secretion, it is certain that the proteases do have the potential to degrade cornea once an infection of Acanthamoeba has been established, and it is believed that the proteolytic activity is a key fact in Acanthamoeba keratitis (see Acanthamoeba pathogenesis). It has been suggested that the cytopathogenic activity found in culture supernatant from Acanthamoeba that seems to work through induction of apoptosis (Alizadeh et al, 1994) is actually the protease .

The pH optima is reported to be acidic (pH 5.0 - 5.5) (Alfiero et al, 2000; Seo et al, 1991).

Acanthamoeba strain	Mol.Wt. kDa	Type	Name	Reference and comments.
A. castellanii MEEI 0184	230 97 80 55	Serine Serine Metaloprotease Serine	P1 P2 P3 P4	Cao et al, 1998. Proteases were identified by zymography and so there is a question mark over the molecular weights. The strain was designated "A. castellanii" by morphological characteristics and so must await further classification. P3 only appeared if the amoeba were cultured with epithelial cells.
A. polyphaga ATCC30461	66 49 24 22	Serine		Mitro et al, 1994. Proteases were identified by zymography.
A. polyphaga ATCC30461	110 100 75 60 47	Serine		Alfieri et al, 2000. Again by zymography. A range of cysteine proteases (130-43 kDa) were also detected but oligomerization of proteases was suspected.
A. castellanii	12	Serine		Na et al, 2001. This protease was purified to homogeniety and found to have a genuine mol.wt of 12 kDa by SDS-PAGE.
A. healyi	33	Serine	AhSUB AF221523 AF282597	Hong et al, 2000. The first Acanthamoeba protease to be cloned and sequenced (that I know of?) Genbank numbers left.

Table 1

Zymography - A convenient method to study proteases

Most studies of the proteases of Acanthamoeba have used gelatin containing gel systems (Zymograms). This method is attractive since it is rapid easy to perform, sensitive and cheap. However, this method produces very different results from lab to lab (see table 1) and the determination of the molecular weight, (the main identifying feature) is suspect.

I have attempted to use zymograms to classify Acanthamoeba. Several differences in mobility are obvious between strains but if these can be used in all or indeed any case remains to be ascertained. One especially good candidate appears to be A. palestinensis a fairly infrequently identified species, that has an especially distinctively mobile protease activity. I have identified another isolate (GTG) from Edinburgh, Scotland that has a similar pattern.

References:-

Alfieri, S.C., Correia, C.E.B., Motegi, S.A., & Pral, E.M.F. (2000). "Proteinase activities in total extracts and in medium conditioned by Acanthamoeba polyphaga trophozoites." J.Parasitol. 86, 220-227.

Alizadeh, H., Pidherney, M. S., McCulley, J. P. & Niederkorn, J. Y. (1994) Apoptosis as a mechanism of cytolysis of tumor cells by a pathogenic free-living amoeba, Infect.Immun. 62, 1298-1303.

Cao, Z., Jefferson, D. M. & Panjwani, N. (1998) Role of carbohydrate-mediated adherence in cytopathogenic mechanisms of Acanthamoeba, J.Biol.Chem. 273, 15838-15845.

Ferrante, A. & Bates, E. J. (1988) Elastase in the pathogenic free-living amoebae Naegleria and Acanthamoeba, Infect.Immun. 56, 3320-3321.

He, Y.-G., Niederkorn, J. Y., McCulley, J. P., Stewart, G. L., Meyer, D. R., Silvany, R. & Dougherty, J. (1990) In vivo and in vitro collagenolytic activity of Acanthamoeba castellanii, In.Oph.Vis.Sci. 31, 2235-2240.

Hohman, T. C. & Bowers, B. (1984) Hydrolase secretion is a consequence of membrane recycling, J.Cell Biol. 98, 246-252.

Hong, C.-Y., Kong, H.-H., Ock, M.-S., Kim, I.-S., & Chung, D.-I. (2001). "Isolation and characterization of a cDNA encoding a subtilisin-like serine proteinase (AhSUB) from Acanthamoeba healyi". Mol.Biochem.Parasitol. 111, 441-446.

Mitro, K., Bhagavathiammai, A., Zhou, O.-M., Bobbett, G., McKerrow, J. H., Chokshi, R., Chokshi, B. & James, E. R. (1994) Partial characterization of the proteolytic secretions of Acanthamoeba polyphaga, Exp.Parsitol. 78, 377-385.

Na, B.-K., Kim, J.-C. & Song, C.-Y. (2001) Characterization and pathogenetic role of proteinase from Acanthamoeba castellanii., Microbial Pathogenesis. 30, 39-48.

Seo, J.-H., Yong, T.-S. & Im, K.-i. (1991) Biochemical studies on proteinase in Acanthamoeba culbertsoni, Yonsei Rep.Trop.Med. 22, 21-28.