Entamoeba histolytica: Collagenolytic Activity and Virulence1

Several axenic strains of pathogenic and nonpathogenic Entamoeba histolytica were tested for their capacity to digest native radioactive type I collagen gels and to produce liver abscesses when injected into the liver of newborn hamsters. The results demonstrate that the pathogenic strains of amebas (HM1:IMSS, HM3:IMSS, HM38:IMSS, and HK9) have a collagenolytic activity that closely correlates with their in vivo capacity to produce liver lesions. The nonpathogenic isolate (Laredo) did not show collagenolytic activity and failed to produce lesions in the liver of newborn hamsters. The results also demonstrate that type I collagen obtained from rodents and cats is degraded less by amebic collagenase than is bovine collagen, which is similar to human collagen. These findings suggest that species susceptibility to invasive infection may depend, among other factors, on the characteristics of the extracellular components of host tissues.     

Entamoeba histolytica: electron-dense granule secretion, collagenase activity and virulence are altered in the cytoskeleton mutant BG-3

HM-1:IMSS, a pathogenic strain of Entamoeba histolytica, and its mutant BG-3, Identified by resistance to cytochalasin D, were tested for their capacity to: (i) secrete electron-dense granules; (ii) adhere and digest native type I collagen gels; and (iii) produce liver abscesses in new-born hamsters. The results demonstrate that the mutant has low adherence to collagen, low electron-dense granule secretion and collagenolytic activity, and low capacity to produce liver lesions in vivo, compared with the parental strain HM1:IMSS.     

Entamoeba histolytica: virulence potential and sensitivity to metronidazole and emetine of four isolates possessing nonpathogenic zymodemes

The pathogenic potential of four Entamoeba histolytica isolates obtained from asymptomatic carriers and possessing nonpathogenic zymodemes was compared to four E. histolytica strains obtained from invasive cases of amebiasis and having pathogenic zymodemes. Both xenic and axenic cultures of a number of strains were tested. Determinations of cytopathogenicity were done in vitro by measuring the rates of destruction of tissue cultured monolayers of baby hamster kidney cells by intact amebae or by its cell-free extracts. The in vivo virulence was tested by assessing their capacity to form hepatic abscesses in hamsters or cecal ulcerations in rats. The results obtained show that two of the isolates from asymptomatic carriers (strains SAW 1734R clAR and WI:0385:191) were as virulent as three of the invasive ones (HM-1:IMSS, 200:NIH, and SAW 408). Two other isolates from asymptomatic carriers and one from a dysentery case were avirulent. All the E. histolytica isolates tested were similarly sensitive to metronidazole and emetine (IC50 1-10 micrograms/ml). The results indicate that the pathogenic potential of E. histolytica varies between isolates and can be affected by culture conditions and by the presence or absence of bacterial cells. These findings suggest that virulence does not necessarily correlate with a pathogenic zymodeme.     

Pulmonary fibroblasts mobilize the membrane-tethered matrix metalloprotease, MT1-MMP, to destructively remodel and invade interstitial type I collagen barriers

In acute and chronic lung disease, widespread disruption of tissue architecture underlies compromised pulmonary function. Pulmonary fibroblasts have been implicated as critical effectors of tissue-destructive extracellular matrix (ECM) remodeling by mobilizing a spectrum of proteolytic enzymes. Although efforts to date have focused on the catabolism of type I collagen, the predominant component of the lung interstitial matrix, the key collagenolytic enzymes employed by pulmonary fibroblasts remain unidentified. Herein, membrane type-1 matrix metalloprotease (MT1-MMP) is identified as the dominant and direct-acting protease responsible for the type I collagenolytic activity mediated by both mouse and human pulmonary fibroblasts. Furthermore, MT1-MMP is shown to be essential for pulmonary fibroblast migration within three-dimensional (3-D) hydrogels of cross-linked type I collagen that recapitulate ECM barriers encountered in the in vivo environment. Together, these findings demonstrate that MT1-MMP serves as a key effector of type I collagenolytic activity in pulmonary fibroblasts and earmark this pericellular collagenase as a potential target for therapeutic intervention.     

Entamoeba histolytica: cytopathogenicity and lectin activity of avirulent mutants

Three clones of Entamoeba histolytica (L-6, C93, C919) were isolated by mutagenesis with ethyl methanesulfonate from the axenic strain HM1:IMSS and were studied for adherence, cytolytic, and soluble galactose inhibitable lectin activity. Avirulent clones adhered to and killed fewer Chinese hamster ovary cells than HM1:IMSS (P less than 0.01). However, only C919 was deficient in adherence to red blood cells. Galactose (1.0 g) completely inhibited adherence of all the mutants to Chinese hamster ovary cells; however, adherence to erythrocytes was only partially inhibitable by galactose. Avirulent mutants were more susceptible to being killed by human neutrophils in vitro (P less than 0.01 compared to HM1:IMSS). Soluble protein preparations from all the avirulent mutants were markedly less mitogenic for human lymphocytes and had lower lectin activity for Chinese hamster ovary cells compared to the HM1:IMSS wild type (P less than 0.01 for each activity with each mutant). Indirect immunofluorescence with a monoclonal antibody (F-14) that recognizes the Gal/GalNAc lectin was positive for L-6 and C919. These findings utilizing avirulent mutants of E. histolytica further support a role for the amebic galactose inhibitable lectin in the in vivo pathogenesis of amebiasis.     

Antisense inhibition of expression of the light subunit (35 kDa) of the Gal/GalNac lectin complex inhibits Entamoeba histolytica virulence

One of the under-represented genes identified by cDNA representational difference analysis (RDA) between avirulent Entamoeba histolytica strain Rahman and virulent strain HM-1:IMSS was the amoebic light (35 kDa) subunit of the Gal/GalNac lectin complex. This lectin complex, which mediates the adhesion of the parasite to the target cell, also contains a heavy (170 kDa) subunit, which has the carbohydrate-binding domain. Stable transfectants of the virulent strain in which the expression of the 35 kDa subunit was inhibited by antisense RNA were not significantly affected in their adhesion activity to mammalian or bacterial cells but were strongly inhibited in their cytopathic activity, cytotoxic activity and in their ability to induce the formation of liver lesions in hamsters. These findings suggest that the 35 kDa subunit may have a specific function in the pathogenic pathway and provides a new insight into the role of this component of the Gal/GalNac lectin complex in amoebic virulence.     

Matrix metalloproteinase-1 takes advantage of the induced fit mechanism to cleave the triple-helical type I collagen molecule

The collagenases are members of the matrix metalloproteinase family (MMP) that degrade native triple-helical type I collagen. To understand the mechanism by which these enzymes recognize and cleave this substrate, we studied the substrate specificity of a modified form of MMP-1 (FC) in which its active site region (amino acids 212-254) had been replaced with that of MMP-9 (amino acids 395-437). Although this substitution increased the activity of the enzyme toward gelatin and the peptide substrate Mca-PLGL(Dpa)AR-NH2 by approximately 3- and approximately 11-fold, respectively, it decreased the type I collagenolytic activity of the enzyme to 0.13%. The replacement of Gly233, the only amino acid in this region of FC that is conserved in all collagenase family members, with the corresponding Glu residue in MMP-9 resulted in a substantial decrease in the type I collagenolytic activity of the enzyme without affecting its general proteolytic activities. The kinetic parameters of the FC/G233E mutant for the collagen substrate were similar to those of the chimeric enzyme. In addition, substituting Gly233 for Glu in the chimera increased the collagenolytic activity of the enzyme by 12-fold. Interestingly, replacing Glu415 in MMP-9 with Gly, its corresponding residue in FC, endowed the enzyme with type I collagenolytic activity. The catalytic activity of the MMP-9 mutant toward triple-helical type I collagen was 2-fold higher than that of the collagenase chimera. These data in conjunction with the X-ray crystal structure of FC indicate that Gly233 provides the flexibility necessary for the enzyme active site to change conformation upon substrate binding. The flexibility provided by the Gly residue is essential for type I collagenolytic activity.     

Structural and mechanistic insights into collagen degradation by a bacterial collagenolytic serine protease in the subtilisin family

A number of proteases in the subtilisin family derived from environmental or pathogenic microorganisms have been reported to be collagenolytic serine proteases. However, their collagen degradation mechanisms remain unclear. Here, the degradation mechanism of type I collagen fibres by the S8 collagenolytic protease MCP‐01, from Pseudoalteromonas sp. SM9913, was studied. Atomic force microscopy observation and biochemical analysis confirmed that MCP‐01 progressively released single fibrils from collagen fibres and released collagen monomers from fibrils mainly by hydrolysing proteoglycans and telopeptides in the collagen fibres. Structural and mutational analyses indicated that an enlarged substrate‐binding pocket, mainly composed of loops 7, 9 and 11, is necessary for collagen recognition and that the acidic and aromatic residues on these loops form a negatively charged, hydrophobic environment for collagen binding. MCP‐01 displayed a non‐strict preference for peptide bonds with Pro or basic residues at the P1 site and/or Gly at the P1’ site in collagen. His211 is a key residue for the P1‐basic‐residue preference of MCP‐01. Our study gives structural and mechanistic insights into collagen degradation of the S8 collagenolytic protease, which is helpful in developing therapeutics for diseases with S8 collagenolytic proteases as pathogenic factors and in studying environmental organic nitrogen degradation mechanisms.     

Entamoeba histolytica: generation and characterization of hybrid clones

Transference of DNA to Entamoeba histolytica was carried out by polyethylene glycol fusion of two amebic clones with different phenotypes. Clone C9, strain HM1:IMSS, was the donor. It is emetine-resistant, highly phagocytic and virulent, and grows in soft agar. Clone L6, strain HM1:IMSS, the recipient, is emetine-sensitive, phagocytic, and virulence-deficient, and it does not grow in soft agar. Clones L6 and C9 have shown high stability in their virulence phenotypes since their isolation more than 5 years ago. Before fusion experiments, clone C9 was incubated in 20 micrograms/ml bromodeoxyuridine for 24 hr, and then, irradiated with 310 nm light to complete inactivation. Controls ensured that all irradiated trophozoites died after 24 hr of incubation at 37 degrees C. Irradiated C9 trophozoites were fused with L6 trophozoites, and hybrids were selected by their ability to grow in the presence of emetine. All hybrids, independently generated, grew poorly in soft agar and showed both an intermediate emetine-resistance and rate of phagocytosis. Some of them destroyed efficiently cell monolayers, but interestingly, they showed differences in their ability to produce hepatic abscesses in hamsters.     

Collagenase of hepatocytes and sinusoidal liver cells in the reversibility of experimental cirrhosis of the liver

In order to explore the cellular source(s) and the behaviour of the collagenolytic activity previously described in rat liver homogenates, in the reversibility of experimental cirrhosis of the liver, enriched suspensions of hepatocytes and of sinusoidal liver cells were obtained by a procedure which employs low EDTA concentrations and no bacterial collagenase. Cell suspensions were prepared from three different groups of animals: 1) normal controls, 2) rats with CCl4-induced cirrhosis of the liver, and 3) rats with swine serum-induced cirrhosis of the liver. Animals were sacrificed in each group upon completion of treatment and also after 3, 6 and 12 months. In each liver wet weight and collagen concentration were determined, and collagenolytic activity of both enriched cell suspensions was measured separately. In addition, histological studies of liver tissue and ultrastructural examination of cell suspensions were performed by standard procedures. Enriched suspensions of both normal hepatocytes and sinusoidal liver cells display Ca2(+)-dependent collagenolytic activities. Both cell suspensions obtained from each of the two types of cirrhotic livers show normal or slightly increased average levels of collagenase activity at the time of treatment discontinuation, when average liver collagen content ranges from 6 to 10-fold over normal, suggesting that the normal collagenase/collagen ratio is disturbed and that collagenolytic activity is deeply decreased in relation to the actual liver collagen load.(ABSTRACT TRUNCATED AT 250 WORDS)     

Entamoeba histolytica: Expression and localization of Gal/GalNAc lectin in virulent and non-virulent variants from HM1:IMSS strain

We have purified Gal/GalNAc lectin from Entamoeba histolytica by electroelution. The purified protein was used to immunize rabbits and obtain polyclonal IgG’s anti-lectin. These antibodies were used as tools to analyze the expression and localization of the amoebic lectin in both virulent (vEh) and non-virulent (nvEh) variants of axenically cultured HM1:IMSS strain. vEh is able to induce liver abscesses in hamsters, whereas nvEh has lost this ability. In vitro, amoebic trophozoites from both variants equally express this protein as shown by densitometric analysis of the corresponding band in Western blots from lysates. In both types of trophozoites, the pattern of distribution of the lectin was mainly on the surface. We have also compared by immunohistochemistry the presence and distribution of lectin in the in vivo liver lesions produced in hamsters. In order to prolong the survival of nvEh to analyze both variants in an in vivo model, hamsters inoculated with nvEh were treated with methyl prednisolone. Our results suggest that the Gal/GalNAc lectin is equally expressed in both nvEh and vEh.     

Tumor-associated trypsinogen-2 (trypsinogen-2) activates procollagenases (MMP-1, -8, -13) and stromelysin-1 (MMP-3) and degrades type I collagen

A critical step in cancer growth and metastasis is the dissolution of the extracellular matrix surrounding the malignant tumor, which leads to tumor cell invasion and dissemination. Type I collagen degradation involves the initial action of collagenolytic matrix metalloproteinases (MMP-1, -8, and -13) activated by MMP-3 (stromelysin-1). The role of interactive matrix serine proteinases (MSPs), including tumor-associated trypsinogens, has been unclear in collagenolysis. Now, we provide evidence that the major isoenzyme of human tumor-associated trypsinogens, trypsin-2, can directly activate three collagenolytic proMMPs as well as proMMP-3. These proMMP activations are inhibited by tumor-associated trypsin inhibitor (TATI). Furthermore, we demonstrate that trypsin-2 efficiently degrades native soluble type I collagen, which can be inhibited by TATI. However, cell culture studies showed that trypsin-2 transfection into the HSC-3 cell line did not result in MMP-1, -3, -8, and -13 activation but affected MMP-3 and -8 production at the protein level. These findings indicate that human trypsin-2 can be regarded as a potent tumor-associated matrix serine protease capable of being the initial activator of the collagenolytic MMP activation network as well as directly attacking type I collagen.     

Degradation of collagen substrates by a trypsin-like serine protease from the fiddler crab Uca pugilator

The collagenolytic properties of a trypsin-like protease from the hepatopancreas of the fiddler crab Uca pugilator have been examined. All collagen types, I-V, were attacked by this enzyme. Types III and IV were degraded much more rapidly than types I, II, and V. Crab protease produced multiple cleavages in the triple helix of each collagen at 25 degrees C; only in the case of type III collagen, however, was a major cleavage observed at a 3/4:1/4 locus that corresponded to the region of collagen susceptibility to mammalian collagenase action. Additionally, both the affinity and the specific activity of the crab protease for native collagen were lower than those which characterize mammalian collagenase. The results of this study, in conjunction with a previous report on the collagenolytic activity of another serine protease from the fiddler crab [Welgus, H. G., Grant, G. A., Jeffrey, J. J., & Eisen, A. Z. (1982) Biochemistry 21, 5183], suggest that the following properties distinguish the action of these invertebrate collagenolytic enzymes from the metalloenzyme collagenases of mammals: (1) broad substrate specificity, including both noncollagenous proteins and collagen types I-V; (2) ability to cleave the native triple helix of collagen at multiple loci; (3) reduced affinity or higher Km for collagen; and (4) lower specific activity on collagen fibrils.     

Degradation of type I collagen by rat mucosal keratinocytes. Evidence for secretion of a specific epithelial collagenase

Feeder-cell-independent serially propagating keratinocytes from rat oral mucosa (tongue) dissolved reconstituted type I [3H]collagen fibrils, although rather slowly. Analysis of the conditioned medium from such cultures revealed secretion of a Mr = 65,000 collagenase which remained almost entirely latent in the absence of exogenous protease activity. Addition of trypsin (0.1-1.0 microgram/ml) or plasmin (1.0-4.0 micrograms/ml) resulted in substantial acceleration of the collagenolytic process in stimulated secretion of latent collagenase and, at higher concentrations, in conversion of the latent enzyme to the catalytic form. The keratinocyte collagenase was indistinguishable from interstitial, fibroblast-type collagenases by several criteria including: cleavage of native type I collagen in solution at the characteristic collagenase-sensitive locus at 22 degrees C and dissolution of reconstituted type I collagen fibrils at 35 degrees C; activation by trypsin and by organomercurials and inhibition by Zn2+ and Ca2+ chelators; and cross-reaction with antibody to fibroblast-type procollagenase. Expression of collagenolytic activity in keratinocyte cultures was effectively regulated by cell density. The activity (on a per cell basis) was maximal at 10-20% confluence and was more than 95% "contact-inhibited" at subconfluent and early confluent densities (2-4 X 10(5)/cm2). Our findings show that mucosal keratinocytes possess a potent enzymatic apparatus for degradation of interstitial collagen fibrils which includes a classical vertebrate collagenase.     

Unexpected crucial role of residue 272 in substrate specificity of fibroblast collagenase

Degradation of type I collagen by collagenases is an important part of extracellular remodeling. To understand the role of the hinge region of fibroblast collagenase in its collagenolytic activity, we individually substituted the 10 conserved amino acid residues at positions 264, 266, 268, 296, 272, 277, 284, 289, 307, and 313 in this region of the enzyme by their corresponding residues in MMP-3, a noncollagenolytic matrix metalloproteinase. The general proteolytic and triple helicase activities of all of the enzymes were determined, and their abilities to bind to type I collagen were assessed. Among the mutants, only G272D mutant enzyme exhibited a significant change in type I collagenolysis. The alteration of the Gly(272) to Asp reduced the collagenolytic activity of the enzyme to 13% without affecting its general proteolytic activity, substrate specificity, or the collagen binding ability. The catalytic efficiency of the G272D mutant for the triple helical peptide substrate [C(6)-(GP- Hyp)(4)GPL(Mca)GPQGLRGQL(DPN)GVR(GP-HYP)(4)-NH(2)](3) and the peptide substrate Mca-PLGL(Dpa)AR-NH(2) and its dissociation constant for the triple helical collagen were similar to that of the wild type enzyme, indicating that the presence of this residue in fibroblast collagenase is particularly important for the efficient cleavage of type I collagen. Gly(272) is evidently responsible for the hinge-bending motion that is essential for allowing the COOH-terminal domain to present the collagen to the active site.     

Inhibition of type I collagen film degradation by tumour cells using a specific antibody to collagenase and the specific tissue inhibitor of metalloproteinases (TIMP)

Rabbit VX2 tumour cells in culture produced a collagenolytic activity which was shown to be immunologically identical to collagenase from rabbit articular chondrocytes and bone. VX2 cells degraded type I collagen films spontaneously and did not produce detectable levels of the tissue inhibitor of metalloproteinases (TIMP). Chondrocytes, however, required both stimulation of collagenase synthesis and activation to effect lysis and were observed to make appreciable amounts of TIMP. The degradation of type I collagen films by VX2 tumour cells was significantly inhibited by both a specific antibody to rabbit collagenase and by purified TIMP, thus demonstrating the unequivocal role of collagenase in this model system.