Decreasing effect of an anti-Nfa1 polyclonal antibody on the in vitro cytotoxicity of pathogenic Naegleria fowleri

The nfa1 gene was cloned from a cDNA library of pathogenic Naegleria fowleri by immunoscreening; it consisted of 360 bp and produced a 13.1 kDa recombinant protein (rNfa1) that showed the pseudopodia-specific localization by immunocytochemistry in the previous study. Based on the idea that the pseudopodia-specific Nfa1 protein mentioned above seems to be involved in the pathogenicity of N. fowleri, we observed the effect of an anti-Nfa1 antibody on the proliferation of N. fowleri trophozoites and the cytotoxicity of N. fowleri trophozoites on the target cells. The proliferation of N. fowleri trophozoites was inhibited after being treated with an anti-Nfa1 polyclonal antibody in a dose-dependent manner for 48 hrs. By a light microscope, CHO cells co-cultured with N. fowleri trophozoites (group I) for 48 hrs showed severe morphological destruction. On the contrary, CHO cells co-cultured with N. fowleri trophozoites and anti-Nfa1 polyclonal antibody (1:100 dilution) (group II) showed less destruction. In the LDH release assay results, group I showed 50.6% cytotoxicity, and group II showed 39.3%. Consequently, addition of an anti-Nfa1 polyclonal antibody produced a decreasing effect of in vitro cytotoxicity of N. fowleri in a dose-dependent manner.     

The protective effects of monoclonal antibodies in mice from Naegleria fowleri infection]

Protective effects of monoclonal antibodies against N. fowleri were comparatively studied. BALB/c mice were treated with two types of monoclonal antibodies, Nf 2 and Nf 154, before and after the infection with N. fowleri. The mortality and mean survival times were then compared. Also, direct effect of the monoclonal antibodies on the N. fowleri trophozoites in vitro were observed. In vitro protective effects of the monoclonal antibodies were also studied in cells infected with N. fowleri. The observed results are summarized as follows: 1. Among mice pretreated twice before the infection with monoclonal antibody Nf 2(McAb Nf 2), only 15.8% were killed, and the mean survival time was 17.7 days. This was not much different from the mice pretreated once, as the mortality and mean survival time were 16.7% and 17 days. Those effects were compatible with monoclonal antibody Nf 154(McAb Nf 154). The above findings contrast with the mortality and mean survival time of the control mice, which were 22.7% and 14.6 days respectively. 2. Mice which received twice the McAb Nf 2 following N. fowleri infection incurred a 19.4% mortality rate with 13.6 days survival time; 17.9% and 15.8 days with on time administration, in contrast to the 25% and 14.6 days in the control group. 3. Marked agglutination effect of McAb Nf 2 or McAb Nf 154 were observed on N. fowleri trophozoites. 4. When N. fowleri trophozoites were treated with McAb Nf 2 or McAb Nf 154 combined with comments, the proliferation rate was more significantly suppressed than in that the control. 5. N. fowleri trophozoites treated with McAb Nf 2 or McAb Nf 154 showed an increased number of swollen mitochondria, disfigured cisternae, lipid droplets, and osmiophilic granules in the cytoplasm. 6. A remarkable protective effect of monoclonal antibodies was noticed in CHO cells infected with N. fowleri. More than 90.6% of the infected CHO cells survived, contrasted with 27% of untreated cells. The overall results in this study suggest that N. fowleri treated with monoclonal antibodies against N. fowleri reduce the mortality and prolong the survival time of the mice when the antibodies are administered before the infection. The protective effect of the monoclonal antibodies is surmised being caused by agglutination of the trophozoites.     

The production and characterization of anti-Naegleria fowleri monoclonal antibodies.

Naegleria fowleri, a free-living amoeba commonly found in moist soil and fresh water, enters the body via the nasal mucosa and migrates along the olfactory nerve to the brain, where it causes acute amoebic meningoencephalitis. In the present study 7 clones secreting monoclonal antibodies (McAbs) against N. fowleri were produced and the effector function of them was investigated. Their isotypes were IgG1 (Nf 1, Nf 154), IgG3 (Nf 137) and IgA (Nf 1, Nf 2, Nf 256, Nf 279). Five McAbs (McAb Nf 2, Nf 279, Nf 27, Nf 154, Nf 137) were specific for N. fowleri by ELISA and recognized the antigenic determinants located on the trophozoite surface by IFAT and immunoperoxidase stain. These five McAbs had capacity to agglutinate N. fowleri trophozoites and inhibited the growth of the amoeba in culture medium. McAb Nf 2 inhibited proliferation of trophozoites in vitro significantly. Also the cytotoxicity of N. fowleri against CHO cell was reduced in the presence of McAb Nf 2 and McAb Nf 154. From these results McAb Nf 2 was confirmed to weaken the virulence of the amoeba among 7 screened McAbs.     

Membrane carbohydrate characterization of Acanthamoeba astronyxis, A. castellanii and Naegleria fowleri by fluorescein-conjugated lectins

A comparative study of membrane carbohydrate characteristics of pathogenic and non-pathogenic trophozoites and cysts of free-living Acanthamoeba castellanii, Naegleria fowleri and A. astronyxis, respectively from sewage sludge in India was carried out by means of fluorescein-conjugated lectin binding using eight lectins. Two lectins, viz. Concanavalin A and Phytohaemagglutinin P, could bind all free-living amoebae at different concentrations. The most notable feature of the study is that peanut agglutinin (PNA) and wheatgerm agglutinin (WGA) can differentiate between the pathogenic A. castellanii and non-pathogenic A. astronyxis strain, respectively. However, Ulex agglutinin I (UEA I) was the only lectin positive to both pathogenic A. castellanii and N. fowleri. During in vitro conversion from trophozoites to cysts, A. castellanii and N. fowleri cysts gained WGA-specific saccharide whereas A. castellanii; A. astronyxis and N. fowleri lost or reduced Dolichos biflorus agglutinin, PNA; WGA and ConA, and UEA I-specific saccharides, respectively. Neuraminidase could not alter the fluorescein-lectin binding to WGA and PNA. These demonstrated that only two lectins can recognize the factors giving Acanthamoeba their pathogenic (PNA-specific) and non-pathogenic (WGA-specific) status. More interestingly, UEA I can only differentiate between pathogenic and non-pathogenic amoebae. It is also suggested that during stage conversion the surface of the organism exhibited replacement of saccharides.     

Recognition of Entamoeba histolytica 115-kDa surface protein by human secretory immunoglobulin A antibodies from asymptomatic carriers

Entamoeba histolytica is a protozoan parasite that can invade the intestinal mucosa. Infection induces production of secretory immunoglobulin A (SIgA) antibodies that can diminish the adhesion between E. histolytica trophozoites and epithelial cells in vitro and reduce the rate of new infections in children. SIgA antibodies produced by asymptomatic cyst carriers could play a protective role against the damage caused by E. histolytica. To identify membrane antigens capable of inducing SIgA response in E. histolytica cyst carriers, salivary SIgA antibodies were confronted with blotted plasma membrane proteins from amebae. A surface 115-kDa ameba protein was recognized by 62% of the human SIgA antibodies tested. The 115-kDa protein is not a mannose-containing glycoprotein and has no protease activity. Rabbit anti-115-kDa protein antibodies were capable of reducing erythrophagocytosis but were unable to protect culture cells from the cytopathic damage caused by E. histolytica. However, anti-115-kDa protein antibodies induced surface receptor redistribution.     

Evaluation of the Indirect Fluorescent-Antibody Technique for Identification of Naegleria Species

The indirect fluorescent-antibody technique was used to assess a rapid method for identification of amoebae belonging to the genus Naegleria. Thirty-eight Naegleria and eight other limax amoeba strains were examined by using one N. gruberi and two N. fowleri antisera. All pathogenic Naegleriae, most of which originated from fatal cases of primary amoebic meningo-encephalitis, were identified as belonging to the fowleri species. Most of the N. gruberi strains showed irregular fluorescence. Other limax amoebae, such as Vahlkampfia, Acanthamoeba, Hartmannella, and Schizopyrenus sp. gave negative responses with the prepared antisera. The indirect fluorescent-antibody technique allows the identification of N. fowleri in a mixed culture of both N. fowleri and N. gruberi strains. Twenty-two Naegleria isolated from a suspected stream, other surface waters, and muddy soil could be excluded from the fowleri species with the indirect fluorescent-antibody technique. The results obtained demonstrate that this immunological technique is a valid method for the rapid identification of N. fowleri trophozoites.     

Naegleria fowleri: nfa1 gene knock-down by double-stranded RNAs

Nfa1 protein expressed by the nfa1 gene that was cloned recently from pathogenic Naegleria fowleri was found in pseudopodia, especially food-cups, and concerned with a mechanism of pathogenicity of N. fowleri. In the present study, N. fowleri nfa1 gene was knocked down using double-stranded RNAs, and the expression of Nfa1 protein was observed. Using synthetic double-stranded RNA of the nfa1 gene in vitro, the nfa1 gene and Nfa1 protein were knocked down about 50.4+/-3.1% and 52+/-2%, respectively. These results suggest that RNA interference (RNAi) may be an effective technique for gene knock-down in N. fowleri trophozoites.     

Identification of multiple cell adhesion receptors for collagen and fibronectin in human fibrosarcoma cells possessing unique alpha and common beta subunits

Using monoclonal antibody technology and affinity chromatography we have identified four distinct classes of cell surface receptors for native collagen on a cultured human fibrosarcoma cell line, HT-1080. Two classes of monoclonal antibodies prepared against HT-1080 cells inhibited adhesion to extracellular matrix components. Class I antibodies inhibited cell adhesion to collagen, fibronectin, and laminin. These antibodies immunoprecipitated two noncovalently linked proteins (subunits) with molecular masses of 147 and 125 kD, termed alpha and beta, respectively. Class II antibodies inhibited cell adhesion to native collagen only and not fibronectin or laminin. Class II antibodies immunoprecipitated a single cell surface protein containing two noncovalently linked subunits with molecular masses of 145 and 125 kD, termed alpha and beta, respectively. The two classes of antibodies did not cross-react with the same cell surface protein and recognized epitopes present on the alpha subunits. Pulse-chase labeling studies with [35S]methionine indicated that neither class I nor II antigen was a metabolic precursor of the other. Comparison of the alpha and beta subunits of the class I and II antigens by peptide mapping indicated that the beta subunits were identical while the alpha subunits were distinct. In affinity chromatography experiments HT-1080 cells were extracted with Triton X-100 or octylglucoside detergents and chromatographed on insoluble fibronectin or native type I or VI collagens. A single membrane protein with the biochemical characteristics of the class I antigen was isolated on fibronectin-Sepharose and could be immunoprecipitated with the class I monoclonal antibody. The class I antigen also specifically bound to type I and VI collagens, consistent with the observation that the class I antibodies inhibit cell adhesion to types VI and I collagen and fibronectin. The class II antigen, however, did not bind to collagen (or fibronectin) even though class II monoclonal antibodies completely inhibited adhesion of HT-1080 cells to types I and III-VI collagen. The class I beta and II beta subunits were structurally related to the beta subunit of the fibronectin receptor described by others. However, none of these receptors shared the same alpha subunits. Additional membrane glycoprotein(s) with molecular mass ranges of 80-90 and 35-45 kD, termed the class III and IV receptors, respectively, bound to types I and VI collagen but not to fibronectin. Monoclonal antibodies prepared against the class III receptor had no consistent effect on cell attachment or spreading, suggesting that it is not directly involved in adhesion to collagen-coated substrates.(ABSTRACT TRUNCATED AT 400 WORDS)     

Resistance to Naegleria fowleri infection passively acquired from immunized splenocyte, serum or milk

A pathogenic free-living amoeba, Naegleria fowleri, causes primary amoebic meningoencephalitis to human and experimental animals. This infection is rare, but the mortality is very high. Nowadays, drug treatment or active immunization of human or mice are being tried with partial effectiveness. This study shows passive immunization effect by transfer of immunized spleen cells, serum, or milk from immunized mother in mouse experimental model. Young BALB/c mice were immunized intraperitoneally with 2-3 X 10(6) trophozoites of N. fowleri, and spleen cells and sera were collected for injection to recipient mice. There were seven transfer groups, i.e., immunized mouse serum, spleen cells, serum and spleen cells, normal mouse serum, spleen cells, serum and spleen cells, and control group. Three days later, BALB/c mice were inoculated with 1 x 10(4) trophozoites of N. fowleri intranasally. After infection, decreased mortality and prolonged survival time of mice were noted in immunized groups compared with non-immunized control group. The groups injected with immunized spleen cells or normal serum showed lower mortality than that of controls but showed no changes of serum IgG level. The groups injected with immunized serum or normal spleen cells showed increased serum IgG level after immunization but hundred percent mortality was observed. Mother mice were immunized intraperitoneally with 2-3 X 10(6) trophozoites of N. fowleri at the end of pregnancy and weaning period. Soon after the delivery, litters born of non-immunized mother were matched with immunized mother for feeding immune milk. After three weeks, the litters were infected with 1 X 10(4) trophozoites of N. fowleri or sacrificed for serum collection to measure the IgG levels.(ABSTRACT TRUNCATED AT 250 WORDS)     

Entamoeba histolytica trophozoites transfer lipophosphopeptidoglycans to enteric cell layers

Transfer of antigens frequently follows adhesion of protozoan parasites to host cells. We were interested in such transfer from the Entamoeba surface to enterocytes following adhesion of trophozoites. Therefore, cocultures of enterocytes in vitro and ex vivo with Entamoeba histolytica (strain HM-1:IMSS) or Entamoeba dispar (strain SAW760) trophozoites were processed for immunocytochemistry. The EH5 monoclonal antibody against amoebic proteophosphoglycans marked a dotted pattern on the apical side of enterocytes in in vitro cocultures with HM-1:IMSS and SAW760 trophozoites. Basolateral staining was present in cocultures following dysfunction of tight junctions, or when trophozoites made direct contact with the basolateral side of enterocytes in in vitro and ex vivo cocultures. Based on the molecular mass in Western blot, the transferred proteophosphoglycan was identified as a lipophosphopeptidoglycan. In conclusion, trophozoites transfer LPPG to the apical side of enterocytes following adhesion and prior to dysfunction of tight junctions.     

Isolation of a unique membrane protein from Naegleria fowleri

Naegleria fowleri, an amoeboflagellate, is the causative agent of Primary Amoebic Meningoencephalitis, a fulminating disease of the central nervous system. In order to elucidate the mechanisms of pathogenicity of this amoeba, a cDNA expression library was prepared from N. fowleri RNA. A specific protein was found to be expressed from a cDNA clone designated Mp2CL5. Northern blot analysis showed that the Mp2CL5 mRNA was expressed in pathogenic N. fowleri but was not expressed in non-pathogenic Naegleria species nor in Acanthamoeba. Western blot analysis using anti-N. fowleri antiserum demonstrated that IPTG-induced Escherichia coli Mp2CL5 expressed a 23-kDa recombinant protein. The Mp2CL5 recombinant protein was histidine-tagged and purified to homogeneity from E. coli. A polyclonal rabbit antiserum was prepared against the purified Mp2CL5 recombinant protein. This antibody was used to further characterize the Mp2CL5 native protein expressed by N. fowleri. Western blot analysis in conjunction with immunofluorescence microscopy demonstrated the presence of a native protein of 17 kDa on the plasma membrane of N. fowleri trophozoites. The native N. fowleri protein was expressed in the logarithmic phase of trophozoite growth and the production of this protein increased through the stationary phase of growth. Studies are in progress to examine further its role as a virulence factor.     

In vitro and in vivo interaction of Entamoeba histolytica Gal/GalNAc lectin with various target cells: an immunocytochemical analysis

The Gal/GalNAc lectin of Entamoeba histolytica trophozoites plays an important role in adhesion. The distribution and final destiny of the lectin during the interaction with host cells are poorly understood. Using monoclonal and polyclonal antibodies against the lectin we studied by immunocytochemistry the in vitro and in vivo interaction of E. histolytica trophozoites with human and hamster hepatocytes. We also analyzed the presence and distribution of the lectin in a mouse model of intestinal amoebiasis. In all cases, trophozoites were highly labeled by anti-lectin antibodies. Cultured human and hamster hepatocytes in contact with, or localized at the vicinity of parasites were also labeled by anti-lectin antibodies. Most of the labeled hepatocytes showed variable degrees of cell damage. Hepatocytes distantly localized from the parasites were also stained with the anti-lectin antibodies. Immunolabeling of tissue sections from different stages of the development of experimental amoebic liver abscess in hamsters showed inflammatory foci containing lectin-labeled trophozoites, hepatocytes, and sinusoidal and inflammatory cells. Lectin-containing hepatocytes had vacuolated cytoplasm with some nuclei with a condensed appearance. Damaged intestinal epithelium also was labeled with anti-lectin antibodies in a mouse model of intestinal amoebiasis. Electron microscopy of axenically cultured trophozoites using gold-labeled monoclonal and polyclonal anti-lectin antibody showed that plasma membrane, vacuole membranes and areas of cell cytosol were labeled. Higher deposits of gold particles in plasma membrane suggestive of cell secretion were observed. Our results demonstrated that Gal/GalNAc lectin was bound and captured by different target cells, and that host cells containing the lectin showed signs of cell damage. The contribution of lectin transfer to host cells in adherence and cell injury remains to be determined.     

A cell surface chondroitin sulfate proteoglycan, immunologically related to CD44, is involved in type I collagen-mediated melanoma cell motility and invasion

The metastatic spread of tumor cells occurs through a complex series of events, one of which involves the adhesion of tumor cells to extracellular matrix (ECM) components. Multiple interactions between cell surface receptors of an adherent tumor cell and the surrounding ECM contribute to cell motility and invasion. The current studies evaluate the role of a cell surface chondroitin sulfate proteoglycan (CSPG) in the adhesion, motility, and invasive behavior of a highly metastatic mouse melanoma cell line (K1735 M4) on type I collagen matrices. By blocking mouse melanoma cell production of CSPG with p-nitrophenyl beta-D-xylopyranoside (beta-D-xyloside), a compound that uncouples chondroitin sulfate from CSPG core protein synthesis, we observed a corresponding decrease in melanoma cell motility on type I collagen and invasive behavior into type I collagen gels. Melanoma cell motility on type I collagen could also be inhibited by removing cell surface chondroitin sulfate with chondroitinase. In contrast, type I collagen-mediated melanoma cell adhesion and spreading were not affected by either beta-D-xyloside or chondroitinase treatments. These results suggest that mouse melanoma CSPG is not a primary cell adhesion receptor, but may play a role in melanoma cell motility and invasion at the level of cellular translocation. Furthermore, purified mouse melanoma cell surface CSPG was shown, by affinity chromatography and in solid phase binding assays, to bind to type I collagen and this interaction was shown to be mediated, at least in part, by chondroitin sulfate. Additionally we have determined that mouse melanoma CSPG is composed of a 110-kD core protein that is recognized by anti-CD44 antibodies on Western blots. Collectively, our data suggests that interactions between a cell surface CD44-related CSPG and type I collagen in the ECM may play an important role in mouse melanoma cell motility and invasion, and that the chondroitin sulfate portion of the proteoglycan seems to be a critical component in mediating this effect.     

Characterization of epitopes of human secretory component on free secretory component, secretory IgA, and membrane-associated secretory component

We have compared the epitopes present in various forms of human secretory component by using a panel of hybridoma-derived antibodies elicited by immunizing mice with free secretory component (FSC) or secretory IgA (sIgA). Enzyme-linked immunosorbent binding assays (ELISA) were used to assess antibody binding to FSC- and SC-containing antigens, including sIgA isolated from milk, reduced and alkylated sIgA, and sIgA assembled in vitro by incubating dimeric IgA with FSC. Immunofluorescence assays were also used to assess binding to a human epithelial tumor cell line (HT29) that expresses secretory component as an integral protein of the plasma membrane. The results can be summarized as follows. 1) Most antibodies from fusions in which sIgA was the immunizing antigen bound preferentially to sIgA. 2) Most antibodies from fusions in which FSC was the immunizing antigen bound preferentially to FSC. 3) Antibodies that bound preferentially to sIgA invariably bound sIgA assembled in vitro; antibodies that bound preferentially to FSC invariably did not. 4) Antibodies that bound readily to both sIgA and FSC were rare in all fusions. 5) The monoclonal antibodies defined at least six classes of epitopes on SC, including epitopes that were a) FSC specific and reduction sensitive, b) FSC specific and reduction insensitive, c) sIgA specific and reduction-sensitive, d) sIgA specific and reduction insensitive, e) shared by FSC and sIgA and reduction-sensitive, and f) shared by FSC and sIgA and reduction-insensitive. 6) Antibodies that mediated intense immunofluorescent staining of secretory component on HT29 cell membranes were rare and constituted a distinct subset of those which recognized epitopes shared by FSC, reduced and alkylated sIgA, and some preparations of native sIgA. Results of these antibody-binding studies indicate that most SC epitopes are not shared by FSC and sIgA. Most SC-related epitopes on sIgA appear to be generated by the physical interaction of SC with dimeric IgA, whereas most epitopes on FSC are masked or altered by this interaction. Finally, epitopes that are shared by membrane SC and FSC and/or sIgA represent a minor and immunochemically distinct subset of epitopes on SC. The high proportion of unique epitopes on the different physical forms of SC suggest that the epitopes of this molecule are highly sensitive to its molecular environment. The monoclonal reagents described here will be useful in studying the structure and function of SC; quantitating FSC, sIgA, and membrane SC; purifying various molecular forms of SC by immunoaffinity chromatography; and localizing SC in human tissues and cultured cells by immunocytochemical techniques.     

A comparative study of the membrane proteins from Naegleria species: A 23-kDa protein participates in the virulence of Naegleria fowleri

The plasma membrane is essential in the pathogenicity of several microorganisms. However, to date, there are few studies related to the plasma membrane proteins in Naegleria fowleri; this amoeba produces a fatal disease called primary amoebic meningoencephalitis. In the present study, we analyzed the electrophoretic pattern of the membrane proteins of N. fowleri and compared it with the nonpathogenic N. lovaniensis and N. gruberi. We detected a 23-kDa protein (Nf23) present at a higher level in N. fowleri than in the nonpathogenic amoebae. The mass spectrometry analysis showed that the Nf23 protein has a sequence of 229 amino acids that corresponds to a membrane protein. The mRNA level of nf23 was overexpressed 4-fold and 40,000-fold in N. fowleri compared with N. lovaniensis and N. gruberi, respectively. Moreover, we found a 5-fold overexpression of nf23 in N. fowleri trophozoites recovered from mouse brains compared with trophozoites axenically cultivated. In addition, the cytopathic effect on Madin-Darby Canine Kidney cells coincubated with N. fowleri diminished in the presence of antibodies against Nf23; nevertheless, the nonpathogenic amoebae did not produce damage to the monolayer cells. These results suggest that the plasma membrane protein Nf23 is probably involved in the virulence of N. fowleri.     

Studies on the cell-mediated immunity in experimental Naegleria spp. infections

Observations were made on the differences in cell-mediated immune responses in the mice infected with strongly pathogenic Naegleria fowleri ITMAP 359, weakly pathogenic Naegleria jadini 0400, or non-pathogenic Naegleria gruberi EGB, respectively. Variations in cell-mediated responses and changes in antibody titers according to the duration after infection were noted. Infections were done by dropping 5 microliters saline suspension containing 10 x 10(4) trophozoites cultured axenically in the CGVS medium into the right nasal cavity of ICR mice aging about 6-7 weeks, under the anesthesia by intraperitoneal injection of secobarbital. Following infection, delayed type hypersensitivity(DTH) responses in the footpad and blastogenic responses of the mouse spleen cells using [3H]-thymidine were observed on the day 1, 4, 7, 10 and 14 after infection. For the preparation of amoeba lysates, each of cultured trophozoites were homogenized with an ultrasonicator, and centrifugated at 20,000 g. The supernatants of amoeba lysates were used as the mitogen and antigen for ELISA. Concanavalin A(Con. A) and lipopolysaccharide(LPS) were also used as mitogens in the blastogenic response. 1. The mice infected with N. fowleri showed the mortality rate of 75.7%. The rate was 6.2% for the N. jadini infected group, while no dead mouse was observed for N. gruberi infections. 2. In regard to DTH responses in the N. fowleri infected mice, the level increased in comparison to the control group but declined after 7 days. An increase was also noted for the N. jadini group after 1 day, but gradual decreases were observed through the infection period. In addition, no difference was noted between the N. gruberi infected and control groups. 3. Concerning the blastogenic response of the splenocytes, it increased after 10 days in the experimental group of N. fowleri infection, but the differences were not statistically significant compared with control group. It was evident that N. jadini group was not different from control group either, while there was a tendency of decrease in N. gruberi infected group. In regard to the blastogenic response of the splenocytes by LPS, it was found that the N. fowleri, N. jadini and N. gruberi infected groups had no differences from the control group. 4. The serum antibody titer of N. fowleri and N. jadini infected mice increased from the day 7 and 14 after infection respectively, while the N. gruberi infected mice showed no increase.(ABSTRACT TRUNCATED AT 400 WORDS)     

Interaction between chondroitin-6-sulfate and Entamoeba histolytica as revealed by force spectroscopy

We have observed by atomic force microscopy (AFM) the amoeba surface and probed the interaction force between Entamoeba histolytica and chondroitin-6-sulphate (C6S). We have used several substrates to adhere trophozoites. The best reproducibility in sample preparation was obtained with fibronectin-coated coverslips and when the cells were fixed with paraformaldehyde. The images obtained with the AFM showed that the trophozoite exhibits an irregular surface. Pseudopods and waving adhesion plaques could be observed. Force spectroscopy analysis showed that the trophozoite surface strongly interacts with C6S-functionalized tips. During cantilever retraction, attractive force peaks were observed at distances up to 1.3 microm above the trophozoite surface. Statistical analysis of the force distributions collected for five samples shown a reproducible 2.2 nN mean adhesion force. We observed a reduction of the adhesion force and of the interaction distance after addition of galactose to the buffer solution suggesting that the observed interaction is also Gal/GalNAc-lectin-mediated.     

Integrin alpha(2)I domain recognizes type I and type IV collagens by different mechanisms

The collagens are recognized by the alphaI domains of the collagen receptor integrins. A common structural feature in the collagen-binding alphaI domains is the presence of an extra helix, named helix alphaC. However, its participation in collagen binding has not been shown. Here, we have deleted the helix alphaC in the alpha(2)I domain and tested the function of the resultant recombinant protein (DeltaalphaCalpha(2)I) by using a real-time biosensor. The DeltaalphaCalpha(2)I domain had reduced affinity for type I collagen (430 +/- 90 nM) when compared with wild-type alpha(2)I domain (90 +/- 30 nM), indicating both the importance of helix alphaC in type I collagen binding and that the collagen binding surface in alpha(2)I domain is located near the metal ion-dependent adhesion site. Previous studies have suggested that the charged amino acid residues, surrounding the metal ion-dependent adhesion site but not interacting with Mg(2+), may play an important role in the recognition of type I collagen. Direct evidence indicating the participation of these residues in collagen recognition has been missing. To test this idea, we produced a set of recombinant alpha(2)I domains with mutations, namely D219A, D219N, D219R, E256Q, D259N, D292N, and E299Q. Mutations in amino acids Asp(219), Asp(259), Asp(292), and Glu(299) resulted in weakened affinity for type I collagen. When alpha(2) D219N and D292N mutations were introduced separately into alpha(2)beta(1) integrin expressed on Chinese hamster ovary cells, no alterations in the cell spreading on type I collagen were detected. However, Chinese hamster ovary cells expressing double mutated alpha(2) D219N/D292N integrin showed remarkably slower spreading on type I collagen, while spreading on type IV collagen was not affected. The data indicate that alpha(2)I domain binds to type I collagen with a different mechanism than to type IV collagen.     

Selective adhesion of macrophages to denatured forms of type I collagen is mediated by scavenger receptors.

Macrophages (Mφs) are multifunctional immune cells which are involved in the regulation of immune and inflammatory responses, as well as in tissue repair and remodeling. In tissues, Mφs reside in areas which are rich in extracellular matrix (ECM), the structural component which also plays an essential role in regulating a variety of cellular functions. A major ECM protein encountered by Mφs is type I collagen, the most abundant of the fibril-forming collagens. In this study, the adhesion of RAW 264.7 murine Mphis to native fibrillar, monomeric, and denatured type I collagen was investigated. Using atomic force microscopy, structural differences between fibrillar and monomeric type I collagen were clearly resolved. When cultured on fibrillar type I collagen, Mphis adhered poorly. In contrast, they adhered significantly to monomeric, heat-denatured, or collagenase-modified type I collagen. Studies utilizing anti-beta1 and -beta2 integrin adhesion-blocking antibodies, RGD-containing peptides, or divalent cation-free conditions did not inhibit Mphi; adhesion to monomeric or denatured type I collagen. However, macrophage scavenger receptor (MSR) ligands and anti-MSR antibodies significantly blocked Mphi; adhesion to denatured and monomeric type I collagen strongly suggesting the involvement of the MSR as an adhesion molecule for denatured type I collagen. Further analysis by Western blot identified the MSR as the primary receptor for denatured type I collagen among Mphi; proteins purified from a heat-denatured type I collagen affinity column. These findings indicate that Mphis adhere selectively to denatured forms of type I collagen, but not the native fibrillar conformation, via their scavenger receptors.     

Subcellular Distribution of Hydrolases in Naegleria fowleri1

The presence and particle association of various hydrolytic enzymes in Naegleria fowleri has been studied in whole cell extracts of trophozoites in an effort to establish authentic markers for surface membrane and lysosomal components. Evidence from the experiments reported here indicates that in N. fowleri a) acid proteinase, N-acetylglucosaminidase, and acid phosphatase are associated with cytoplasmic granules closely resembling lysosomes; b) 5'-nucleotidase is associated with the surface membrane, probably on the external surface; c) aspartate aminotransferase is associated with mitochondria; d) a-D-glucosidase and an aminopeptidase have bimodal distributions, activity being associated with both the surface membrane and lysosomal particles.