Strains HM1 Research Articles

Biodegradation of used engine oil by novel strains of Ochrobactrum anthropi HM-1 and Citrobacter freundii HM-2 isolated from oil-contaminated soil.

Used engine oil (UEO) constitutes a serious environmental problem due to the difficulty of disposal off or reuse. Ten bacterial strains with biodegradation potential were isolated from UEO-contaminated soil sample using enrichment technique. Two strains which exhibited the highest degradation %, 51 ± 1.2 and 48 ± 1.5, respectively, were selected. Based on the morphological, biochemical characteristics and 16S rRNA sequence analysis, they were identified as Ochrobactrum anthropi HM-1 (accession no: KR360745) and Citrobacter freundii HM-2 (accession no: KR360746). The different conditions which may influence their biodegradation activity, including UEO concentration (1–6 %, v/v), inoculum size (0.5–4 %, v/v), initial pH (6–8), incubation temperature (25–45 °C), and rotation speed (0–200 rpm), were evaluated. The optimum conditions were found to be 2 % UEO, 2 % inoculum size, pH 7.5, incubation temperature 37 °C, and 150 rpm. Under the optimized conditions, strains HM-1, HM-2, and their mixture efficiently degraded UEO, they achieved 65 ± 2.2, 58 ± 2.1, and 80 ± 1.9 %, respectively, after 21 days of incubation. Biodegradation of UEO was confirmed by employing gas chromatography analysis. Gamma radiation (1.5 kGy) enhanced the degradation efficiency of irradiated bacterial mixture (95 ± 2.1 %) as compared to non-irradiated (79 ± 1.6 %). Therefore, strains HM-1 and HM-2 can be employed to develop a cost-effective method for bioremediation of used engine-oil-polluted soil.

Luminal bacteria and proteases together decrease adherence of Entamoeba histolytica trophozoites to Chinese hamster ovary epithelial cells: a novel host defence against an enteric pathogen.

Factors that prevent colonic mucosal invasion by pathogenic Entamoeba histolytica are not understood. A key initial step in pathogenesis of injury induced by amoeba is adherence to target cells mediated by a surface glycoprotein lectin on E histolytica. Mucin degrading bacteria normally present in the colon lumen produce glycosidases that degrade soluble or cell surface glycoconjugates. To determine whether glycosidases produced by mucin degrading bacteria, alone or in combination with proteases present in colon lumen, can decrease E histolytica adherence to target epithelial cells by degrading E histolytica adherence lectin. The effects of exposure of E histolytica trophozoites strains HM1:IMSS and 200:NIH to faecal culture supernatant fluids, culture supernatant preparations of mucin degrading bacteria, and luminal proteases on their adherence to Chinese hamster ovary (CHO) cells were determined. The amount of surface adherence lectin on E histolytica trophozoites before and after treatment with glycosidases and proteases was determined by immunofluorescence. The effect of glycosidases and proteases on purified E histolytica lectin was determined by gel electrophoresis. Incubation of E histolytica with culture supernatant preparations or proteases alone did not modify their CHO cell adherence. However, 24 hour incubation of trophozoites with culture supernatant preparations together with pancreatic proteases decreased CHO cell adherence of HM1:IMSS strain by 71.1% (p < 0.001) and of 200: NIH strain by 95% (p < 0.05). Incubation of trophozoites for 24 hours with faecal extracts which contain bacterial and host hydrolases decreased the adherence of the HM1:IMSS strain by 69.2% (p < 0.01) and of the 200: NIH strain by 83.0%. Reduction of trophozoite adherence to CHO cells by hydrolases was promoted by 7.5 mM cycloheximide, and was reversible on incubation in an enzyme free medium. Decrease in CHO cell adherence of trophozoites was associated with decreased lectin on trophozoites as determined by immunofluorescence using a monoclonal antibody to the lectin. Purified lectin was degraded by the mixture of faecal culture supernant preparations and proteases, but not by either alone. Mucin degrading bacterial glycosidases and colonic luminal proteases together, but not alone, degrade the key adherence lectin on E histolytica trophozoites resulting in decreased epithelial cell adherence. These in vitro findings suggest a potential novel host defence mechanism in the human colon wherein the invasiveness of a pathogen could be curtailed by the combined actions of bacterial and host hydrolases. This mechanism may be responsible for preventing mucosal invasion by pathogenic E histolytica.

Lipophosphoglycan is present in distinctly different form in different Entamoeba histolytica strains and absent in Entamoeba moshkovskii and Entamoeba invadens.

Lipophosphoglycan has recently been demonstrated on the cell surface of Entamoeba histolytica strain HM-1:IMSS. A monoclonal antibody against this molecule had failed to react with some other strains of E. histolytica, including the strain Rahman. To determine if a structurally distinct lipophosphoglycan existed in Rahman, [3H]galactose-labeled glycoconjugates were electrophoresed through sodium dodecyl sulfate polyacrylamide gel electrophoresis. The electrophoretic pattern in Rahman was very different compared to that obtained with strains HM-1:IMSS and 200:NIH. A number of experiments including sensitivity to mild acid, nitrous acid and phosphoinositol-specific phospholipase C suggest that the Rahman glycoconjugate is indeed a lipophosphoglycan-like molecule but distinctly different from that of HM-1:IMSS. Mild acid-treated glycoconjugates from Rahman and HM-1:IMSS revealed the presence of neutral trisaccharides and monosaccharides in Rahman but not in HM-1:IMSS. Human immune sera from amoebiasis patients and a polyclonal antibody against HM-1:IMSS lipophosphoglycan both recognized Rahman glycoconjugate. Thus, while lipophosphoglycan molecules from the two strains share common epitopes, they are clearly distinct from each other. Molecules bearing resemblance to lipophosphoglycan could not be detected in other Entamoeba species, namely Entamoeba invadens and Entamoeba moshkovskii.

Nuclear Behavior in Artificially Induced Multi-Nuclear Cells of Dictyostelium discoideum: (cellular slime mold/Dictyostelium discoideum/Polysphondylium pallidum/artificially induced cell fusion/nuclear fusion).

The culture medium of the strain CK-8 of the cellular slime mold Polysphondylium pallidum contains a cell-fusion induction factor. Cells of the two opposite mating type strains NC-4 and HM1 of Dictyostelium discoideum were treated to induce cell fusion with the diluted fraction of CK-8 cultures, F2, which contains the factor and consequently numerous multinuclear cells were produced. NC-4 and HM1 usually fuse in the sexual cycle and form large multinuclear cells, called giant cells, which develop into macrocysts. These cells are very similar in morphology to the multinuclear cells produced following F2 treatment, however, the latter cells did not develop into macrocysts. In the sexually formed multinuclear cells, only two haploid nuclei fused to form a diploid nucleus and all others degenerate as previously reported. However, in the artificially produced multinuclear cells, no nuclear-fusion and degeneration took place. They stayed as heterokaryons and seem to lyse within 20 h incubation.

Reactivity of Monoclonal Antibodies to Species‐Specific Antigens of Entamoeba histolytica

Twenty monoclonal antibodies were produced against trophozoites of Entamoeba histolytica strains HK-9 and HM-1: IMSS. When reactivity to various enteric protozoa was examined by an indirect fluorescence antibody test, 15 of the monoclonal antibodies were strongly reactive with E. histolytica trophozoites. Species-specific antigens recognized by these monoclonal antibodies were located on the plasma membrane, nucleus, cytoplasm, and cytoskeletal structures of the trophozoites. Two of the remaining five monoclonals reacted strongly with trophozoites of the E. histolytica-like Laredo strain. The determinant antigen was located in the cytoplasm. The three remaining monoclonal antibodies were found to recognize cross-reactive antigens between E. histolytica and E. histolytica-like Laredo, E. hartmanni, E. coli, Dientamoeba fragilis, Giardia lamblia, and Trichomonas hominis. These three antibodies were also reactive with T. vaginalis and mammalian cells such as HeLa cells. Thus, the combined use of monoclonal antibodies seems capable of distinguishing E. histolytica and/or E. histolytica-like Laredo from other enteric protozoa.

Antigens in electron-dense granules from Entamoeba histolytica as possible markers for pathogenicity

In vitro interaction of Entamoeba histolytica with collagen induces intracellular formation and release of electron-dense granules (EDG) and stimulation of collagenolytic activity. Purified EDG contain 1.66 U of collagenase per mg of protein. Thus, EDG may participate in tissue destruction during invasive amebiasis. Monoclonal antibodies (MAbs) L1.1 and L7.1 reacted specifically with EDG in enzyme-linked immunosorbent assay (ELISA) and immunofluorescence and immunoelectron microscopy. MAb L7.1 immunoprecipitated three polypeptides with molecular weights of 95,000, 68,000, and 28,000 from lysates of biosynthetically labeled E. histolytica. Both MAbs recognized the pathogenic E. histolytica axenic strains HM1:IMSS, HM38:IMSS, and HK-9 but failed to react in ELISA with Entamoeba moshkovskii, Entamoeba invadens, and E. histolytica-like Laredo. In addition, MAb L7.1 reacted with one E. histolytica isolate from a symptomatic patient but did not react with four of five isolates from asymptomatic patients. EDG antigens were detected by a MAb L7.1-based ELISA in E. histolytica-containing fecal samples from symptomatic, but not asymptomatic, individuals. These results suggest that the EDG antigen detected with MAb L7.1 may be differentially expressed in pathogenic and nonpathogenic E. histolytica.

Species-specific and cross-reactive antigens of Entamoeba

Specific and cross-reactive antigens were defined in four species of Entamoeba: invadens, moshkovskii, Laredo and histolytica strains HM1, HM3, HM38 and HK9. Among these species extensive common reactivities were observed by immunoblot. Eight E. histolytica antigenic markers were revealed after blocking common specificities with antigens of other Entamoeba species. A monoclonal antibody (mAb) defined two protein markers of E. histolytica, M, 29 and 25 kDa. The four strains of E. histolytica, which varied in virulence as determined by the development of liver abscesses in hamsters, showed the same antigenic patterns with the mAb and with polyclonal antibodies.

Identification of the cell surface molecule involved in sexual cell fusion of Dictyostelium discoideum

Abstract Dictyostelium discoideum was used as a model system for elucidating the molecular mechanism of sexual cell fusion. In heterothallic strains NC4 and HM1 of D. discoideum, complements in mating type, amoeboid cells acquire fusion competence only under certain environmental conditions, such as the presence of excess water and a certain period of darkness, to fuse sexually. The surface of cells which acquired fusion competence was found to possess specific antigens. Monovalent antibodies prepared from rabbit antiserum against fusion-competent NC4 cells inhibit the sexual cell fusion of these cells completely. Two specific antigenic proteins, 39 and 138 k Da in relative molecular mass and specific for fusion-competent cells, were detected. Only one, the 138-k Da protein, was capable of neutralizing the fusion-inhibitory activity of the monovalent antibody. These results show that the 139-k Da protein is the one involved in the sexual cell fusion of NC4 and HM1 strains in D. discoideum

Environmental factors inducing sexual development inDictyostelium discoideum

In the heterothallic strains NC4 and HM1 ofDictyostelium discoideum, sexual development is initiated by the formation of diploid zygotic giant cells produced through the fusion of these two opposite mating-type haploid cells. For sexual cell fusion, amoeboid cells must first acquire fusion competence, which requires culture under certain environmental conditions, such as darkness, excessive water, and sufficient bacteria as food. However, in the subsequent stages of cell fusion and development of the giant cells into mature macrocysts, cells do not require the above conditions. Cell fusion and development into macrocysts were able to occur even in light with minimum water and in the absence of bacteria. For cell fusion calcium ions were required.

Effects of Panmede and various horse serum concentrations on the axenic cultivation ofEntamoeba histolytica strains in TPS-1 medium

We analyzed the influence of Panmede and horse serum concentrations on the growth of five Entamoeba histolytica strains (HK9, HM1, HM2, HM3, and HM38) axenically cultivated in TPS-1 medium. Panmede was evaluated by comparing the growth of strain HM1 in medium prepared with each of 15 Panmede lots; the yields of E. histolytica trophozoites depended on the lot quality of Panmede, and their maximal values ranged from 8 x 10(3) to 8.9 x 10(4) amoebae/ml. The growth-promoting effect of eight lots of horse serum on strains HK9 and HM1 were studied using a single Panmede lot of good quality. Yields obtained with strain HK9 ranged from 8 x 10(4) to 1.8 x 10(5) amoebae/ml, whereas yields obtained with HM1 ranged from 3 x 10(4) to 1.2 x 10(5) amoebae/ml. Thus, the optimal serum concentration in TPS-1 medium that caused maximal growth of E. histolytica cultures depended on the quality of the serum lot and proved to be specific for each of the five E. histolytica strains investigated. It ranged from 18% (v/v) for strain HM2 to 28% (v/v) for strain HM1. Our results reveal that the growth of E. histolytica trophozoites in TPS-1 medium can be distinctly improved by selecting appropriate lots of Panmede and horse serum and using optimal serum concentrations.

A membrane protein with possible relevance to sexual cell fusion in Dictyostelium discoideum

The molecular mechanism of sexual cell fusion in Dictyostelium discoideum was studied using the heterothallic strains HM1 and NC4. Monovalent antibodies (Fab) prepared from rabbit antiserum against a crude membrane preparation of fusion-competent HM1 cells inhibited fusion between HM1 and NC4 cells. Six out of 43 antigenic proteins were found in fusion-competent HM1 cells but not in fusion-incompetent cells. Among them, only one protein with a molecular mass of 70 kDa was able to neutralize the fusion-inhibiting activity of Fab, suggesting its possible participation in sexual cell fusion.

Intestinal lumen and mucosal microclimate H+ and NH3 concentrations as factors in the etiology of experimental amebiasis.

Axenically cultivated Entamoeba histolytica, trophozoites, strains HM-1:IMSS and HM-38, were suspended in solutions of NaCl, 330 mOsm, of varying pH and ammonium concentrations. Short-term viability was inversely proportional to the ammonia concentration of the medium and was independent of the ammonium concentration and pH. The NH3-induced ameba killing was associated with a cellular alkalosis and cell swelling. While short-term trophozoite viability was unaffected by changes in the medium pH over the range 5.5-8.0, long-term viability was reduced by high pH, and of three pH values tested (6.27, 7.27, and 8.27), trophozoite growth was greatest at the lower value. Chemotaxis was observed in media over the pH range 5.5-8.0, and attenuated chemotaxis was observed in trophozoites in media containing NH3 (0.1 mM). The cecal content total ammonia concentration and pH and the in vivo mucosal microclimate pH were measured in young adult male rats, hamsters, and gerbils. Ceca of the three rodent species were also inoculated with HM-38 trophozoites and 7 days later the cecal contents were studied for signs of amebic infection. Infections were absent in the rat, the species with highest luminal total ammonia concentration and mucosal microclimate pH. All gerbils were infected. This species had the lowest mucosal microclimate pH. The hamster, with the intermediate microclimate pH, had a low infection rate (1 of 5). It is proposed that when ammonium diffuses from the large intestinal lumen into a more basic mucosal microclimate, it is converted to ammonia, and the combination of this ammonia and the high microclimate pH threatens Entamoeba trophozoite viability and reduces the probability of a given ameba penetrating the mucus blanket and invading the mucosal epithelium.

Fusion of cell ghosts with sexually opposite type cells in Dictyostelium discoideum

In the sexual cycle of Dictyostelium discoideum, haploid cells of two opposite mating types, strains HM1 and NC4, acquire fusion-competence under certain conditions, such as suspension culture in the dark, and fuse specifically to form giant zygote cells. Each giant cell engulfs the surrounding cells, gradually increases in size, and finally develops into a macrocyst that is a sexual structure in D. discoideum. Fusion-competent HM1 cells suspended in a solution were frozen and thawed to make cell ghosts. When cell ghosts were introduced into fusion-competent and -incompetent intact NC4 cells, the cell ghosts killed them in a short time, but the fusion-competent cells were killed in preference to the fusion-incompetent cells. This killing occurred through the fusion of the cell ghosts directly to intact cell membranes. Since the fusion was specific, the fusion between ghosts and cells appears to be essentially the same as that between intact cells during the sexual cycle in molecular mechanisms.

Analysis of antigens from membrane and soluble fractions of Entamoeba histolytica.

Axenic cultures of Entamoeba histolytica strains HK-9, HM-1, and Rahman were fractionated to provide plasma membranes, internal, vesiculated membranes, and a soluble cytosol. Each particulate fraction was solubilized and all fractions were analyzed by techniques designed to demonstrate molecular complexity and serologic reactivity. The cytosol contained more antigenic moieties than either membrane fraction; however, the antigens associated with the membranes had very high reactivity and lower nonspecific activity than the cytosol.

Crown galls induced by octopine-degrading biotype 3 strains of Agrobacterium tumefaciens contain a new form of lysopine dehydrogenase

Extracts of crown gall tumours induced on Kalanchoe daigremontiana by the biotype 3 strains AB-3, AT-6 and Hm-1 were compared with extracts of tumours induced by the wide host range octopine strain B6S3 for their ability to convert lysine, arginine, histidine and methionine into the corresponding opines: lysopine, octopine, histopine and methiopine. In contrast to B6S3 tumour extracts which readily converted all precursors into opines, extracts of tumour tissue induced by biotype 3 strains produced mainly lysopine and octopine. It can therefore be concluded that the enzymes found in tumours induced by AB-3, AT-6 and Hm-1 are different from the well-known lysopine dehydrogenase from B6S3 tumours.

Virulence of Entamoeba histolytica trophozoites. Effects of bacteria, microaerobic conditions, and metronidazole.

The association of axenically grown trophozoites of Entamoeba histolytica strains HK-9 or HM-1:IMSS with various types of gram-negative bacteria for relatively short periods markedly increased their virulence, as evidenced by their ability to destroy monolayers of tissue-cultured cells. Interaction of trophozoites with bacteria that were heat inactivated, glutaraldehyde fixed, or disrupted by sonication, or bacteria treated with inhibitors of protein synthesis, did not augment amebic virulence. Lethally irradiated bacteria, however, retained their stimulative properties and trophozoites that ingested bacteria were protected from the toxic effects of added hydrogen peroxide. An increase in virulent properties of amebae was also found in experiments carried out under microaerobic conditions (5% O2, 10% CO2). The augmentation of amebic virulence due to association with bacteria was specifically blocked by metronidazole, but not by tetracycline or aminoglycosides, and the rate of metronidazole uptake in stimulated trophozoites was two to three times higher. The results obtained suggest that virulence of axenically grown E. histolytica trophozoites may depend to a considerable extent on the cell's reducing power. Both microaerobic conditions and the association with bacteria apparently stimulate the electron transport system of the ameba. Bacteria may function as broad range scavengers for oxidized molecules and metabolites through the contribution of enzymatic systems, components, or products.

Entamoeba histolytica and Giardia lamblia: Growth responses to reducing agents

The growth responses of Entamoeba histolytica strains HM-1:IMSS and HK-9 to a variety of reducing agents were tested for one subculture in TYI-S-33 medium, prepared with no cysteine or ascorbic acid. Amoebae did not grow in this medium. Addition of l-ascorbic acid, d- or l-cysteine, or l-cystine each permitted the maximum growth observed. Dithiothreitol supported 68% maximum growth of HK-9 amoebae, but only 12% of HM-1. In contrast, growth of both strains was greatly diminished (0–13% growth) with 11 other compounds tested including glutathione, thiomalic acid, thioglycolic acid, and methionine. The growth responses of Giardia lamblia were similarly tested in TYI-S-33, as well as in TP-S-1 media. If l-cysteine was omitted from either medium, trophozoites did not grow, and eventually lysed. In TYI-S-33 medium, the requirement for l-cysteine was specific, whereas in TP-S-1 medium, other sulfhydryl compounds were partially effective and lower concentrations of l-cysteine satisfied the requirement. Ascorbic acid or l-cystine alone was totally ineffective; however, in combination, 30 to 60% of maximum growth was achieved. Once added to either medium, cysteine was rapidly oxidized. Amino acid analysis of the growth media revealed that the broth components of TP-S-1 medium contained 2.8 m M and TYI-S-33 broth 2.1 m M endogenous levels of cysteine (or half-cystine), with an additional 3 m M contributed by 10% serum.