Spherical particles of halophilic archaea correlate with exposure to low water activity--implications for microbial survival in fluid inclusions of ancient halite.

Viable extremely halophilic archaea (haloarchaea) have been isolated from million-year-old salt deposits around the world; however, an explanation of their supposed longevity remains a fundamental challenge. Recently small roundish particles in fluid inclusions of 22 000- to 34 000-year-old halite were identified as haloarchaea capable of proliferation (Schubert BA, Lowenstein TK, Timofeeff MN, Parker MA, 2010, Environmental Microbiology, 12, 440-454). Searching for a method to produce such particles in the laboratory, we exposed rod-shaped cells of Halobacterium species to reduced external water activity (a(w)). Gradual formation of spheres of about 0.4 µm diameter occurred in 4 M NaCl buffer of a(w) ≤ 0.75, but exposure to buffered 4 M LiCl (a(w) ≤ 0.73) split cells into spheres within seconds, with concomitant release of several proteins. From one rod, three or four spheres emerged, which re-grew to normal rods in nutrient media. Biochemical properties of rods and spheres were similar, except for a markedly reduced ATP content (about 50-fold) and an increased lag phase of spheres, as is known from dormant bacteria. The presence of viable particles of similar sizes in ancient fluid inclusions suggested that spheres might represent dormant states of haloarchaea. The easy production of spheres by lowering a(w) should facilitate their investigation and could help to understand the mechanisms for microbial survival over geological times.

Radiation sensitivity of N. pharaonis in comparison with E. coli K12 strains.

To investigate the radiation sensitivity of the natronobacterium Natronomonas pharaonis in comparison with Escherichia coli strains (N. pharaonis DSM 2160T, E. coli strains AB1157 and K12 lambda s) were exposed to gamma-radiation (60Co-gamma-source, 100 Gy min-1) in the presence of oxygen (air) and under strongly reduced oxygen conditions (argon-saturated medium). After irradiation, the colony-forming ability (dose-survival curves) and the D37 dose were determined. The oxygen content of the solutions containing high NaCl concentrations was measured with an oxygen electrode (Clark electrode). It was found that N. pharaonis can tolerate a remarkably higher irradiation dose than the two E. coli strains (approx. 1.5-fold of K12 lambda s and approx. 4-fold of AB1157). The oxygen enhancement ratio (OER) is 2.8 for N. pharaonis and 2.6 for both E. coli strains. Therefore the higher radiation resistance of the N. pharaonis is not due to the low oxygen content of the cell solution (high salt concentration) but is probably related to the higher DNA repair ability of this archaebacteria strain.

Novel haloarchaeal 16S rRNA gene sequences from Alpine Permo-Triassic rock salt.

Prokaryotic diversity in Alpine salt sediments was investigated by polymerase chain reaction (PCR) amplification of 16S rRNA genes, sequencing of cloned products, and comparisons with culturable strains. DNA was extracted from the residue following filtration of dissolved Permo-Triassic rock salt. Fifty-four haloarchaeal sequences were obtained, which could be grouped into at least five distinct clusters. Similarity values of three clusters to known 16S rRNA genes were less than 90%-95%, suggesting the presence of uncultured novel taxa; two clusters were 98% and 99% similar to isolates from Permo-Triassic or Miocene salt from England and Poland, and to Halobacterium salinarum, respectively. Some rock salt samples, including drilling cores, yielded no amplifiable DNA and no cells or only a few culturable cells. This result suggested a variable distribution of haloarchaea within different strata, probably consistent with the known geologic heterogeneity of Alpine salt deposits. We recently reported identical culturable Halococcus salifodinae strains in Permo-Triassic salt sediments from England, Germany, and Austria; together with the data presented here, those results suggest one plausible scenario to be an ancient continuous hypersaline ocean (Zechstein sea) populated by haloarchaea, whose descendants are found today in the salt sediments. The novelty of the sequences also suggested avoidance of haloarchaeal contaminants during our isolation of strains, preparation of DNA, and PCR reactions.

Overexpression of cysteine proteinase 2 in Entamoeba histolytica or Entamoeba dispar increases amoeba-induced monolayer destruction in vitro but does not augment amoebic liver abscess formation in gerbils.

To study the role of cysteine proteinases in the pathogenicity of Entamoeba histolytica, we have attempted to overexpress the three main cysteine proteinases (EhCP1, EhCP2, EhCP5) of this parasite in trophozoites of E. histolytica as well as in non-pathogenic Entamoeba dispar by episomal transfection. Although each of the corresponding coding sequences were cloned in identical expression plasmids, we were unable to overexpress EhCP1 and EhCP5, respectively, but could substantially induce expression of EhCP2 in both amoeba species by sevenfold, leading to a threefold increase in total cysteine proteinase activity. Overexpression of EhCP2 did not influence expression of other cysteine proteinases and could be attributed to an increase of a single 35 kDa activity band in substrate gel electrophoresis. In contrast to previous findings, which indicated that amoeba cysteine proteinases are involved in erythrophagocytosis and liver abscess formation, cells overexpressing EhCP2 showed no difference in erythrophagocytosis or liver abscess formation compared with respective controls. However, overexpression of EhCP2 in both amoeba species resulted in a marked increase of in vitro monolayer destruction.

Protection of gerbils from amebic liver abscess by vaccination with a 25-mer peptide derived from the cysteine-rich region of Entamoeba histolytica galactose-specific adherence lectin.

The protozoan parasite Entamoeba histolytica causes extensive morbidity and mortality through intestinal infection and amebic liver abscess. Here we show that immunization of gerbils with a single keyhole limpet hemocyanin-coupled 25-mer peptide derived from the 170-kDa subunit of the E. histolytica galactose-binding adhesin is sufficient to confer substantial protection against experimentally induced amebic liver abscesses. Vaccination provided total protection in 5 of 15 immunized gerbils, and abscesses were significantly smaller (P < 0.01) in the remaining vaccinated animals. The degree of protection correlated with the titer of antibodies to the peptide, and results of passive transfer experiments performed with SCID mice were consistent with a role for antibodies in protection. In addition, parenteral or oral vaccination of gerbils with 13-amino-acid subfragments of the peptide N-terminally fused to the B subunit of cholera toxin also significantly inhibited liver abscess formation (P < 0.05). These data indicate that small peptides derived from the galactose-binding adhesin administered by the parenteral or oral route can provide protection against amebic liver abscess and should be considered as components of a subunit vaccine against invasive amoebiasis.

Isolation of a chymotrypsinogen B-like enzyme from the archaeon Natronomonas pharaonis and other halobacteria.

A protease of a molecular mass of approximately 30kDa was isolated and purified from the haloalkaliphilic archaeon Natronomonas (formerly Natronobacterium) pharaonis. The enzyme hydrolyzed synthetic peptides, preferentially at the carboxyl terminus of phenylalanine or leucine, as well as large proteins. Hydrolysis occurred over the range of pH from 6 to 12, with an optimum at pH 10. The temperature optimum was 61 degrees C. The enzyme was nearly equally active over the range of salt concentration from 0.5 to 4M (NaCl or KCl). A strong cross-reaction with a polyclonal antiserum against human chymotrypsin was observed. Enzymatic activity was inhibited by typical serine protease inhibitors. There was significant homology between N-terminal and internal sequences from autolytic fragments and the sequence of bovine chymotrypsinogen B; the overall amino acid composition was similar to that of vertebrate chymotrypsinogens. Evidence for a zymogen-like processing of the protease was obtained. Cell extracts from other halobacteria exhibited similar proteolytic activity and immunoreactivity. The data suggested a widespread distribution of a chymotrypsinogen B-like protease among halo- and haloalkaliphilic Archaea.

Marinobacter aquaeolei sp. nov., a halophilic bacterium isolated from a Vietnamese oil-producing well.

Several strains of moderately halophilic and mesophilic bacteria were isolated at the head of an oil-producing well on an offshore platform in southern Vietnam. Cells were Gram-negative, non-spore-forming, rod-shaped and motile by means of a polar flagellum. Growth occurred at NaCl concentrations between 0 and 20%; the optimum was 5% NaCl. One strain, which was designated VT8T, could degrade n-hexadecane, pristane and some crude oil components. It grew anaerobically in the presence of nitrate on succinate, citrate or acetate, but not on glucose. Several organic acids and amino acids were utilized as sole carbon and energy sources. The major components of its cellular fatty acids were C12:0 3-OH, C16:1, omega 9c, C16:0 and C18:1 omega 9c. The DNA G + C content was 55.7 mol%. 16S rDNA sequence analysis indicated that strain VT8T was closely related to Marinobacter sp. strain CAB (99.8% similarity) and Marinobaster hydrocarbonoclasticus (99.4% similarity). Its antibiotic resistance, isoprenoid quinones and fatty acids were similar to those of Marinobacter hydrocarbonoclasticus and Pseudomonas nautica. However, the whole-cell protein pattern of VT8T differed from that of other halophilic marine isolates, including P. nautica. DNA-DNA hybridization indicated that the level of relatedness to Marinobacter hydrocarbonoclasticus was 65% and that to P. nautica was 75%. Further differences were apparent in Fourier-transformed IR spectra of cells and lipopolysaccharide composition. It is proposed that VT8T should be the type strain of a new species and should be named Marinobacter aquaeolei. P. nautica may have been misclassified, as suggested previously, and may also belong to the genus Marinobacter.

Munchiwarin, a prenylated chalcone from crotalaria trifoliastrum

Munchiwarin, a chalcone with the first 2,2, 6-tri-isoprenyl-cyclohex-5-ene-1,3-dione skeleton, was isolated from Crotalaria trifoliastrum and structurally identified by various NMR techniques in combination with X-ray crystallography.

F-and V-ATPases in the genus Thermus and related species.

The discovery of a V-type ATPase in the gram-negative bacterium Thermus thermophilus HB8 (YOKOYAMA et al., J. Biol. Chem. 265, 21946, 1990) was unexpected, since only eukaryotic endomembranes and archaea were thought to contain this enzyme complex, and horizontal gene transfer was suggested to explain the finding. We examined membrane-associated ATPases from representatives of several groups of the genus Thermus. The enzymes were extracted with chloroform and purified by ion exchange chromatography or native gel electrophoresis. One novel Islandic isolate, T. scotoductus SE-1, as well as strain T. filiformis from New Zealand, possessed F-ATPases, as judged by the typical five subunit composition of the F1-moiety, sensitivity to azide, insensitivity to nitrate and a strong crossreaction with antibodies against the F1-ATPase from E. coli. In addition, N-terminal amino acid sequencing of the beta subunit from T. scotoductus SE-1 confirmed its homology with beta subunits from known F-ATPases. In contrast, the same extraction procedure released a V-ATPase from the membranes of T. thermophilus HB27 and T. aquaticus YT-1. The related species Meiothermus (formerly Thermus) chliarophilus ALT-8 also possessed a V-ATPase. All V-ATPases examined in this study contained larger major subunits than F-ATPases, crossreacted with antiserum against subunit A of the V-ATPase from the archaeon Halobacterium saccharovorum, and the N-terminal sequences of their major subunits were homologous to those of other V-ATPases. Sequences of the 16S rRNA gene clearly placed T. scotoductus SE-1, along with other non-pigmented Thermus strains, as a distinct species close to T. aquaticus. Our results suggested that at least two members of the genus, T. scotoductus SE-1 and T. filiformis, contain an F-ATPase, whereas several others possess a V-ATPase. These data could indicate a greater diversity of the genus Thermus than was previously thought. Alternatively, the genus may consist of species where horizontal gene transfer has occurred and others, where it has not.

Identification of an epitope on the Entamoeba histolytica 170-kD lectin conferring antibody-mediated protection against invasive amebiasis.

The emergence of multidrug-resistant organisms and the failure to eradicate infection by a number of important pathogens has led to increased efforts to develop vaccines to prevent infectious diseases. However, the nature of the immune response to vaccination with a given antigen can be complex and unpredictable. An example is the galactose- and N-acetylgalactosamine-inhibitable lectin, a surface antigen of Entamoeba histolytica that has been identified as a major candidate in a vaccine to prevent amebiasis. Vaccination with the lectin can induce protective immunity to amebic liver abscess in some animals, but others of the same species exhibit exacerbations of disease after vaccination. To better understand this phenomenon, we used recombinant proteins corresponding to four distinct domains of the molecule, and synthetic peptides to localize both protective and exacerbative epitopes of the heavy chain subunit of the lectin. We show that protective immunity after vaccination can be correlated with the development of an antibody response to a region of 25 amino acid residues of the lectin, and have confirmed the importance of the antibody response to this region by passive immunization studies. In addition, we show that exacerbation of disease can be linked to the development of antibodies that bind to an NH2-terminal domain of the lectin. These findings are clinically relevant, as individuals who are colonized with E. histolytica but are resistant to invasive disease have a high prevalence of antibodies to the protective epitope(s), compared to individuals with a history of invasive amebiasis. These studies should enable us to develop an improved vaccine for amebiasis, and provide a model for the identification of protective and exacerbative epitopes of complex antigens.

The galactose-inhibitable surface lectin of Entamoeba histolytica, a possible candidate for a subunit vaccine to prevent amoebiasis.

Invasive amoebiasis, a spectrum of diseases caused by the enteric protozoan parasite Entamoeba histolytica, constitutes a major health problem mainly in tropical and subtropical countries with poor sanitary conditions. The different forms of the disease are characterized by massive tissue lesions. Amoeba-induced tissue destruction requires an intimate contact between E. histolytica trophozoites and host cells. This contact is predominantly mediated by a galactose-inhibitable lectin located on the surface of the amoebae. Therefore, the lectin is considered a prime candidate for the development of a vaccine to prevent amoebiasis. This communication reports on recent developments in characterizing the structure and function of the E. histolytica surface lectin and its use as a subunit vaccine.

Cyclooxygenase inhibitory constituents from Houttuynia cordata.

The n-hexane extract of Houttuynia cordata was shown to inhibit prostaglandin synthase in vitro. Phytochemical examination led to the identification of five fatty acids (linolenic, linoleic, oleic, palmitic and stearic), cepharanone B, phytol and stigmast-4-ene-3,6-dione. The inhibitory effect of the extract on prostaglandin formation in vitro could be attributed mainly to linoleic and linolenic acid.

Evaluation of three serological tests for the detection of antiamebic antibodies applied to sera of patients from an area endemic for amebiasis.

Two enzyme immuno assays based on a single recombinant Entamoeba histolytica antigen (P1-EIA) or soluble E. histolytica extract (SA-EIA) as well as a latex agglutination test using an E. histolytica membrane fraction (M-LA) were evaluated for its use to detect anti-amebic serum antibodies in patients from Durban, South Africa, an area endemic for amebiasis. In a previous study, all three test systems were found to be reliable in terms of sensitivity and specificity when applied to sera of European individuals. By analysing a total of 167 serum samples of patients from the Durban area, suffering from invasive amebiasis (n = 76) or miscellaneous diseases unrelated to E. histolytica infection (n = 91), the present study revealed sensitivity for the detection of anti-amebic antibodies of 97.4% for SA-EIA, 86.8% for P1-EIA and 96.1% for M-LA, respectively. Specificity was high for P1-EIA (96.7%) and M-LA (92.3%) but substantially lower for SA-EIA (62.6%). In addition, antibody responses to the recombinant P1 antigen were analysed in 16 patients with amebic liver abscess before and after anti-amebic treatment. The results indicated that most of the patients lost their specific antibody response within 7 month of follow up. Therefore, P1-EIA seems to be a valuable test for distinguishing between present and past E. histolytica infections.

Crude or recombinant proteins applied to latex agglutination, complement fixation and enzyme-linked immunosorbent assays for the serodiagnosis of invasive amebiasis.

Three different agglutination tests were developed for the detection of serum antibodies against Entamoeba histolytica. These tests are based on carboxylated polystyrene beads loaded either with a purified recombinant E. histolytica protein, designated recEh-P1, or a soluble fraction, or a membrane fraction (M-LA) both prepared from E. histolytica trophozoites. The three agglutination tests were compared with an enzyme-linked immunosorbent assay and a complement fixation test based on crude soluble E. histolytica antigens as well as with an enzyme-linked immunosorbent assay using recEh-P1 as antigen (P1-EIA). Serum samples from patients with invasive amebiasis (n = 30), or infectious diseases unrelated to E. histolytica (n = 57), as well as sera of apparently healthy individuals (n = 25) including some with noninvasive amebiasis (n = 5) were analysed by all six methods. Depending on the assay used, the results obtained, revealed sensitivities ranging from 83% to 100% and specificities ranging from 93% to 100%. P1-EIA and M-LA exhibited best results, both with a sensitivity of 100% and a specificity of 98%.

Diversionary role of hoofed game in the transmission of Lyme disease spirochetes.

To determine whether the presence of ungulates may inhibit transmission of the agent of Lyme disease (Borrelia burgdorferi) while promoting the abundance of its European vector tick (Ixodes ricinus), we compared the feeding density of subadult ticks on roe deer (Capreolus capreolus), red deer (Cervus elaphus), fallow deer (Dama dama), and wild sheep (Ovis ammon) near Berlin and in Brandenburg State, Germany. The prevalence of spirochetal infection in these ticks was compared with that in ticks swept from nearby vegetation. Spirochetes are present in nearly one-fifth of nonfed, questing nymphal and adult wood ticks in the region. Many ungulates in this intensely enzootic region fail to mount a detectable humoral response against the agent of Lyme disease, even when exposed to numerous infected ticks. During the height of the summer, each ungulate may support the feeding of hundreds of subadult ticks. Larvae feed lower on the bodies of hoofed game than do nymphs. Few ticks retain infection by the Lyme disease spirochete after feeding on hoofed game animals. We conclude that numerous I. ricinus ticks feed on ungulates, but that such host-contact fails to infect these ticks while eliminating pre-existing spirochetal infection.

Comparison of membrane ATPases from extreme halophiles isolated from ancient salt deposits.

Halophilic microorganisms were isolated from Triassic and Permian salt deposits. Two were rods and grew as red colonies; another was a coccus and produced pink colonies. The rods lysed in solutions that lacked added sodium chloride. Growth of all isolates was inhibited by aphidicolin and their bulk proteins were acidic as judged from isoelectric focusing. Therefore, these organisms were tentatively identified as extreme halophiles. Whole cell proteins patterns of the isolates following gel electrophoresis were distinct and differed from those of representative type strains of halophilic bacteria. The membrane ATPases from the rods were similar to the enzyme from Halobacterium saccharovorum with respect to subunit composition, enzymatic properties and immunological cross-reaction, but differed slightly in amino acid composition. If the age of the microbial isolated is similar to that of the salt deposits, they can be considered repositories of molecular information of great evolutionary interest.

Crystal structure of artemisinic acid: a possible biogenetic precursor of antimalarial artemisinin from Artemisia annua.

Artemisinic acid [1], a possible biogenetic precursor of the antimalarial artemisinin [2], was isolated from the hexane extract of Artemisia annua. X-ray crystallography of the dimer of artemisinic acid shows that the cyclization during intermolecular hydrogen bonding occurs by the opposite orientation of the alpha, beta-methylene group in each molecule. Complete spectroscopic data of 1 are also given.

Sensitive and specific serodiagnosis of invasive amebiasis by using a recombinant surface protein of pathogenic Entamoeba histolytica.

A recombinantly expressed protein, recEh-P1, representing part of an immunodominant surface antigen of pathogenic Entamoeba histolytica, was used for serodiagnosis of invasive amebiasis. Expression was performed under the control of a T7-RNA promoter by using a modified procaryotic expression vector, designated pHisT7. This vector allowed high-yield expression of recEh-P1 fused to a stretch of sequence containing eight histidine residues, which facilitated purification by metal chelate affinity chromatography on Ni2+ columns under highly denatured conditions. Purified recEh-P1 was found to be water soluble after prolonged dialysis and was used as the antigen for the detection of antiamebic serum antibodies by immunoblotting and enzyme-linked immunosorbent assay. In both tests all sera of patients with invasive amebiasis reacted to recEh-P1 whereas none of those collected from healthy controls, including individuals with noninvasive amebiasis, or from patients suffering from bacterial or protozoan infections unrelated to E. histolytica did so.