Girishchandra B. Patel

Ammonia toxicity in pure cultures of methanogenic bacteria

Summary The toxicity of ammonia to the growth of several methanogenic bacteria was evaluated in terms of an ammonia/potassium exchange reaction and in terms of inhibition of methanogenesis. Growth of Methanobrevibacter smithii, Methanobrevibacter arboriphilus and Methanobacterium strain G2R was normal in media containing up to 400 mM NH 4 Cl, and neither the exchange reaction nor inhibition of methane synthesis occurred. Exchange and inhibition of CH 4 synthesis by ammonia was found in shortterm studies with cell suspensions of Methanospirillum hungatei, Methanosarcina barkeri and Methanothrix concilii . Certain cations countered the toxic effects which ammonia had on methane synthesis, notably Ca 2+ in M. concilii and Na + in M. barkeri . During growth in media containing increasing amounts of NH 4 Cl, the cytoplasmic K + concentration declined dramatically only in M. hungatei , but this was not the mechanism of toxicity. Evidently the presence of counterions in the growth medium caused an enhanced tolerance of certain strains to ammonia. Methanobacterium bryantii displayed an appreciable decline in cytoplasmic K + content and grew slowly in media containing 300 mM NH 4 Cl. Ammoniatoxicity to the growth of all methanogenic bacteria tested can be correlated to alterations in their K + /NH 3 content and/or to inhibitions of CH 4 synthesis.

Archaeosome Vaccine Adjuvants Induce Strong Humoral, Cell-Mediated, and Memory Responses: Comparison to Conventional Liposomes and Alum

ABSTRACT Ether glycerolipids extracted from various archaeobacteria were formulated into liposomes (archaeosomes) possessing strong adjuvant properties. Mice of varying genetic backgrounds, immunized by different parenteral routes with bovine serum albumin (BSA) entrapped in archaeosomes (∼200-nm vesicles), demonstrated markedly enhanced serum anti-BSA antibody titers. These titers were often comparable to those achieved with Freunds adjuvant and considerably more than those with alum or conventional liposomes (phosphatidylcholine-phosphatidylglycerol-cholesterol, 1.8:0.2:1.5 molar ratio). Furthermore, antigen-specific immunoglobulin G1 (IgG1), IgG2a, and IgG2b isotype antibodies were all induced. Association of BSA with the lipid vesicles was required for induction of a strong response, and >80% of the protein was internalized within most archaeosome types, suggesting efficient release of antigen in vivo. Encapsulation of ovalbumin and hen egg lysozyme within archaeosomes showed similar immune responses. Antigen-archaeosome immunizations also induced a strong cell-mediated immune response: antigen-dependent proliferation and substantial production of cytokines gamma interferon (Th1) and interleukin-4 (IL-4) (Th2) by spleen cells in vitro. In contrast, conventional liposomes induced little cell-mediated immunity, whereas alum stimulated only an IL-4 response. In contrast to alum and Freunds adjuvant, archaeosomes composed of Thermoplasma acidophilum lipids evoked a dramatic memory antibody response to the encapsulated protein (at ∼300 days) after only two initial immunizations (days 0 and 14). This correlated with increased antigen-specific cell cycling of CD4+ T cells: increase in synthetic (S) and mitotic (G2/M) and decrease in resting (G1) phases. Thus, archaeosomes may be potent vaccine carriers capable of facilitating strong primary and memory humoral, and cell-mediated immune responses to the entrapped antigen.

Methanosaeta concilii gen. nov. sp. nov. ("Methanothrix concilii") and Methanosaeta thermoacetophila nom. rev., comb. nov.?

Methanosaeta concilii gen. nov., sp. nov. (“Methanothrix concilii”) is described. Cells of species in the genus Methanosaeta are obligately anaerobic, gram-negative, nonmotile rods (length, 2.5 to 6.0 μm) with flat ends. The cells are enclosed within a proteinaceous, cross-striated sheath. Growth can occur as long filaments which represent chains of individual cells separated by spacer plugs and continuously enclosed by the tubular sheath. Acetic acid is used as the sole source of energy; its metabolism results in the production of about equimolar amounts of CH4 and CO2. Acetic acid and CO2 are carbon sources for growth. The description of Methanosaeta concilii, the type species, is based on type strain GP6 (= DSM 3671 = OGC 69 = NRC 2989 = ATCC 35969).

Stability of pressure-extruded liposomes made from archaeobacterial ether lipids

Ether lipids were obtained from a wide range of archaeobacteria grown at extremes of pH, temperature, and salt concentration. With the exception ofSulfolobus acidocaldarius, unilamellar and/or multilamellar liposomes could be prepared from emulsions of total polar lipid extracts by pressure extrusion through filters of various pore sizes. Dynamic light scattering, and electron microscopy revealed homogeneous liposome populations with sizes varying from 40 to 230 nm, depending on both the lipid source and the pore size of the filters. Leakage rates of entrapped fluorescent or radioactive compounds established that those archaeobacterial liposomes that contained tetraether lipids were the most stable to high temperatures, alkaline pH, and serum proteins. Most ether liposomes were stable to phospholipase A2, phospholipase B and pancreatic lipase. These properties of archaeobacterial liposomes make them attractive for applications in biotechnology.

Archaeosomes Induce Long-Term CD8+ Cytotoxic T Cell Response to Entrapped Soluble Protein by the Exogenous Cytosolic Pathway, in the Absence of CD4+ T Cell Help

The unique ether glycerolipids of Archaea can be formulated into vesicles (archaeosomes) with strong adjuvant activity for MHC class II presentation. Herein, we assess the ability of archaeosomes to facilitate MHC class I presentation of entrapped protein Ag. Immunization of mice with OVA entrapped in archaeosomes resulted in a potent Ag-specific CD8+ T cell response, as measured by IFN-γ production and cytolytic activity toward the immunodominant CTL epitope OVA257–264. In contrast, administration of OVA with aluminum hydroxide or entrapped in conventional ester-phospholipid liposomes failed to evoke significant CTL response. The archaeosome-mediated CD8+ T cell response was primarily perforin dependent because CTL activity was undetectable in perforin-deficient mice. Interestingly, a long-term CTL response was generated with a low Ag dose even in CD4+ T cell deficient mice, indicating that the archaeosomes could mediate a potent T helper cell-independent CD8+ T cell response. Macrophages incubated in vitro with OVA archaeosomes strongly stimulated cytokine production by OVA-specific CD8+ T cells, indicating that archaeosomes efficiently delivered entrapped protein for MHC class I presentation. This processing of Ag was Brefeldin A sensitive, suggesting that the peptides were transported through the endoplasmic reticulum and presented by the cytosolic MHC class I pathway. Finally, archaeosomes induced a potent memory CTL response to OVA even 154 days after immunization. This correlated to strong Ag-specific up-regulation of CD44 on splenic CD8+ T cells. Thus, delivery of proteins in self-adjuvanting archaeosomes represents a novel strategy for targeting exogenous Ags to the MHC class I pathway for induction of CTL response.

Isolation and Characterization of an Anaerobic, Cellulolytic Microorganism, Acetivibrio cellulolyticus gen. nov., sp. nov.†

A new genus of cellulolytic, gram-negative, nonsporeforming, anaerobic bacteria is described. The colonies produced by these bacteria on cellulose agar were round, clear, translucent, and cream-colored and had an undulate margin. Single cells of the bacterium were straight to slightly curved rods 0.5 to 0.8 μm wide by 4 to 10 μm long and were motile by means of a single flagellum located one-third of the distance from the end of the cell. Among the various substrates tested, only cellulose, cellobiose, and salicin were able to support growth. The major fermentation products of cellobiose or cellulose degradation were acetic acid, hydrogen, and carbon dioxide. The deoxyribonucleic acid base composition of the type species was 38 mol% guanine plus cytosine. The name Acetivibrio is proposed for this new genus, which is placed in the family Bacteroidaceae. The type species, Acetivibrio cellulolyticus sp. nov., is named on the basis of its cellulolytic activity. The type strain of A. cellulolyticus is CD2 (= NRC 2248).

In vitro assessment of archaeosome stability for developing oral delivery systems

The in vitro stability of archaeosomes made from the total polar lipids of Methanosarcina mazei, Methanobacterium espanolae or Thermoplasma acidophilum, was evaluated under conditions encountered in the human gastrointestinal tract. At acidic pH, multilamellar vesicles (MLV) prepared from T. acidophilum lipids were the most stable, releasing approximately 80, 20, 10 and 5% of encapsulated 14C-sucrose at pH 1.5, 2.0, 2.5 and 6.2, respectively, after 90 min at 37 degrees C. Archaeosomes from M. mazei lipids were the least stable. For each type of total polar lipid, unilamellar vesicles (ULV) were less stable than the corresponding MLV vesicles. Pancreatic lipase had relatively minor effect on the stability of archaeosomes made from either of the three types of total polar lipids, causing the release of 12-27% of the encapsulated 5(6)-carboxyfluorescein (CF) from ULV and MLV after 90 min at 37 degrees C. In simulated human bile at pH 6.2, MLV from M. mazei total polar lipids lost 100% of the encapsulated CF after 90 min at 37 degrees C, whereas those from the polar lipids of M. espanolae or T. acidophilum lost approximately 85% of the marker. Pancreatic lipase and simulated human bile had no synergistic effect on the release of carboxyfluorescein from ULV or MLV prepared from any of the total polar lipids. After 90 min in the combined presence of these two stressors at pH 6.2, the leakage of fluorescein conjugated bovine serum albumin from MLV prepared from T. acidophilum lipids was similar to that of CF, and 13% of the initially present vesicles appeared to be intact. These results indicate that archaeosomes show stability properties indicative of potential advantages in developing applications as an oral delivery system.

The Potent Adjuvant Activity of Archaeosomes Correlates to the Recruitment and Activation of Macrophages and Dendritic Cells In Vivo

The unique glycerolipids of Archaea can be formulated into vesicles (archaeosomes) with potent adjuvant activity. We studied the effect of archaeosomes on APCs to elucidate the mechanism(s) of adjuvant action. Exposure of J774A.1 macrophages to archaeosomes in vitro resulted in up-regulation of B7.1, B7.2, and MHC class II molecules to an extent comparable to that achieved with LPS. Similarly, incubation of bone marrow-derived DCs with archaeosomes resulted in enhanced expression of MHC class II and B7.2 molecules. In contrast, conventional liposomes made from ester phospholipids failed to modulate the expression of these activation markers. APCs treated with archaeosomes exhibited increased TNF production and functional ability to stimulate allogenic T cell proliferation. More interestingly, archaeosomes enhanced APC recruitment and activation in vivo. Intraperitoneal injection of archaeosomes into mice led to recruitment of Mac1α+, F4/80+ and CD11c+ cells. The expression of MHC class II on the surface of peritoneal cells was also enhanced. Furthermore, peritoneal cells from archaeosome-injected mice strongly enhanced allo-T cell proliferation and cytokine production. The ability of archaeosome-treated APCs to stimulate T cells was restricted to Mac1αhigh, B220− cells in the peritoneum. These Mac1αhigh cells in the presence of GM-CSF gave rise to both F4/80+ (macrophage) and CD11c+ (dendritic) populations. Overall, the activation of APCs correlated to the ability of archaeosomes to induce strong humoral, T helper, and CTL responses to entrapped Ag. Thus, the recruitment and activation of professional APCs by archaeosomes constitutes an efficient self-adjuvanting process for induction of Ag-specific responses to encapsulated Ags.

Identification of β-l-gulose as the sugar moiety of the main polar lipid of Thermoplasma acidophilum

Abstract The main polar lipid (MPL) of Thermoplasma acidophilum has been purified and its structure determined. NMR, mass spectrometry, and capillary gas chromatography-mass spectrometry experiments have shown that the previously unidentified sugar moiety of MPL is the rare sugar l -gulose. MPL is thus a tetraether lipid with cyclopentane rings and head groups of phosphoglycerol, as previously reported, and β- l -gulopyranose. Further, MPL is also the dominant lipid found in lipid extracts from another species of the Thermoplasma genus, T. volcanium, suggesting that l -gulose may represent a dominant sugar moiety of the polar lipids biosynthesized by this archaeobacterial genus. Minor phospholipids were tentatively identified as diether and hydroxydiether analogs of phosphatidylglycerol, and phosphatidylinositol.

Immunization of mice with lipopeptide antigens encapsulated in novel liposomes prepared from the polar lipids of various Archaeobacteria elicits rapid and prolonged specific protective immunity against infection with the facultative intracellular pathogen, Listeria monocytogenes.

Protective immunity to intracellular bacterial pathogens usually requires the participation of specific CD8+ T cells. Natural exposure of the host to sublethal infection, or vaccination with attenuated live vaccines are the most effective means of eliciting prolonged protective cell-mediated immunity against this class of pathogens. The ability to replace these immunization strategies with defined sub-unit vaccines would represent a major advance for clinical vaccinology. The present study examines the ability of novel liposomes, termed archaeosomes, made from the polar lipids of various Archaeobacteria to act as self-adjuvanting vaccine delivery vehicles for such defined acellular antigens. Using infection of mice with Listeria monocytogenes as a model system, this study clearly demonstrates the ability of defined, archaeosome-entrapped antigens to elicit rapid and prolonged specific immunity against a prototypical intracellular pathogen. In this regard, all of the tested archaeosomes were superior to conventional liposomes.