Influence of pH and the degree of protonation on the inhibitory effect of fatty acids in the ruminal methanogen Methanobrevibacter ruminantium strain M1.

AIMS: To investigate the relationship between the protonation of medium-chain fatty acids (MCFA) and their inhibitory effect on a ruminal methanogen species. METHODS AND RESULTS: Cell suspensions of Methanobrevibacter ruminantium M1 in 1 mg dry matter (DM) ml(-1) were supplemented with lauric acid (C12 ) and myristic acid (C14 ) at a concentration of 8 µg ml(-1) with different pH levels of the potassium-free buffer, where the calculated degrees of protonation of C12 and C14 varied from 0·3 to 50% and from 1 to 76% respectively. Methane formation, ATP efflux, potassium leakage and cell viability were monitored 15, 30 and 45 min after the reaction started. Declining methane formation rate, increasing ATP efflux and potassium leakage, and decreasing survival of M. ruminantium were observed with increasing degrees of protonation, i.e. with decreasing pH. CONCLUSIONS: The inhibition of methanogenesis by C12 and C14 is more efficient at a pH of 5-6 as compared to pH 7. SIGNIFICANCE AND IMPACT OF THE STUDY: Methane mitigation strategies in ruminants which use supplementation of feed with MCFA such as C12 and C14 may be more effective in a low rumen pH environment. This finding is helpful in designing diets to effectively decrease methane emissions by ruminants.

Inhibition of methyl-CoM Reductase from Methanobrevibacter ruminantium by 2-bromoethanesulfonate.

Cattle husbandry is a major contributor to atmospheric methane, which is considered as an important greenhouse gas. Moreover, the generation of methane in the intestine of domestic ruminants by methanogenic bacteria is a drag on feed efficacy. Studies on methanogenesis have typically implied model organisms that are, however, not relevant in the ruminant gut. This paper shows that methyl-CoM reductase catalyzing the final step of methanogenesis in Methanobrevibacter ruminantium, a major participant in methane production by cattle, is inhibited by 2-bromoethanesulfonate, a compound often used as a model in animal agriculture, with an apparent IC50 of 0.4 ± 0.04 µM.

Archaeosome adjuvant overcomes tolerance to tumor-associated melanoma antigens inducing protective CD8 T cell responses.

Vesicles comprised of the ether glycerolipids of the archaeon Methanobrevibacter smithii (archaeosomes) are potent adjuvants for evoking CD8(+) T cell responses. We therefore explored the ability of archaeosomes to overcome immunologic tolerance to self-antigens. Priming and boosting of mice with archaeosome-antigen evoked comparable CD8(+) T cell response and tumor protection to an alternate boosting strategy utilizing live bacterial vectors for antigen delivery. Vaccination with melanoma antigenic peptides TRP(181-189) and Gp100(25-33) delivered in archaeosomes resulted in IFN-γ producing antigen-specific CD8(+) T cells with strong cytolytic capability and protection against subcutaneous B16 melanoma. Targeting responses against multiple antigens afforded prolonged median survival against melanoma challenge. Entrapment of multiple peptides within the same vesicle or admixed formulations were both effective at evoking CD8(+) T cells against each antigen. Melanoma-antigen archaeosome formulations also afforded therapeutic protection against established B16 tumors when combined with depletion of T-regulatory cells. Overall, we demonstrate that archaeosome adjuvants constitute an effective choice for formulating cancer vaccines.

Structural characterization of archaeal lipid mucosal vaccine adjuvant and delivery (AMVAD) formulations prepared by different protocols and their efficacy upon intranasal immunization of mice.

Intranasal administration of ovalbumin (OVA) formulated in an archaeal lipid mucosal vaccine adjuvant and delivery (AMVAD) system prepared by the addition of CaCl2 to small unilamellar archaeosomes (liposomes made from archaeal polar lipids) containing encapsulated OVA, was recently shown to elicit strong and sustained OVA-specific mucosal and systemic immune responses. In this study, we show that the centrifugation/washing and antigen quantization steps required in the standard protocol for obtaining OVA/AMVAD model vaccine formulations can be eliminated by using simpler protocols such as admixing OVA with preformed empty archaeosomes, or by changing the starting ratio (w/w) of archaeal lipid to antigen at the archaeosome preparation stage, prior to the addition of CaCl2 to convert to the AMVAD structures. Irrespective of the vaccine preparation protocol, the AMVAD particle typically comprised of larger spherical structures that had aggregated like a bunch of grapes, and it contained aqueous compartment(s). The anti-OVA IgA antibody responses in vaginal wash, nasal wash, serum, and bile samples, and the anti-OVA IgG antibody responses in sera, in mice intranasally immunized with the OVA/AMVAD formulations prepared by the simplified or the standard protocols, were comparable.