T cell-mediated regulation of the microbiota protects against obesity.

The microbiota influences obesity, yet organisms that protect from disease remain unknown. During studies interrogating host-microbiota interactions, we observed the development of age-associated metabolic syndrome (MetS). Expansion of Desulfovibrio and loss of Clostridia were key features associated with obesity in this model and are present in humans with MetS. T cell-dependent events were required to prevent disease, and replacement of Clostridia rescued obesity. Inappropriate immunoglobulin A targeting of Clostridia and increased Desulfovibrio antagonized the colonization of beneficial Clostridia. Transcriptional and metabolic analysis revealed enhanced lipid absorption in the obese host. Colonization of germ-free mice with Clostridia, but not Desulfovibrio, down-regulated genes that control lipid absorption and reduced adiposity. Thus, immune control of the microbiota maintains beneficial microbial populations that constrain lipid metabolism to prevent MetS.

Immunological alteration and changes of gut microbiota after dextran sulfate sodium (DSS) administration in mice.

Ulcerative colitis (UC) is characterized by chronic inflammation of the colonic mucosa. Administration of dextran sulfate sodium (DSS) to animals is a frequently used model to mimic human colitis. Deregulation of the immune response to the enteric microflora or pathogens as well as increased intestinal permeability have been proposed as disease-driving mechanisms. To enlarge the understanding of the pathogenesis, we have studied the effect of DSS on the immune system and gut microbiota in mice. Intestinal inflammation was verified through histological evaluation and myeloperoxidase activity. Immunological changes were assessed by flow cytometry in spleen, Peyer's patches and mesenteric lymph nodes and through multiplex cytokine profiling. In addition, quantification of the total amount of bacteria on colonic mucosa as well as the total amount of lactobacilli, Akkermansia, Desulfovibrio and Enterobacteriaceae was performed by the use of quantitative PCR. Diversity and community structure were analysed by terminal restriction fragment length polymorphism (T-RFLP) patterns, and principal component analysis was utilized on immunological and T-RFLP patterns. DSS-induced colitis show clinical and histological similarities to UC. The composition of the colonic microflora was profoundly changed and correlated with several alterations of the immune system. The results demonstrate a relationship between multiple immunological changes and alterations of the gut microbiota after DSS administration. These data highlight and improve the definition of the immunological basis of the disease and suggest a role for dysregulation of the gut microbiota in the pathogenesis of colitis.

The effect of culture media on antigenic expression in sulfate-reducing bacteria.

The importance of sulfate-reducing bacteria (SRB) in nature has been widely recognized for many years. However, little is known about the ecology of SRB. The problem has been detecting, classifying, and quantifying these organisms. There are many shortcomings in the use of culture media for this purpose. As an alternative, fluorescent antibody (FA) techniques were considered as a method for the detection and identification of SRB. Antisera were prepared against whole cells of different species of SRB and evaluated for detection and identification of these organisms. Surface antigens of SRB were species specific. In addition, culture conditions influenced the expression of surface antigens, causing the antisera to be extremely specific. These results were confirmed by the sodium dodecyl sulfate-polyacrylamide-gel electrophoresis (SDS-PAGE) profiles of membrane proteins. On the basis of this specificity, the application of FA produced against culture collection strains would have limited application for detecting, identifying, and enumerating these organisms in nature.

Application of antisera raised against sulfate-reducing bacteria for indirect immunofluorescent detection of immunoreactive bacteria in sediment from the German Baltic Sea.

Polyclonal rabbit antisera raised against sulfate-reducing bacteria (SRB) could detect several distinct populations of bacteria in sediment from the German Baltic Sea. The depth distribution of immunoreactive bacteria was determined by an indirect immunofluorescence filter method. Anti-Desulfovibrio desulfuricans DSM 1926 serum showed maximum bacterial numbers at a depth of 18 cm, with a concentration of 60 x 10(6) cells cm-3. With anti-Desulfovibrio baculatus DSM 2555 serum, counts were highest at the same depth, approaching 0.7 x 10(6) cells cm-3. Other significantly smaller populations were observed. Anti-SRBStrain 1 (lactate,vibrio) maxima were at 0 to 4 cm and at 17 to 18 cm. Anti-SRBStrain 2 (lactate,vibrio) serum showed several local maxima. Anti-SRBStrain 3 (lactate,oval) serum detected one single peak at a depth of 10 to 12 cm. Also determined were rates of sulfate reduction, total bacterial counts by acridine orange staining, and the viable counts by dilution series on anaerobic lactate medium. The total bacterial counts were highest (180 x 10(6) cells cm-3) at 3 to 4 cm and dropped to 24 x 10(6) cells cm-3 at 10 to 11 cm but showed additional local maxima reaching 140 x 10(6) cells cm-3 at a depth of 17 to 18 cm. Viable counts probable number) were above 10(5) CFU cm-3 at 0 to 3.6 cm but remained below 10(3) CFU at 7.2 to 18 cm. The sulfate reduction rate was maximal (107 nmol cm-3 day-1) at a depth of 1 to 2 cm, dropped to 10 nmol cm-3 day-1 at 12 to 13 cm, and reached 38 nmol cm-3 day-1 at 17 to 18 cm.

The nucleotide sequence of the Desulfovibrio gigas desulforedoxin gene indicates that the Desulfovibrio vulgaris rbo gene originated from a gene fusion event.

Expression of the rbo gene from Desulfovibrio vulgaris Hildenborough in Escherichia coli minicells and Western blotting (immunoblotting) of Desulfovibrio cell extracts with antibodies raised against a synthetic peptide indicated the presence of a 14-kDa polypeptide product, as expected from the gene sequence. Cloning and sequencing of the gene (dsr) for desulforedoxin, a 4-kDa redox protein from Desulfovibrio gigas, showed that it is formed by expression of an autonomous gene of 111 bp, not by processing of a 14-kDa protein. The results indicate that the rbo gene product, which has a 4-kDa desulforedoxin domain as the NH2 terminus, may have arisen by gene fusion. Shuffling and fusion of genes for redox protein domains can explain the large variety of redox proteins found in sulfate-reducing bacteria.

Interaction of cellular hydrogenase, cytochrome c3, and desulfoviridin in Desulfovibrio vulgaris Miyazaki with their antibodies.

Anti-sera for hydrogenase, cytochrome c3, and desulfoviridin (abbreviated as anti-hyd, anti-c3, and anti-dvn, respectively) were raised in mice, and used to locate these antigens in cells of Desulfovibrio vulgaris Miyazaki. The activity of the intact cells to absorb H2 with methyl viologen or sulfite as an electron acceptor was cumulatively inhibited by treating the cells with anti-hyd and anti-c3 but unaffected by anti-dvn treatment. The activity of the intact cells to produce H2 from formate was also inhibited by anti-c3 treatment, but the inhibition by anti-hyd treatment was not significant. The fluorescent antibody technique applied to intact cells of D. vulgaris Miyazaki indicated that both hydrogenase and cytochrome c3 are localized on the surface of the cell. These results are not exactly in conformity with the hydrogen-cycling hypothesis for proton gradient formation in the energy metabolism in Desulfovibrio. The procedure described in the present paper provides a new technique to elucidate the roles of proteins by applying anti-sera to intact cells without destroying the cellular structure.

Serological characteristics within the genus Desulfovibrio.

Antisera were prepared against one strain each of Desulfovibrio desulfuricans, D. vulgaris and D. salexigens. The antisera were tested for cross reactivity against 36 heterologous Desulfovibrio strains by both agglutination titration and by double immunodiffusion percipitin plates. Generally no cross-reaction was demonstrated by agglutination even between heterologous strains of the same species, suggesting that the surface antigens of Desulfovibrio are highly specific. In immunodiffusion plates a single apparently genus-specific surface antigen could be shown to be present in all but two of the strains tested. Although other common precipitin bands showed the presence of some antigens common between heterologous strains these appeared to be randomly distributed among the strains tested, with the exception of one band shown to be generally specific to strains of D. salexigens. With this exception no other precipitin band could be shown to be consistently specific to any other species, nor consistently common to more than one species.

Desulfovibrio Africans sp. n., a new dissimilatory sulfate-reducing bacterium.

Campbell, L. Leon (University of Illinois, Urbana), Mary A. Kasprzycki, and John R. Postgate. Desulfovibrio africanus sp. n., a new dissimilatory sulfate-reducing bacterium. J. Bacteriol. 92:1122-1127. 1966.-The strains Benghazi and Walvis Bay can be distinguished from 40 strains of Desulfovibrio and from D. gigas on the basis of morphological and immunological studies. Electron microscopy revealed polar lophotrichous flagellation similar to that of D. gigas but different from the characteristic single polar flagellum of the 40 strains of Desulfovibrio. Immunological evidence shows that the two strains are related to members of the genus Desulfovibrio but possess several common antigenic components not present in the other strains tested. The deoxyribonucleic acid of both strains has a buoyant density of 1.724 g/cc and a guanine plus cytosine content of 60.2%. Cell-free extracts of both organisms show absorption bands of cytochrome c(3) and desulfoviridin, characteristic for Desulfovibrio. The two organisms carry out the sulfate-linked lactate fermentation and neither will grow in the absence of sulfate. Both strains contain the enzymes of the dissimilatory pathway of sulfate reduction. Therefore, these studies have demonstrated that the Benghazi and Walvis Bay strains should be regarded as taxonomically distinct from other species of Desulfovibrio.