Cytotoxic T lymphocyte antigen-4 regulates development of xenogenic graft versus host disease in mice via modulation of host immune responses induced by changes in human T cell engraftment and gene expression.

Graft versus host disease (GvHD) is a major clinical problem with a significant unmet medical need. We examined the role of cytotoxic T lymphocyte antigen-4 (CTLA-4) in a xenogenic GvHD (xeno-GvHD) model induced by injection of human peripheral mononuclear cells (hPBMC) into irradiated non-obese diabetic (NOD) SCID gamma (NSG) mice. Targeting the CTLA-4 pathway by treatment with CTLA-4 immunoglobulin (Ig) prevented xeno-GvHD, while anti-CTLA-4 antibody treatment exacerbated the lethality and morbidity associated with GvHD. Xeno-GvHD is associated with infiltration of hPBMCs into the lungs, spleen, stomach, liver and colon and an increase in human proinflammatory cytokines, including interferon (IFN)-γ, tumor necrosis factor (TNF)-α and interleukin (IL)-5. Infiltration of donor cells and increases in cytokines were attenuated by treatment with CTLA-4 Ig, but remained either unaffected or enhanced by anti-CTLA-4 antibody. Further, splenic human T cell phenotyping showed that CTLA-4 Ig treatment prevented the engraftment of human CD45+ cells, while anti-CTLA-4 antibody enhanced donor T cell expansion, particularly CD4+ (CD45RO+ ) subsets, including T box transcription factor TBX21 (Tbet)+ CXCR3+ and CD25+ forkhead box protein 3 (FoxP3) cells. Comprehensive analysis of transcriptional profiling of human cells isolated from mouse spleen identified a set of 417 differentially expressed genes (DEGs) by CTLA-4 Ig treatment and 13 DEGs by anti-CTLA-4 antibody treatment. The CTLA-4 Ig regulated DEGs mapped to down-regulated apoptosis, inflammasome, T helper type 17 (Th17) and regulatory T cell (Treg ) pathways and enhanced Toll-like receptor (TLR) receptor signaling, TNF family signaling, complement system and epigenetic and transcriptional regulation, whereas anti-CTLA-4 antibody produced minimal to no impact on these gene pathways. Our results show an important role of co-inhibitory CTLA-4 signaling in xeno-GvHD and suggest the therapeutic utility of other immune checkpoint co-inhibitory pathways in the treatment of immune-mediated diseases driven by hyperactive T cells.

Distinct roles of histamine H1- and H2-receptor signaling pathways in inflammation-associated colonic tumorigenesis.

Inflammatory bowel disease (IBD) is a well-known risk factor for the development of colorectal cancer. Prior studies have demonstrated that microbial histamine can ameliorate intestinal inflammation in mice. We tested the hypothesis whether microbe-derived luminal histamine suppresses inflammation-associated colon cancer in Apcmin/+ mice. Mice were colonized with the human-derived Lactobacillus reuteri. Chronic inflammation was induced by repeated cycles of low-dose dextran sulfate sodium (DSS). Mice that were given histamine-producing L. reuteri via oral gavage developed fewer colonic tumors, despite the presence of a complex mouse gut microbiome. We further demonstrated that administration of a histamine H1-receptor (H1R) antagonist suppressed tumorigenesis, while administration of histamine H2-receptor (H2R) antagonist significantly increased both tumor number and size. The bimodal functions of histamine include protumorigenic effects through H1R and antitumorigenic effects via H2R, and these results were supported by gene expression profiling studies on tumor specimens of patients with colorectal cancer. Greater ratios of gene expression of H2R ( HRH2) vs. H1R ( HRH1) were correlated with improved overall survival outcomes in patients with colorectal cancer. Additionally, activation of H2R suppressed phosphorylation of mitogen-activated protein kinases (MAPKs) and inhibited chemokine gene expression induced by H1R activation in colorectal cancer cells. Moreover, the combination of a H1R antagonist and a H2R agonist yielded potent suppression of lipopolysaccharide-induced MAPK signaling in macrophages. Given the impact on intestinal epithelial and immune cells, simultaneous modulation of H1R and H2R signaling pathways may be a promising therapeutic target for the prevention and treatment of inflammation-associated colorectal cancer. NEW & NOTEWORTHY Histamine-producing Lactobacillus reuteri can suppress development of inflammation-associated colon cancer in an established mouse model. The net effects of histamine may depend on the relative activity of H1R and H2R signaling pathways in the intestinal mucosa. Our findings suggest that treatment with H1R or H2R antagonists could yield opposite effects. However, by harnessing the ability to block H1R signaling while stimulating H2R signaling, novel strategies for suppression of intestinal inflammation and colorectal neoplasia could be developed.

Gut Microbe-Mediated Suppression of Inflammation-Associated Colon Carcinogenesis by Luminal Histamine Production.

Microbiome-mediated suppression of carcinogenesis may open new avenues for identification of therapeutic targets and prevention strategies in oncology. Histidine decarboxylase (HDC) deficiency has been shown to promote inflammation-associated colorectal cancer by accumulation of CD11b+Gr-1+ immature myeloid cells, indicating a potential antitumorigenic effect of histamine. Here, we demonstrate that administration of hdc+Lactobacillus reuteri in the gut resulted in luminal hdc gene expression and histamine production in the intestines of Hdc-/- mice. This histamine-producing probiotic decreased the number and size of colon tumors and colonic uptake of [18F]-fluorodeoxyglucose by positron emission tomography in Hdc-/- mice. Administration of L. reuteri suppressed keratinocyte chemoattractant (KC), Il22, Il6, Tnf, and IL1α gene expression in the colonic mucosa and reduced the amounts of proinflammatory, cancer-associated cytokines, keratinocyte chemoattractant, IL-22, and IL-6, in plasma. Histamine-generating L. reuteri also decreased the relative numbers of splenic CD11b+Gr-1+ immature myeloid cells. Furthermore, an isogenic HDC-deficient L. reuteri mutant that was unable to generate histamine did not suppress carcinogenesis, indicating a significant role of the cometabolite, histamine, in suppression of chronic intestinal inflammation and colorectal tumorigenesis. These findings link luminal conversion of amino acids to biogenic amines by gut microbes and probiotic-mediated suppression of colorectal neoplasia.

Compositional and functional features of the gastrointestinal microbiome and their effects on human health.

The human gastrointestinal tract contains distinct microbial communities that differ in composition and function based on their location, as well as age, sex, race/ethnicity, and diet of their host. We describe the bacterial taxa present in different locations of the GI tract, and their specific metabolic features. The distinct features of these specific microbial communities might affect human health and disease. Several bacterial taxa and metabolic modules (biochemical functions) have been associated with human health and the absence of disease. Core features of the healthy microbiome might be defined and targeted to prevent disease and optimize human health.

Rapid identification of heterozygous mutations in Drosophila melanogaster using genomic capture sequencing.

One of the key advantages of using Drosophila melanogaster as a genetic model organism is the ability to conduct saturation mutagenesis screens to identify genes and pathways underlying a given phenotype. Despite the large number of genetic tools developed to facilitate downstream cloning of mutations obtained from such screens, the current procedure remains labor intensive, time consuming, and costly. To address this issue, we designed an efficient strategy for rapid identification of heterozygous mutations in the fly genome by combining rough genetic mapping, targeted DNA capture, and second generation sequencing technology. We first tested this method on heterozygous flies carrying either a previously characterized dac(5) or sens(E2) mutation. Targeted amplification of genomic regions near these two loci was used to enrich DNA for sequencing, and both point mutations were successfully identified. When this method was applied to uncharacterized twr mutant flies, the underlying mutation was identified as a single-base mutation in the gene Spase18-21. This targeted-genome-sequencing method reduces time and effort required for mutation cloning by up to 80% compared with the current approach and lowers the cost to <$1000 for each mutant. Introduction of this and other sequencing-based methods for mutation cloning will enable broader usage of forward genetics screens and have significant impacts in the field of model organisms such as Drosophila.

Butyrate-Producing Bacteria and Insulin Homeostasis: The Microbiome and Insulin Longitudinal Evaluation Study (MILES).

Gut microbiome studies have documented depletion of butyrate-producing taxa in type 2 diabetes. We analyzed associations between butyrate-producing taxa and detailed measures of insulin homeostasis, whose dysfunction underlies diabetes in 224 non-Hispanic Whites and 129 African Americans, all of whom completed an oral glucose tolerance test. Stool microbiome was assessed by whole-metagenome shotgun sequencing with taxonomic profiling. We examined associations among 36 butyrate-producing taxa (n = 7 genera and 29 species) and insulin sensitivity, insulin secretion, disposition index, insulin clearance, and prevalence of dysglycemia (prediabetes plus diabetes, 46% of cohort), adjusting for age, sex, BMI, and race. The genus Coprococcus was associated with higher insulin sensitivity (ß = 0.14; P = 0.002) and disposition index (ß = 0.12; P = 0.012) and a lower rate of dysglycemia (odds ratio [OR] 0.91; 95% CI 0.85-0.97; P = 0.0025). In contrast, Flavonifractor was associated with lower insulin sensitivity (ß = -0.13; P = 0.004) and disposition index (ß = -0.11; P = 0.04) and higher prevalence of dysglycemia (OR 1.22; 95% CI 1.08-1.38; P = 0.0013). Species-level analyses found 10 bacteria associated with beneficial directions of effects and two bacteria with adverse associations on insulin homeostasis and dysglycemia. Although most butyrate producers analyzed appear to be metabolically beneficial, this is not the case for all such bacteria, suggesting that microbiome-directed therapeutic measures to prevent or treat diabetes should be targeted to specific butyrate-producing taxa rather than all butyrate producers.

The nos gene cluster from gram-positive bacterium Geobacillus thermodenitrificans NG80-2 and functional characterization of the recombinant NosZ.

The nos gene cluster encoding the activity of nitrous oxide reductase (N2OR) for the final step of the denitrification pathway has been well studied in gram-negative bacteria. Our previous study on the genome of Geobacillus thermodenitrificans NG80-2 revealed the presence of the nos gene cluster in this gram-positive bacterium. In this follow-up study, the nos gene cluster of G. thermodenitrificans NG80-2 was further analyzed and compared with those of other origins. The structural gene nosZ was heterologously expressed in Escherichia coli and the product was purified as a His-tagged fusion protein. The recombinant NosZ as prepared showed detectable N2OR activity, and the activity was enhanced by preincubation of the protein under argon and with copper compounds. The recombinant NosZ contains 2.5 atoms of copper per dimer and exhibits weak spectral features in the visible range, indicating that spontaneous incorporation of copper compounds into the NG80-2 NosZ can result in some but not full activity of the authentic NG80-2 N2OR. The enzymatic properties of the NosZ were also investigated. This is the first functional characterization of nosZ gene from gram-positive bacteria. This study indicates that the molecular mechanism for N2O reduction is conserved between gram-negative and gram-positive bacteria.

[Molecular characterization of O-antigen gene cluster of Escherichia coli O23 reference strain and identification of UDP-N-acetylglucosamine 4-epimerase].

Lipopolysaccharide (LPS) is one of the major components of the outer membrane of gram-negative bacteria. It is an amphipathic molecule composing of lipid A, a core oligosaccharide and an O-specific antigen. O-antigen, which is a repeat unit polysaccharide, is a major contribution to the antigenic variability of the bacterial cell surface. Genes involved in O-antigen biosynthesis are generally found to be clustered between the housekeeping genes galF and gnd on the chromosome of E. coli. E. coli O23 is one of the enterotoxigenic E. coli causing pediatric diarrhea in the developing world. The O-antigen gene cluster of E. coli O23 type strain was amplified by long-range PCR using primers based on galF and gnd and then sequenced. Except for galF and gnd, seven open reading frames were identified and assigned functions on the basis of their similarity to those from available databases. The seven genes include a UDP-N-acetylglucosamine 4-epimerase gene (gne), the O-antigen polymerase gene (wzy), the O-antigen transferase gene (wzx) and four glycosyltransferase genes (orf2, orf4, orf5, orf6). The UDP-N-acetylglucosamine 4-epimerase (Gne) was identified by mutation and complementation complement tests. The structure of Gne was predicted by the homology modeling method, and the active sites were also analyzed. The phylogenetic and structural analysis showed that the Gne derived from the common ancestor with E. coli O23 Gne were UDP-GlcNAc/UDP-GalNAc epimerases. The specific DNA used for rapid molecular genotyping for E. coli O23 was also identified.

FolC2-mediated folate metabolism contributes to suppression of inflammation by probiotic Lactobacillus reuteri.

Bacterial-derived compounds from the intestinal microbiome modulate host mucosal immunity. Identification and mechanistic studies of these compounds provide insights into host-microbial mutualism. Specific Lactobacillus reuteri strains suppress production of the proinflammatory cytokine, tumor necrosis factor (TNF), and are protective in a mouse model of colitis. Human-derived L. reuteri strain ATCC PTA 6475 suppresses intestinal inflammation and produces 5,10-methenyltetrahydrofolic acid polyglutamates. Insertional mutagenesis identified the bifunctional dihydrofolate synthase/folylpolyglutamate synthase type 2 (folC2) gene as essential for 5,10-methenyltetrahydrofolic acid polyglutamate biosynthesis, as well as for suppression of TNF production by activated human monocytes, and for the anti-inflammatory effect of L. reuteri 6475 in a trinitrobenzene sulfonic acid-induced mouse model of acute colitis. In contrast, folC encodes the enzyme responsible for folate polyglutamylation but does not impact TNF suppression by L. reuteri. Comparative transcriptomics between wild-type and mutant L. reuteri strains revealed additional genes involved in immunomodulation, including previously identified hdc genes involved in histidine to histamine conversion. The folC2 mutant yielded diminished hdc gene cluster expression and diminished histamine production, suggesting a link between folate and histadine/histamine metabolism. The identification of genes and gene networks regulating production of bacterial-derived immunoregulatory molecules may lead to improved anti-inflammatory strategies for digestive diseases.

Histamine H2 Receptor-Mediated Suppression of Intestinal Inflammation by Probiotic Lactobacillus reuteri.

UNLABELLED: Probiotics and commensal intestinal microbes suppress mammalian cytokine production and intestinal inflammation in various experimental model systems. Limited information exists regarding potential mechanisms of probiotic-mediated immunomodulation in vivo. In this report, we demonstrate that specific probiotic strains of Lactobacillus reuteri suppress intestinal inflammation in a trinitrobenzene sulfonic acid (TNBS)-induced mouse colitis model. Only strains that possess the hdc gene cluster, including the histidine decarboxylase and histidine-histamine antiporter genes, can suppress colitis and mucosal cytokine (interleukin-6 [IL-6] and IL-1ß in the colon) gene expression. Suppression of acute colitis in mice was documented by diminished weight loss, colonic injury, serum amyloid A (SAA) protein concentrations, and reduced uptake of [(18)F]fluorodeoxyglucose ([(18)F]FDG) in the colon by positron emission tomography (PET). The ability of probiotic L. reuteri to suppress colitis depends on the presence of a bacterial histidine decarboxylase gene(s) in the intestinal microbiome, consumption of a histidine-containing diet, and signaling via the histamine H2 receptor (H2R). Collectively, luminal conversion of l-histidine to histamine by hdc(+) L. reuteri activates H2R, and H2R signaling results in suppression of acute inflammation within the mouse colon. IMPORTANCE: Probiotics are microorganisms that when administered in adequate amounts confer beneficial effects on the host. Supplementation with probiotic strains was shown to suppress intestinal inflammation in patients with inflammatory bowel disease and in rodent colitis models. However, the mechanisms of probiosis are not clear. Our current studies suggest that supplementation with hdc(+) L. reuteri, which can convert l-histidine to histamine in the gut, resulted in suppression of colonic inflammation. These findings link luminal conversion of dietary components (amino acid metabolism) by gut microbes and probiotic-mediated suppression of colonic inflammation. The effective combination of diet, gut bacteria, and host receptor-mediated signaling may result in opportunities for therapeutic microbiology and provide clues for discovery and development of next-generation probiotics.

Characterization of two long-chain fatty acid CoA ligases in the Gram-positive bacterium Geobacillus thermodenitrificans NG80-2.

The functions of two long-chain fatty acid CoA ligase genes (facl) in crude oil-degrading Geobacillus thermodenitrificans NG80-2 were characterized. Facl1 and Facl2 encoded by GTNG_0892 and GTNG_1447 were expressed in Escherichia coli and purified as His-tagged fusion proteins. Both enzymes utilized a broad range of fatty acids ranging from acetic acid (C(2)) to melissic acid (C(30)). The most preferred substrates were capric acid (C(10)) for Facl1 and palmitic acid (C(16)) for Facl2, respectively. Both enzymes had an optimal temperature of 60°C, an optimal pH of 7.5, and required ATP as a cofactor. Thermostability of the enzymes and effects of metal ions, EDTA, SDS and Triton X-100 on the enzyme activity were also investigated. When NG80-2 was cultured with crude oil rather than sucrose as the sole carbon source, upregulation of facl1 and facl2 mRNA was observed by real time RT-PCR. This is the first time that the activity of fatty acid CoA ligases toward long-chain fatty acids up to at least C(30) has been demonstrated in bacteria.

A recalibrated molecular clock and independent origins for the cholera pandemic clones.

Cholera, caused by Vibrio cholerae, erupted globally from South Asia in 7 pandemics, but there were also local outbreaks between the 6(th) (1899-1923) and 7(th) (1961-present) pandemics. All the above are serotype O1, whereas environmental or invertebrate isolates are antigenically diverse. The pre 7th pandemic isolates mentioned above, and other minor pathogenic clones, are related to the 7(th) pandemic clone, while the 6(th) pandemic clone is in the same lineage but more distantly related, and non-pathogenic isolates show no clonal structure. To understand the origins and relationships of the pandemic clones, we sequenced the genomes of a 1937 prepandemic strain and a 6(th) pandemic isolate, and compared them with the published 7(th) pandemic genome. We distinguished mutational and recombinational events, and allocated these and other events, to specific branches in the evolutionary tree. There were more mutational than recombinational events, but more genes, and 44 times more base pairs, changed by recombination. We used the mutational single-nucleotide polymorphisms and known isolation dates of the prepandemic and 7(th) pandemic isolates to estimate the mutation rate, and found it to be 100 fold higher than usually assumed. We then used this to estimate the divergence date of the 6(th) and 7(th) pandemic clones to be about 1880. While there is a large margin of error, this is far more realistic than the 10,000-50,000 years ago estimated using the usual assumptions. We conclude that the 2 pandemic clones gained pandemic potential independently, and overall there were 29 insertions or deletions of one or more genes. There were also substantial changes in the major integron, attributed to gain of individual cassettes including copying from within, or loss of blocks of cassettes. The approaches used open up new avenues for analysing the origin and history of other important pathogens.

Genome and proteome of long-chain alkane degrading Geobacillus thermodenitrificans NG80-2 isolated from a deep-subsurface oil reservoir.

The complete genome sequence of Geobacillus thermodenitrificans NG80-2, a thermophilic bacillus isolated from a deep oil reservoir in Northern China, consists of a 3,550,319-bp chromosome and a 57,693-bp plasmid. The genome reveals that NG80-2 is well equipped for adaptation into a wide variety of environmental niches, including oil reservoirs, by possessing genes for utilization of a broad range of energy sources, genes encoding various transporters for efficient nutrient uptake and detoxification, and genes for a flexible respiration system including an aerobic branch comprising five terminal oxidases and an anaerobic branch comprising a complete denitrification pathway for quick response to dissolved oxygen fluctuation. The identification of a nitrous oxide reductase gene has not been previously described in Gram-positive bacteria. The proteome further reveals the presence of a long-chain alkane degradation pathway; and the function of the key enzyme in the pathway, the long-chain alkane monooxygenase LadA, is confirmed by in vivo and in vitro experiments. The thermophilic soluble monomeric LadA is an ideal candidate for treatment of environmental oil pollutions and biosynthesis of complex molecules.