A gnotobiotic mouse model demonstrates that dietary fiber protects against colorectal tumorigenesis in a microbiota- and butyrate-dependent manner.

UNLABELLED: Whether dietary fiber protects against colorectal cancer is controversial because of conflicting results from human epidemiologic studies. However, these studies and mouse models of colorectal cancer have not controlled the composition of gut microbiota, which ferment fiber into short-chain fatty acids such as butyrate. Butyrate is noteworthy because it has energetic and epigenetic functions in colonocytes and tumor-suppressive properties in colorectal cancer cell lines. We used gnotobiotic mouse models colonized with wild-type or mutant strains of a butyrate-producing bacterium to demonstrate that fiber does have a potent tumor-suppressive effect but in a microbiota- and butyrate-dependent manner. Furthermore, due to the Warburg effect, butyrate was metabolized less in tumors where it accumulated and functioned as a histone deacetylase (HDAC) inhibitor to stimulate histone acetylation and affect apoptosis and cell proliferation. To support the relevance of this mechanism in human cancer, we demonstrate that butyrate and histone-acetylation levels are elevated in colorectal adenocarcinomas compared with normal colonic tissues. SIGNIFICANCE: These results, which link diet and microbiota to a tumor-suppressive metabolite, provide insight into conflicting epidemiologic findings and suggest that probiotic/prebiotic strategies can modulate an endogenous HDAC inhibitor for anticancer chemoprevention without the adverse effects associated with synthetic HDAC inhibitors used in chemotherapy.

Identification and Characterization of an Invasion Antigen B Gene from the Oral Pathogen Campylobacter rectus.

The oral bacterium, Campylobacter rectus, is an etiological agent of periodontitis. The virulence genes of C. rectus are largely unknown. The aim of this study was to query C. rectus for the presence of an invasion antigen B (ciaB) gene, which is needed for cell invasion by the related species Campylobacter jejuni. PCR and PCR-walking identified a ciaB from C. rectus. In silico analyses of C. rectus 314 ciaB (Cr-ciaB) revealed an ORF of 1,830 base pairs. The Cr-CiaB protein shared significant sequence identity (BLASTx and phylogeny) with CiaB from related campylobacters. Cr-CiaB is predicted to lack membrane helices, signal peptides, and localizes to the cytoplasm; which are consistent with CiaB proteins. Expression of Cr-ciaB was confirmed with RT-PCR; and potential ciaB genes were detected in eight additional strains of C. rectus. Cr-ciaB is the first CiaB identified from the oral campylobacters.

Identification and Characterization of a Putative Chemotaxis Protein, CheY, from the Oral Pathogen Campylobacter rectus.

Campylobacter rectus is an understudied oral bacterium that contributes to periodontitis. Processes that contribute to the disease-causing capabilities of pathogens, such as chemotaxis, are largely unknown in C. rectus. The aim of this study was to better understand C. rectus chemotaxis, by examining the C. rectus genome for the presence of a cheY gene. CheY proteins play a part in chemotaxis by acting as two-component response regulators. Significantly, CheY proteins from several pathogens, including the related species Campylobacter jejuni, have been shown to contribute to bacterial virulence. Degenerate PCR, RT-PCR, sequence analyses, and structural modeling showed that C. rectus encodes a gene (Cr-CheY) which shares significant homology with previously characterized CheY proteins. Functional studies of a recombinant form of the protein supports a likely role of Cr-CheY in C. rectus chemotaxis. Cr-CheY is the first CheY characterized from the oral campylobacters.

Campylobacter jejuni induces colitis through activation of mammalian target of rapamycin signaling.

BACKGROUND & AIMS: Campylobacter jejuni is the worldwide leading cause of bacterial-induced enteritis. The molecular and cellular events that lead to campylobacteriosis are poorly understood. We identify mammalian target of rapamycin (mTOR) as a signaling pathway that leads to C jejuni-induced intestinal inflammation. METHODS: Germ-free (control) or conventionally derived Il10(-/-) mice that express enhanced green fluorescent protein (EGFP) under the control of nuclear factor κB (Il10(-/-); NF-κB(EGFP) mice) were infected with C jejuni (10(9) colony-forming units/mouse) for 12 days; their responses were determined using histologic, semiquantitative reverse-transcription polymerase chain reaction, fluorescence in situ hybridization, transmission electron microscopy, and tissue culture analyses. mTOR signaling was blocked by daily intraperitoneal injections of the pharmacologic inhibitor rapamycin (1.5 mg/kg). CD4(+) T cells were depleted by intraperitoneal injections of antibodies against CD4 (0.5 mg/mouse every 3 days). Bacterial survival in splenocytes was measured using a gentamycin killing assay. RESULTS: C jejuni induced intestinal inflammation, which correlated with activation of mTOR signaling and neutrophil infiltration. The inflamed intestines of these mice had increased levels of interleukin-1ß, Cxcl2, interleukin-17a, and EGFP; C jejuni localized to colons and extraintestinal tissues of infected Il10(-/-); NF-κB(EGFP) mice compared with controls. Rapamycin, administered before or after introduction of C jejuni, blocked C jejuni-induced intestinal inflammation and bacterial accumulation. LC3II processing and killing of C jejuni were increased in splenocytes incubated with rapamycin compared with controls. CONCLUSIONS: mTOR signaling mediates C jejuni-induced colitis in Il10(-/-) mice, independently of T-cell activation. Factors involved in mTOR signaling might be therapeutic targets for campylobacteriosis.

Gnotobiotic IL-10; NF-kappaB mice develop rapid and severe colitis following Campylobacter jejuni infection.

Limited information is available on the molecular mechanisms associated with Campylobacter jejuni (C. jejuni) induced food-borne diarrheal illnesses. In this study, we investigated the function of TLR/NF-kappaB signaling in C. jejuni induced pathogenesis using gnotobiotic IL-10(-/-); NF-kappaB(EGFP) mice. In vitro analysis showed that C. jejuni induced IkappaB phosphorylation, followed by enhanced NF-kappaB transcriptional activity and increased IL-6, MIP-2alpha and NOD2 mRNA accumulation in infected-mouse colonic epithelial cells CMT93. Importantly, these events were blocked by molecular delivery of an IkappaB inhibitor (Ad5IkappaBAA). NF-kappaB signalling was also important for C.jejuni-induced cytokine gene expression in bone marrow-derived dendritic cells. Importantly, C. jejuni associated IL-10(-/-); NF-kappaB(EGFP) mice developed mild (day 5) and severe (day 14) ulcerating colonic inflammation and bloody diarrhea as assessed by colonoscopy and histological analysis. Macroscopic analysis showed elevated EGFP expression indicating NF-kappaB activation throughout the colon of C. jejuni associated IL-10(-/-); NF-kappaB(EGFP) mice, while fluorescence microscopy revealed EGFP positive cells to be exclusively located in lamina propria mononuclear cells. Pharmacological NF-kappaB inhibition using Bay 11-7085 did not ameliorate C. jejuni induced colonic inflammation. Our findings indicate that C. jejuni induces rapid and severe intestinal inflammation in a susceptible host that correlates with enhanced NF-kappaB activity from lamina propria immune cells.

The gastrointestinal microbiome: a malleable, third genome of mammals.

The nonpathogenic, mutualistic bacteria of the mammalian gastrointestinal tract provide a number of benefits to the host. Recent reports have shown how the aggregate genomes of gastrointestinal bacteria provide novel benefits by functioning as the third major genome in mammals along with the nuclear and mitochondrial genomes. Consequently, efforts are underway to elucidate the complexity of the organisms comprising the unique ecosystem of the gastrointestinal tract, as well as those associated with other epidermal surfaces. The current knowledge of the gastrointestinal microbiome, its relationship to human health and disease with a particular focus on mammalian physiology, and efforts to alter its composition as a novel therapeutic approach are reviewed.

Anti-inflammatory properties of Lactobacillus gasseri expressing manganese superoxide dismutase using the interleukin 10-deficient mouse model of colitis.

Emerging evidence has implicated reactive oxygen species (ROS) in the pathogenesis of inflammatory bowel disease (IBD). Although intestinal epithelial cells produce the ROS-neutralizing enzyme superoxide dismutase (SOD), the protein and activity levels of copper/zinc (Cu/Zn) and manganese (Mn) SOD are perturbed in inflamed tissues of IBD patients. Thus we investigated the ability of MnSOD from Streptococcus thermophilus to reduce colitis symptoms in interleukin (IL) 10-deficient mice using Lactobacillus gasseri as a delivery vehicle. Cohorts of 13-15 IL-10-deficient mice were left untreated or supplemented with native L. gasseri or L. gasseri expressing MnSOD for 4 wk. Colonic tissue was collected and inflammation was histologically scored. The presence of innate immune cells was investigated by immunohistochemistry and the host antioxidant response was determined by quantitative PCR. It was demonstrated that L. gasseri was stably maintained in mice for at least 3 days. L. gasseri producing MnSOD significantly reduced inflammation in IL-10-deficient mice compared with untreated controls (P < 0.05), whereas the anti-inflammatory effects of both native and MnSOD producing L. gasseri were more pronounced in males. The anti-inflammatory effects of L. gasseri were associated with a reduction in the infiltration of neutrophils and macrophages. Transcripts of antioxidant genes were equivalent in colonic tissues obtained from control and probiotic-treated IL-10-deficient mice. This study demonstrates that L. gasseri producing MnSOD has significant anti-inflammatory activity that reduces the severity of colitis in the IL-10-deficient mouse.

Human epithelial-specific response to pathogenic Campylobacter jejuni.

The gastrointestinal epithelia of mammals are tolerant of their resident gut microbiota but are usually highly responsive to entero-pathogens; the host-specific responses have not been well characterized. To this end, the transcriptional responses of cultured human (Caco-2) and murine (CT-26) colonic epithelial cells were compared after exposure with the microfloral bacterium Lactobacillus reuteri or the human gastrointestinal pathogen Campylobacter jejuni. When in bacterial broth, both species elicit a stronger differential gene expression response in human colonic cells compared with mouse colonic cells. However, when these data are adjusted to remove bacterial broth effects, only human colonic epithelia exposed to C. jejuni show altered gene expression, suggesting that the human pathogen C. jejuni induces a host-specific response. The genes with altered expression are involved in growth, transcription, and steroid biosynthesis. Interestingly, human genes involved in cell polarity and water transport were significantly changed in response to C. jejuni exposure, linking infection with gastrointestinal disease. This study demonstrates that mouse and human colonic epithelia remain relatively unresponsive to commensal bacterial challenge, while the human pathogen C. jejuni elicits a host-specific response.

Genomic diversity in Campylobacter jejuni: identification of C. jejuni 81-176-specific genes.

Since the publication of the complete genomic sequence of Campylobacter jejuni NCTC 11168 in February 2000, evidence has been compiling that suggests C. jejuni strains exhibit high genomic diversity. In order to investigate this diversity, the unique genomic DNA sequences from a nonsequenced Campylobacter strain, C. jejuni 81-176, were identified by comparison with C. jejuni NCTC 11168 by using a shotgun DNA microarray approach. Up to 63 kb of new chromosomal DNA sequences unique to this pathogen were obtained. Eighty-six open reading frames were identified by the presence of uninterrupted coding regions encoding a minimum of 40 amino acids. In addition, this study shows that the whole-plasmid shotgun microarray approach is effective and provides a comprehensive coverage of DNA regions that differ between two closely related genomes. The two plasmids harbored by this Campylobacter strain, pTet and pVir, were also sequenced, with coverages of 2.5- and 2.9-fold, respectively, representing 72 and 92% of their complete nucleotide sequences. The unique chromosomal genes encode proteins involved in capsule and lipooligosaccharide biosynthesis, restriction and modification systems, and respiratory metabolism. Several of these unique genes are likely associated with C. jejuni 81-176 fitness and virulence. Interestingly, the comparison of C. jejuni 81-176 unique genes with those of C. jejuni ATCC 43431 revealed a single gene which encodes a probable TraG-like protein. The product of this gene might be associated with the mechanism of C. jejuni invasion into epithelial cells. In conclusion, this study extends the repertoire of C. jejuni genes and thus will permit the construction of a composite and more comprehensive microarray of C. jejuni.

Iron acquisition and regulation in Campylobacter jejuni.

Iron affects the physiology of bacteria in two different ways: as a micronutrient for bacterial growth and as a catalyst for the formation of hydroxyl radicals. In this study, we used DNA microarrays to identify the C. jejuni genes that have their transcript abundance affected by iron availability. The transcript levels of 647 genes were affected after the addition of iron to iron-limited C. jejuni cells. Several classes of affected genes were revealed within 15 min, including immediate-early response genes as well as those specific to iron acquisition and metabolism. In contrast, only 208 genes were differentially expressed during steady-state experiments comparing iron-rich and iron-limited growth conditions. As expected, genes annotated as being involved in either iron acquisition or oxidative stress defense were downregulated during both time course and steady-state experiments, while genes encoding proteins involved in energy metabolism were upregulated. Because the level of protein glycosylation increased with iron limitation, iron may modulate the level of C. jejuni virulence by affecting the degree of protein glycosylation. Since iron homeostasis has been shown to be Fur regulated in C. jejuni, an isogenic fur mutant was used to define the Fur regulon by transcriptome profiling. A total of 53 genes were Fur regulated, including many genes not previously associated with Fur regulation. A putative Fur binding consensus sequence was identified in the promoter region of most iron-repressed and Fur-regulated genes. Interestingly, a fur mutant was found to be significantly affected in its ability to colonize the gastrointestinal tract of chicks, highlighting the importance of iron homeostasis in vivo. Directed mutagenesis of other genes identified by the microarray analyses allowed the characterization of the ferric enterobactin receptor, previously named CfrA. Chick colonization assays indicated that mutants defective in enterobactin-mediated iron acquisition were unable to colonize the gastrointestinal tract. In addition, a mutation in a receptor (Cj0178) for an uncharacterized iron source also resulted in reduced colonization potential. Overall, this work documents the complex response of C. jejuni to iron availability, describes the genetic network between the Fur and iron regulons, and provides insight regarding the role of iron in C. jejuni colonization in vivo.

Identification of Campylobacter jejuni ATCC 43431-specific genes by whole microbial genome comparisons.

This study describes a novel approach to identify unique genomic DNA sequences from the unsequenced strain C. jejuni ATCC 43431 by comparison with the sequenced strain C. jejuni NCTC 11168. A shotgun DNA microarray was constructed by arraying 9,600 individual DNA fragments from a C. jejuni ATCC 43431 genomic library onto a glass slide. DNA fragments unique to C. jejuni ATCC 43431 were identified by competitive hybridization to the array with genomic DNA of C. jejuni NCTC 11168. The plasmids containing unique DNA fragments were sequenced, allowing the identification of up to 130 complete and incomplete genes. Potential biological roles were assigned to 66% of the unique open reading frames. The mean G+C content of these unique genes (26%) differs significantly from the G+C content of the entire C. jejuni genome (30.6%). This suggests that they may have been acquired through horizontal gene transfer from an organism with a G+C content lower than that of C. jejuni. Because the two C. jejuni strains differ by Penner serotype, a large proportion of the unique ATCC 43431 genes encode proteins involved in lipooligosaccharide and capsular biosynthesis, as expected. Several unique open reading frames encode enzymes which may contribute to genetic variability, i.e., restriction-modification systems and integrases. Interestingly, many of the unique C. jejuni ATCC 43431 genes show identity with a possible pathogenicity island from Helicobacter hepaticus and components of a potential type IV secretion system. In conclusion, this study provides a valuable resource to further investigate Campylobacter diversity and pathogenesis.