Deborah S. Threadgill

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.

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 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.

Characterization of a set of variable number of tandem repeat markers conserved in Bovidae

Screening purpose-built libraries with minisatellite probes, we have isolated 36 bovine variable number of tandem repeat markers (VNTRs) characterized by a mean heterozygosity of 59.3 within the American Holstein breed. Matching probabilities and exclusion powers were estimated by Monte-Carlo simulation, showing that the top 5 to 10 markers could be used as a very efficient DNA-based system for individual identification and paternity diagnosis. The isolated VNTR systems should contribute significantly to the establishment of a bovine primary DNA marker map. Linkage analysis, use of somatic cell hybrids, and in situ hybridization demonstrate that these bovine VNTRs are scattered throughout the bovine genome, without evidence for proterminal confinement as in the human, and that at least some of them are organized as clusters. Moreover, Southern blot analysis and in situ hybridization demonstrate conservation of sequence and map location of minisatellites within Bovidae.

Genomic Diversity in Campylobacter jejuni: Identification of C. jejuni 81-176-Specific Genes

ABSTRACT 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.

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.

Mitogenic synthetic polynucleotides suppress the antibody response to a bacterial polysaccharide

Unmethylated bacterial DNA containing a high frequency of the CpG motif, is mitogenic and induces T-cell independent, murine B-cell proliferation. These stimulatory effects are also induced by synthetic oligonucleotides that contain one or more unmethylated CpG dinucleotides (CpG oligo). Such mitogenicity is not seen with highly methylated vertebrate DNA, which has a lower prevalence of the CpG motif than bacterial DNA. Due to their stimulatory effects, CpG oligo have been proposed for use as vaccine adjuvants. In order to determine if a synthetic CpG oligo that was stimulatory for B-cell proliferation could augment the murine antibody response to protective bacterial polysaccharide epitopes (Pseudomonas aeruginosa LPS-O polysaccharide side chain; high-molecular-weight polysaccharide or high-MW PS), BALB/c mice were injected with mitogenic doses of CpG oligo simultaneously with high-MW PS, and antibody titers were measured by ELISA weekly for 4 weeks. Controls received PBS, a nonstimulatory control oligo plus PS, CpG alone, or PS alone. Despite evidence of B-cell mitogenicity and an increase in total IgM in CpG oligo-treated mice, CpG oligo treatment plus PS significantly decreased the high-MW PS antibody response compared to PS alone. The blunting of the anti-PS antibody response could be eliminated by vaccinating the animals with PS prior to CpG oligo. We conclude that despite in vitro and in vivo evidence of B-cell proliferation, this CpG oligo reduces PS-specific antibody responses in an animal model when given simultaneously with a bacterial polysaccharide. Based on results in this model, oligonucleotides containing stimulatory unmethylated CpG dinucleotides may not be useful adjuvants when given simultaneously with bacterial PS vaccines.

Gnotobiotic IL-10−/−; NF-κBEGFP 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-κB signaling in C. jejuni induced pathogenesis using gnotobiotic IL-10−/−; NF-κBEGFP mice. In vitro analysis showed that C. jejuni induced IκB phosphorylation, followed by enhanced NF-κB transcriptional activity and increased IL-6, MIP-2α and NOD2 mRNA accumulation in infected-mouse colonic epithelial cells CMT93. Importantly, these events were blocked by molecular delivery of an IκB inhibitor (Ad5IκBAA). NF-κB signalling was also important for C.jejuni-induced cytokine gene expression in bone marrow-derived dendritic cells. Importantly, C. jejuni associated IL-10−/−; NF-κBEGFP 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-κB activation throughout the colon of C. jejuni associated IL-10−/−; NF-κBEGFP mice, while fluorescence microscopy revealed EGFP positive cells to be exclusively located in lamina propria mononuclear cells. Pharmacological NF-κB 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-κB activity from lamina propria immune cells.

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-1β, 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.

Mapping HSA 3 loci in cattle: Additional support for the ancestral synteny of HSA 3 and 21

Homologs to genes residing on human chromosome 3 (HSA 3) map to four mouse chromosomes (MMU) 3, 6, 9, and 16. In the bovine, two syntenic groups that contain HSA 3 homologs, unassigned syntenic groups 10 (U10) and 12 (U12), have been defined. U10 also contains HSA 21 genes, which is similar to the situation seen on MMU 16, whereas U12 apparently contains only HSA 3 homologs. The syntenic arrangement of other HSA 3 homologs in the bovine was investigated by physically mapping five genes through segregation analysis of a bovine-hamster hybrid somatic cell panel. The genes mapped include Friend-murine leukemia virus integration site 3 homolog (FIM3; HSA 3/MMU 3), sucrase-isomaltase (SI) and glutathione peroxidase 1 (GPX1) (HSA 3/MMU ?), murine leukemia viral (v-raf-1) oncogene homolog 1 (RAF1; HSA 3/MMU 6), and ceruloplasmin (CP; HSA 3/MMU 9). FIM3, SI, and CP mapped to bovine syntenic group U10, while RAF1 and GPX1 mapped to U12.