Alain Stintzi

Intestinal Inflammation Targets Cancer-Inducing Activity of the Microbiota

Of Microbes and Cancer Inflammation is a well-established driver of tumorigenesis. For example, patients with inflammatory bowel disease have an elevated risk of developing colorectal cancer (CRC). Whether the gut microbiota also contributes to the development of CRC is less well understood. Arthur et al. (p. 120, published online 16 August; see the Perspective by Schwabe and Wang) now show that the microbiota does indeed promote tumorigenesis in an inflammation-driven model of CRC in mice. Although germ-free mice were protected against developing cancer, colonization of mice with Escherichia coli was sufficient to drive tumorigenesis. Microbes resident in the gut can promote colorectal cancer in mice in an inflammation-independent manner. Inflammation alters host physiology to promote cancer, as seen in colitis-associated colorectal cancer (CRC). Here, we identify the intestinal microbiota as a target of inflammation that affects the progression of CRC. High-throughput sequencing revealed that inflammation modifies gut microbial composition in colitis-susceptible interleukin-10–deficient (Il10−/−) mice. Monocolonization with the commensal Escherichia coli NC101 promoted invasive carcinoma in azoxymethane (AOM)–treated Il10−/− mice. Deletion of the polyketide synthase (pks) genotoxic island from E. coli NC101 decreased tumor multiplicity and invasion in AOM/Il10−/− mice, without altering intestinal inflammation. Mucosa-associated pks+ E. coli were found in a significantly high percentage of inflammatory bowel disease and CRC patients. This suggests that in mice, colitis can promote tumorigenesis by altering microbial composition and inducing the expansion of microorganisms with genotoxic capabilities.

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.

Gene Expression Profile of Campylobacter jejuni in Response to Growth Temperature Variation

The foodborne pathogen Campylobacter jejuni is the primary causative agent of gastroenteritis in humans. In the present study a whole genome microarray of C. jejuni was constructed and validated. These DNA microarrays were used to measure changes in transcription levels over time, as C. jejuni cells responded to a temperature increase from 37 to 42 degrees C. Approximately 20% of the C. jejuni genes were significantly up- or downregulated over a 50-min period after the temperature increase. The global change in C. jejuni transcriptome was found to be essentially transient, with only a small subset of genes still differentially expressed after 50 min. A substantial number of genes with a downregulated coexpression pattern were found to encode for ribosomal proteins. This suggests a short growth arrest upon temperature stress, allowing the bacteria to reshuffle their energy toward survival and adaptation to the new growth temperature. Genes encoding chaperones, chaperonins, and heat shock proteins displayed the most dramatic and rapid upregulation immediately after the temperature change. Interestingly, genes encoding proteins involved in membrane structure modification were differentially expressed, either up- or downregulated, suggesting a different protein membrane makeup at the two different growth temperatures. Overall, these data provide new insights into the primary response of C. jejuni to surmount a sudden temperature upshift, allowing the bacterium to survive and adapt its transcriptome to a new steady state.

Use of Siderophores to Type Pseudomonads: The Three Pseudomonas Aeruginosa Pyoverdine Systems

Eighty-eight Pseudomonas aeruginosa isolates, most of them from the Collection of Bacterial Strains of the Institut Pasteur, Paris, were analysed for their pyoverdine-mediated iron incorporation system by different methods, including pyoverdine isoelectrofocusing analysis, pyoverdine-mediated growth stimulation, immunoblot detection of (ferri)pyoverdine outer-membrane receptor and pyoverdine-facilitated iron uptake. The same grouping of the strains was reached by each of these methods, resulting in the classification of the P. aeruginosa isolates, even those which were devoid of pyoverdine production, into three different siderophore types. Forty-two percent of the strains were identified with the type-strain P. aeruginosa ATCC 15,692 (group I), 42% were identical with the second type-strain P. aeruginosa ATCC 27,853 (group II) and 16% reacted identically with the clinical isolate P. aeruginosa Pa6, whose pyoverdine was recognized in this study to be identical in structure to the pyoverdine produced by a natural isolate, P. aeruginosa strain R. No new pyoverdine species was detected among these strains.

Major Role for FeoB in Campylobacter jejuni Ferrous Iron Acquisition, Gut Colonization, and Intracellular Survival

ABSTRACT To assess the importance of ferrous iron acquisition in Campylobacter physiology and pathogenesis, we disrupted and characterized the Fe2+ iron transporter, FeoB, in Campylobacter jejuni NCTC 11168, 81-176, and ATCC 43431. The feoB mutant was significantly affected in its ability to transport 55Fe2+. It accumulated half the amount of iron than the wild-type strain during growth in an iron-containing medium. The intracellular iron of the feoB mutant was localized in the periplasmic space versus the cytoplasm for the wild-type strain. These results indicate that the feoB gene of C. jejuni encodes a functional ferrous iron transport system. Reverse transcriptase PCR analysis revealed the cotranscription of feoB and Cj1397, which encodes a homolog of Escherichia coli feoA. C. jejuni 81-176 feoB mutants exhibited reduced ability to persist in human INT-407 embryonic intestinal cells and porcine IPEC-1 small intestinal epithelial cells compared to the wild type. C. jejuni NCTC 11168 feoB mutant was outcompeted by the wild type for colonization and/or survival in the rabbit ileal loop. The feoB mutants of the three C. jejuni strains were significantly affected in their ability to colonize the chick cecum. And finally, the three feoB mutants were outcompeted by their respective wild-type strains for infection of the intestinal tracts of colostrum-deprived piglets. Taken together, these results demonstrate that FeoB-mediated ferrous iron acquisition contributes significantly to colonization of the gastrointestinal tract during both commensal and infectious relationship, and thus it plays an important role in Campylobacter pathogenesis.

Use of Genome-Wide Expression Profiling and Mutagenesis To Study the Intestinal Lifestyle of Campylobacter jejuni

ABSTRACT Campylobacter jejuni is the most common bacterial cause of diarrhea worldwide. To colonize the gut and cause infection, C. jejuni must successfully compete with endogenous microbes for nutrients, resist host defenses, persist in the intestine, and ultimately infect the host. These challenges require the expression of a battery of colonization and virulence determinants. In this study, the intestinal lifestyle of C. jejuni was studied using whole-genome microarray, mutagenesis, and a rabbit ileal loop model. Genes associated with a wide range of metabolic, morphological, and pathological processes were expressed in vivo. The in vivo transcriptome of C. jejuni reflected its oxygen-limited, nutrient-poor, and hyperosmotic environment. Strikingly, the expression of several C. jejuni genes was found to be highly variable between individual rabbits. In particular, differential gene expression suggested that C. jejuni extensively remodels its envelope in vivo by differentially expressing its membrane proteins and by modifying its peptidoglycan and glycosylation composition. Furthermore, mutational analysis of seven genes, hspR, hrcA, spoT, Cj0571, Cj0178, Cj0341, and fliD, revealed an important role for the stringent and heat shock response in gut colonization. Overall, this study provides new insights on the mechanisms of gut colonization, as well as possible strategies employed by Campylobacter to resist or evade the host immune responses.

l-Fucose utilization provides Campylobacter jejuni with a competitive advantage

Campylobacter jejuni is a prevalent gastrointestinal pathogen in humans and a common commensal of poultry. When colonizing its hosts, C. jejuni comes into contact with intestinal carbohydrates, including l-fucose, released from mucin glycoproteins. Several strains of C. jejuni possess a genomic island (cj0480c–cj0490) that is up-regulated in the presence of both l-fucose and mucin and allows for the utilization of l-fucose as a substrate for growth. Strains possessing this genomic island show increased growth in the presence of l-fucose and mutation of cj0481, cj0486, and cj0487 results in the loss of the ability to grow on this substrate. Furthermore, mutants in the putative fucose permease (cj0486) are deficient in fucose uptake and demonstrate a competitive disadvantage when colonizing the piglet model of human disease, which is not paralleled in the colonization of poultry. This identifies a previously unrecorded metabolic pathway in select strains of C. jejuni associated with a virulent lifestyle.

Characterization of the oxidative stress stimulon and PerR regulon of Campylobacter jejuni

BackgroundDuring gut colonization, the enteric pathogen Campylobacter jejuni must surmount the toxic effects of reactive oxygen species produced by its own metabolism, the host immune system, and intestinal microflora. Elucidation of C. jejuni oxidative stress defense mechanisms is critical for understanding Campylobacter pathophysiology.ResultsThe mechanisms of oxidative stress defense in C. jejuni were characterized by transcriptional profiling and phenotypic analysis of wild-type and mutant strains. To define the regulon of the peroxide-sensing regulator, PerR, we constructed an isogenic ΔperR mutant and compared its transcriptome profile with that of the wild-type strain. Transcriptome profiling identified 104 genes that belonged to the PerR regulon. PerR appears to regulate gene expression in a manner that both depends on and is independent of the presence of iron and/or H2O2. Mutation of perR significantly reduced motility. A phenotypic analysis using the chick colonization model showed that the ΔperR mutant exhibited attenuated colonization behavior. An analysis of changes in the transcriptome induced by exposure to H2O2, cumene hydroperoxide, or menadione revealed differential expression of genes belonging to a variety of biological pathways, including classical oxidative stress defense systems, heat shock response, DNA repair and metabolism, fatty acid biosynthesis, and multidrug efflux pumps. Mutagenic and phenotypic studies of the superoxide dismutase SodB, the alkyl-hydroxyperoxidase AhpC, and the catalase KatA, revealed a role for these proteins in oxidative stress defense and chick gut colonization.ConclusionThis study reveals an interplay between PerR, Fur, iron metabolism and oxidative stress defense, and highlights the role of these elements in C. jejuni colonization of the chick cecum and/or subsequent survival.

Identification of the Iron-Responsive Genes of Neisseria gonorrhoeae by Microarray Analysis in Defined Medium

To ensure survival, most bacteria must acquire iron, a resource that is sequestered by mammalian hosts. Pathogenic bacteria have therefore evolved intricate systems to sense iron limitation and regulate gene expression appropriately. We used a pan-Neisseria microarray to examine genes regulated in Neisseria gonorrhoeae in response to iron availability in defined medium. Overall, 203 genes varied in expression, 109 up-regulated and 94 down-regulated by iron deprivation. In iron-replete medium, genes essential to rapid bacterial growth were preferentially expressed, while iron transport functions, and predominantly genes of unknown function, were expressed in low-iron medium. Of those TonB-dependent proteins encoded in the FA1090 genome with unknown ligand specificity, expression of three was not controlled by iron availability, suggesting that these receptors may not be high-affinity transporters for iron-containing ligands. Approximately 30% of the operons regulated by iron appeared to be directly under control of Fur. Our data suggest a regulatory cascade where Fur indirectly controls gene expression by affecting the transcription of three secondary regulators. Our data also suggest that a second MerR-like regulator may be directly responding to iron availability and controlling transcription independent of the Fur protein. Comparison of our data with those recently published for Neisseria meningitidis revealed that only a small portion of genes were found to be similarly regulated in these closely related pathogens, while a large number of genes derepressed during iron starvation were unique to each organism.

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.