Unknown,Transcriptomics,Genomics,Proteomics

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POTENTIAL GENETIC TARGETS FOR CONTROLLING CAMPYLOBACTER JEJUNI

ABSTRACT: Campylobacter jejuni is one of the leading causes of bacterial diarrhea worldwide and the most common antecedent in peripheral neuropathies such as Guillain Barré and Miller Fisher syndromes (Blaser et al., 2000). Despite the medical and socioeconomic importance of C. jejuni, the proportion of human disease caused by different sources of infection remains unclear. Indeed, this issue has hindered effective control strategies against reducing C. jejuni levels from the food chain. Up to date, a vast number of phenotypic and genotypic typing systems and a large number of databases have been developed in order to determine the different Campylobacter infection sources. However, traditional methods are unable to discriminate strains from different sources that are responsible for causing disease in humans. Interestingly, DNA microarrays represent a technological alternative to compare entire genomes, allowing the identification of all the genes present or absent in a particular strain by comparison with the genome of the reference strain present in the microarray. Previous comparative genomic hybridazation studies of C. jejuni using DNA microarrays have focused on the genetic variability in C. jejuni (Dorrell et al., 2001), which suggest that this organism has high levels of genetic diversity and low levels of genetic plasticity (Taboada et al., 2004). On that account, the aim of this study was based on a comparison by CGH (Comparative Genomic Hybridization) using DNA microarrays for determining the degree of genomic variability of C. jejuni strains from different geographical areas and sources of isolation. This system enabled us to identify possible genes that could be used as genetic markers predictive of infection source. Furthermore, CGH data provide additional information that could be useful for the formulation of new hypotheses about C. jejuni genome evolution, virulence, pathogenicity, and host specificity.

ORGANISM(S): Campylobacter jejuni

SUBMITTER: Héctor Velasco

PROVIDER: E-MTAB-3550 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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Project description:Background: During gut colonization, the enteric pathogen C. jejuni has to surmount the toxic effects of reactive oxygen species produced by its own metabolism, by the host immune system and by the intestinal microflora. Elucidation of C. jejuni oxidative stress defense mechanisms is critical for understanding Campylobacter pathophysiology. Results: The mechanisms of oxidative stress defenses in Campylobacter jejuni were characterized by transcriptional profiling, genes mutagenesis, and phenotypic analysis. The transcriptome changes, in response to H2O2, cumene hydroperoxide, or menadione exposure, were found to be oxidant specific and revealed the differential expression of genes belonging to a variety of biological pathways, from the classical oxidative stress defense systems, to the heat shock response, DNA repair and metabolism, fatty acid and capsule biosynthesis, and multidrug efflux pumps. To define the peroxide sensing regulator PerR, an isogenic mutant was constructed and its transcriptome profile compared to the wild-type strain. Sixty-six genes were found to belong to the PerR regulon. PerR appear to regulate gene expression both dependently and independently of the presence of iron and/or H2O2. The perR mutant was affected in its motility and attenuated in the chick colonization model. Mutagenic and phenotypic studies of the superoxide disumutase SodB, the alkyl-hydroxyperoxidase AhpC, and the catalase KatA, revealed their role in oxidative stress defenses and chick gut colonization. Conclusion: This study reveals the interplay between PerR, the iron metabolism and the oxidative stress defenses and highlights their role in the colonization and/or survival of C. jejuni in the chick cecum. Keywords: Transcriptional response to 3 oxidants (H2O2, menadione and cumene hydroperoxide) and characterization of the perR regulon (comparison of the transciptomes from the wild-type and perR mutant). To investigate the transcriptional responses of C. jejuni to oxidant exposure, hydrogen peroxide (H2O2), cumene hydroperoxide (CHP), or menadione sodium bisulfite (MND) was added to the 50 ml broth at a final concentration of 1 mM. The same amount of water or DMSO was added to the bacterial culture that served as reference samples for the transcriptional profile study in response to H2O2, MND or CHP. Furthermore, to investigate the transcriptional response of C. jejuni to H2O2 exposure in the presence of excess iron, ferrous sulfate was added to the bacterial culture at a final concentration of 40 µM, 15 min prior to H2O2 exposure. Ten minutes after the addition of the oxidant, total RNA was extracted and processed for microarray hybridization. To identify the PerR regulon, the wild-type strain C. jejuni NCTC 11168 and the perR mutant were grown in 500 ml flasks containing 250 ml of MEMα medium. At mid-log phase, 50 ml of the cultures were transferred to 100 ml flasks and ferrous sulfate and/or H2O2 were added . Ten minutes following the addition of H2O2 the cells were collected and the total RNA extracted.

Project description:Background. The bacterial foodborne pathogen Campylobacter jejuni is a common cause of acute gastroenteritis and is also associated with the postinfectious neuropathies, Guillain-BarrÃ© and Miller Fisher syndromes. This study described the use of multilocus sequence typing and DNA microarrays to examine the genetic content of a collection of South African C. jejuni strains, recovered from patients with enteritis, Guillain-BarrÃ© or Miller Fisher syndromes. Methodology/Principal Findings. The comparative genomic analysis by using multilocus sequence typing and DNA microarrays demonstrated that the South African strains with Penner heat-stable (HS) serotype HS:41 were clearly distinct from the other South African strains. Further analysis of the DNA microarray data demonstrated that the serotype HS:41 strains from South African GBS and enteritis patients are highly similar in gene content. Interestingly, the South African HS:41 strains were distinct in gene content when compared to serotype HS:41 strains from other geographical locations due to the presence of genomic islands, referred to as Campylobacter jejuni integrated elements. Only the genomic integrated element CJIE1, a Campylobacter Mu-like prophage, was present in the South African HS:41 strains whereas absent in the closely-related HS:41 strains from Mexico. A more distantly-related HS:41 strain from Canada possessed both genomic integrated elements CJIE1 and CJIE2. Conclusion/Significance. These findings demonstrated that these C. jejuni integrated elements may contribute to the differentiation of closely-related C. jejuni strains. In addition, the presence of bacteriophage-related genes in CJIE1 may probably contribute to increasing the genomic diversity of these C. jejuni strains. This comparative genomic analysis of the foodborne pathogen C. jejuni provides fundamental information that potentially could lead to improved methods for analyzing the epidemiology of disease outbreaks and their sources. The genomic diversity of 33 South African, 2 Mexican and 1 Canadian Campylobacter jejuni strains from humans were examined by microarray-based comparative genomic indexing (CGI) analysis. The CGI analysis allowed the assessment of CDS content for each C. jejuni strain relative to the C. jejuni DNA microarray, which comprises ORFs from strains NCTC 11168, RM1221. ORFs were spotted in duplicate. Genomic DNA from strains NCTC 11168/RM1221 were used as a reference DNA and competitively hybridized with genomic DNA from each of the other C. jejuni strains. Two or three replicates for each strain were performed. Data normalization was performed as in Parker et al. J Clin Microbiol 2006, 44(11):4125-4135.

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POTENTIAL GENETIC TARGETS FOR CONTROLLING CAMPYLOBACTER JEJUNI

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