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Chromosomal integration vectors allowing flexible expression of foreign genes in Campylobacter jejuni.

ABSTRACT: Campylobacter jejuni is a major cause of human gastroenteritis yet there is limited knowledge of how disease is caused. Molecular genetic approaches are vital for research into the virulence mechanisms of this important pathogen. Vectors that allow expression of genes in C. jejuni via recombination onto the chromosome are particularly useful for genetic complementation of insertional knockout mutants and more generally for expression of genes in particular C. jejuni host backgrounds.A series of three vectors that allow integration of genes onto the C. jejuni chromosome were constructed by standard cloning techniques with expression driven from three different strong promoters. Following integration onto the C. jejuni chromosome expression levels were quantified by fluorescence measurements and cells visualized by fluorescence microscopy.We have created plasmid, pCJC1, designed for recombination-mediated delivery of genes onto the C. jejuni chromosome. This plasmid contains a chloramphenicol resistance cassette (cat) with upstream and downstream restriction sites, flanked by regions of the C. jejuni pseudogene Cj0223. Cloning of genes immediately upstream or downstream of the cat gene allows their subsequent introduction onto the C. jejuni chromosome within the pseudogene. Gene expression can be driven from the native gene promoter if included, or alternatively from the cat promoter if the gene is cloned downstream of, and in the same transcriptional orientation as cat. To provide increased and variable expression of genes from the C. jejuni chromosome we modified pCJC1 through incorporation of three relatively strong promoters from the porA, ureI and flaA genes of C. jejuni, Helicobacter pylori and Helicobacter pullorum respectively. These promoters along with their associated ribosome binding sites were cloned upstream of the cat gene on pCJC1 to create plasmids pCJC2, pCJC3 and pCJC4. To test their effectiveness, a green fluorescent protein (gfp) reporter gene was inserted downstream of each of the three promoters and following integration of promoter-gene fusions onto the C. jejuni host chromosome, expression levels were quantified. Expression from the porA promoter produced the highest fluorescence, from flaA intermediate levels and from ureI the lowest. Expression of gfp from the porA promoter enabled visualization by fluorescent microscopy of intracellular C. jejuni cells following invasion of HeLa cells.The plasmids constructed allow stable chromosomal expression of genes in C. jejuni and, depending on the promoter used, different expression levels were obtained making these plasmids useful tools for genetic complementation and high level expression.

SUBMITTER: Jervis AJ

PROVIDER: S-EPMC4619491 | biostudies-literature | 2015 Oct

REPOSITORIES: biostudies-literature

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Chromosomal integration vectors allowing flexible expression of foreign genes in Campylobacter jejuni.

Jervis Adrian J AJ Butler Jonathan A JA Wren Brendan W BW Linton Dennis D

BMC microbiology 20151024

<h4>Background</h4>Campylobacter jejuni is a major cause of human gastroenteritis yet there is limited knowledge of how disease is caused. Molecular genetic approaches are vital for research into the virulence mechanisms of this important pathogen. Vectors that allow expression of genes in C. jejuni via recombination onto the chromosome are particularly useful for genetic complementation of insertional knockout mutants and more generally for expression of genes in particular C. jejuni host backg ...[more]

PMID: 26497958

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Project description:Campylobacter jejuni carry temperate bacteriophages that can affect the biology or virulence of the host bacterium. Known effects include genomic rearrangements and resistance to DNA transformation. C. jejuni prophage CJIE1 shows sequence variability and variability in the content of morons. Homologs of the CJIE1 prophage enhance both adherence and invasion to cells in culture and increase the expression of a specific subset of bacterial genes. Other C. jejuni temperate phages have so far not been well characterized. In this study we describe investigations into the DNA sequence variability and protein expression in a second prophage, CJIE4. CJIE4 sequences were obtained de novo from DNA sequencing of five C. jejuni isolates, as well as from whole genome sequences submitted to GenBank by other research groups. These CJIE4 DNA sequences were heterogenous, with several different insertions/deletions (indels) in different parts of the prophage genome. Two variants of a 3-4 kb region inserted within CJIE4 had different gene content that distinguished two major conserved CJIE4 prophage families. Additional indels were detected throughout the prophage. Detection of proteins in the five isolates characterized in our laboratory in isobaric Tags for Relative and Absolute Quantitation (iTRAQ) experiments indicated that prophage proteins within each of the two large indel variants were expressed during growth of the bacteria on Mueller Hinton agar plates. These proteins included the extracellular DNase associated with resistance to DNA transformation and prophage repressor proteins. Other proteins associated with known or suspected roles in prophage biology were also expressed from CJIE4, including capsid protein, the phage integrase, and MazF, a type II toxin-antitoxin system protein. Together with the results previously obtained for the CJIE1 prophage these results demonstrate that sequence variability and expression of moron genes are both general properties of temperate bacteriophages in C. jejuni.

Project description:Campylobacter jejuni (C. jejuni) is the most common food-borne pathogen that causes human gastroenteritis in the United States. Consumption of contaminated poultry products is considered as the major source of human Campylobacter infection. An effective vaccine would be a promising alternative to antibiotic supplements to curb C. jejuni colonization in poultry gastrointestinal (GI) tract. However, the genetic diversity among the C. jejuni isolates makes vaccine production more challenging. Despite many attempts, an effective Campylobacter vaccine is not yet available. This study aimed to identify suitable candidates to develop a subunit vaccine against C. jejuni, which could reduce colonization in the GI tract of the poultry. In the current study, 4 C. jejuni strains were isolated from retail chicken meat and poultry litter samples and their genomes were sequenced utilizing next-generation sequencing technology. The genomic sequences of C. jejuni strains were screened to identify potential antigens utilizing the reverse vaccinology approach. In silico genome analysis predicted 3 conserved potential vaccine candidates (phospholipase A [PldA], TonB dependent vitamin B12 transporter [BtuB], and cytolethal distending toxin subunit B [CdtB]) suitable for the development of a vaccine. Furthermore, the expression of predicted genes during host-pathogen interaction was analyzed by an infection study using an avian macrophage-like immortalized cell line (HD11). The HD11 was infected with C. jejuni strains, and the RT-qPCR assay was performed to determine the expression of the predicted genes. The expression difference was analyzed using ΔΔCt methods. The results indicate that all 3 predicted genes, PldA, BtuB, and CdtB, were upregulated in 4 tested C. jejuni strains irrespective of their sources of isolation. In conclusion, in silico prediction and gene expression analysis during host-pathogen interactions identified 3 potential vaccine candidates for C. jejuni.

Project description:BACKGROUND:Campylobacter jejuni and Campylobacter coli are major global causes of bacterial gastroenteritis. Whilst several individual colonisation and virulence factors have been identified, our understanding of their role in the transmission, pathogenesis and ecology of Campylobacter has been hampered by the genotypic and phenotypic diversity within C. jejuni and C. coli. Autotransporter proteins are a family of outer membrane or secreted proteins in Gram-negative bacteria such as Campylobacter, which are associated with virulence functions. In this study we have examined the distribution and predicted functionality of the previously described capC and the newly identified, related capD autotransporter gene families in Campylobacter. RESULTS:Two capC-like autotransporter families, designated capC and capD, were identified by homology searches of genomes of the genus Campylobacter. Each family contained four distinct orthologs of CapC and CapD. The distribution of these autotransporter genes was determined in 5829 C. jejuni and 1347 C. coli genomes. Autotransporter genes were found as intact, complete copies and inactive formats due to premature stop codons and frameshift mutations. Presence of inactive and intact autotransporter genes was associated with C. jejuni and C. coli multi-locus sequence types, but for capC, inactivation was independent from the length of homopolymeric tracts in the region upstream of the capC gene. Inactivation of capC or capD genes appears to represent lineage-specific gene decay of autotransporter genes. Intact capC genes were predominantly associated with the C. jejuni ST-45 and C. coli ST-828 generalist lineages. The capD3 gene was only found in the environmental C. coli Clade 3 lineage. These combined data support a scenario of inter-lineage and interspecies exchange of capC and subsets of capD autotransporters. CONCLUSIONS:In this study we have identified two novel, related autotransporter gene families in the genus Campylobacter, which are not uniformly present and exhibit lineage-specific associations and gene decay. The distribution and decay of the capC and capD genes exemplifies the erosion of species barriers between certain lineages of C. jejuni and C. coli, probably arising through co-habitation. This may have implications for the phenotypic variability of these two pathogens and provide opportunity for new, hybrid genotypes to emerge.

Project description:The characterization of the genetic basis of sulfonamide resistance in Campylobacter jejuni was attempted. The resistance determinant from a sulfonamide-resistant strain of C. jejuni was cloned and was found to show 42% identity with the folP gene (which codes for dihydropteroate synthase, the target of sulfonamides) of the related bacterium Helicobacter pylori. The sequences of the areas surrounding the folP gene in C. jejuni showed similarity to those of the areas surrounding the corresponding gene in H. pylori. The folP gene of C. jejuni, which mediates the resistance, was observed to show particular features when it was compared to other known folP genes. One of these features is the presence of two pairs of direct repeats (15 and 27 bp) within the coding sequence of the gene. Comparison of the C. jejuni folP genes that mediate susceptibility and resistance revealed the occurrence of mutations that changed four amino acid residues. Resistance of C. jejuni to sulfonamides could be associated with one or several of these four mutational substitutions, which all occurred in the five different resistant isolates studied. The codon for one of these changed amino acids was found to be located in the second direct repeat within the coding sequence of the gene. The change made the repeat perfect. The transformation of both the resistance and the susceptibility variants of the gene into an Escherichia coli folP knockout mutant was found to complement the dihydropteroate synthase deficiency, confirming that the characterized sulfonamide resistance determinant codes for the C. jejuni dihydropteroate synthase enzyme. Kinetic measurements established different affinities of sulfonamide for the dihydropteroate synthase enzyme isolated from the resistant and susceptible strains. In conclusion, sulfonamide resistance in C. jejuni was shown to be associated with mutational changes in the chromosomally located gene for dihydropteroate synthase, the target of sulfonamides.

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Chromosomal integration vectors allowing flexible expression of foreign genes in Campylobacter jejuni.

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Chromosomal integration vectors allowing flexible expression of foreign genes in Campylobacter jejuni.

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