Systems of experimental genetics for Campylobacter species.
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Bacteriological Techniques↗
Biomedical subjects
P Guerry
Publications and source records attributed to P Guerry.
Isolation of motile and non-motile insertional mutants of Campylobacter jejuni: the role of motility in adherence and invasion of eukaryotic cells.
A method of insertional mutagenesis for naturally transformable organisms has been adapted from Haemophilus influenzae and applied to the study of the pathogenesis of Campylobacter jejuni. A series of kanamycin-resistant insertional mutants of C. jejuni 81-176 has been generated and screened for loss of ability to invade INT407 cells. Eight noninvasive mutants were identified which showed 18-200-fold reductions in the level of invasion compared with the parent. Three of these eight show defects in motility, and five are fully motile. The three mutants with motility defects were further characterized to evaluate the method. One mutant, K2-32, which is non-adherent and non-invasive, has an insertion of the kanamycin-resistance cassette into the flaA flagellin gene and has greatly reduced motility and a truncated flagellar filament typical of flaA mutants. The adherent non-invasive mutants K2-37 and K2-55 are phenotypically paralysed, i.e. they have a full-length flagellar filament but are non-motile. All three mutants show an aberration in flagellar structure at the point at which the filament attaches to the cell. Mutants K2-37 and K2-55 represent overlapping deletions affecting the same gene, termed pflA (paralysed flagella). This gene encodes a predicted protein of 788 amino acid residues and a molecular weight of 90,977 with no significant homology to known proteins. Site-specific insertional mutants into this open reading frame result in the same paralysed flagellar phenotype and the same invasion defects as the original mutants. The differences in adherence between the two classes of flagellar mutant suggest that flagellin can serve as a secondary adhesion, although other adhesins mediate a motility-dependent internalization process. Characterization of the mutants at the molecular level and in animal models should further contribute to our understanding of the pathogenicity of these organisms.
Amino Acid Sequence↗
Development and characterization of recA mutants of Campylobacter jejuni for inclusion in attenuated vaccines.
Isogenic recA mutants of Campylobacter jejuni have been constructed for evaluation of their usefulness in attenuated vaccines against this major worldwide cause of diarrhea. The recA+ gene of C. jejuni 81-176 was cloned by using degenerate primers to conserved regions of other RecA proteins in a PCR. The C. jejuni recA+ gene encodes a predicted protein with an M(r) of 37,012 with high sequence similarity to other RecA proteins. The termination codon of the recA+ gene overlaps with the initiation codon of another open reading frame which encodes a predicted protein which has > 50% identity with the N terminus of the Escherichia coli enolase protein. A kanamycin resistance gene was inserted into the cloned recA+ gene in E. coli and returned to C. jejuni VC83 by natural transformation, resulting in allelic replacement of the wild-type recA gene. The resulting VC83 recA mutant displayed increased sensitivity to UV light and a defect in generalized recombination as determined by natural transformation frequencies. The mutated recA gene was amplified from VC83 recA by PCR, and the product was used to transfer the mutation by natural transformation into C. jejuni 81-176 and 81-116, resulting in isogenic recA mutants with phenotypes similar to VC83 recA. After oral feeding, strain 81-176 recA colonized rabbits at levels comparable to wild-type 81-176 and was capable of eliciting the same degree of protection as wild-type 81-176 against subsequent homologous challenge in the RITARD (removable intestinal tie adult rabbit diarrhea) model.
Amino Acid Sequence↗
Structural and antigenic characteristics of Campylobacter coli FlaA flagellin.
The polar flagellar filament of Campylobacter coli VC167 is composed of two highly related (98%) flagellin subunit proteins, FlaA and FlaB, whose antigenic specificities result from posttranslational modification. FlaA is the predominant flagellin species, and mutants expressing only FlaA form a full-length flagellar filament. Although the deduced M(r) of type 2 (T2) FlaA is 58,884 and the apparent M(r) by sodium dodecyl sulfate-polyacrylamide gel electrophoresis is 59,500, the solution weight-average M(r) by sedimentation analysis was 63,000. Circular dichroism studies in the presence or absence of 0.1% sodium dodecyl sulfate or 50% trifluorethanol showed that the secondary structure of T2 FlaA flagellin was altered, with alpha-helix structure being increased to 25% in the nonpolar environment. The molecule also contained 35 to 48% beta-sheet and 11 to 29% beta-turn structure. Mimeotope analysis of octapeptides representing the sequence of FlaA together with immunoelectron microscopy and enzyme-linked immunosorbent assay with a panel of antisera indicated that many residues in presumed linear epitopes were inaccessible or nonepitopic in the assembled filament, with the majority being in the N-terminal 337 residues of the 572-residue flagellin. Residues at the carboxy-terminal end of the T2 FlaA subunit also become inaccessible upon assembly. Digestion with trypsin, chymotrypsin, and endoproteinase Glu-C revealed a protease-resistant domain with an approximate M(r) of 18,700 between residues 193 and 375. Digestion with endoproteinase Arg-C and endoproteinase Lys-C allowed the mapping of a segment of surface-exposed FlaA sequence which contributes serospecificity to the VC167 T2 flagellar filament at residues between 421 and 480.
Amino Acid Sequence↗
Phylogenetic and molecular characterization of a 23S rRNA gene positions the genus Campylobacter in the epsilon subdivision of the Proteobacteria and shows that the presence of transcribed spacers is common in Campylobacter spp.
The nucleotide sequence of a 23S rRNA gene of Campylobacter coli VC167 was determined. The primary sequence of the C. coli 23S rRNA was deduced, and a secondary-structure model was constructed. Comparison with Escherichia coli 23S rRNA showed a major difference in the C. coli rRNA at approximately position 1170 (E. coli numbering) in the form of an extra sequence block approximately 147 bp long. PCR analysis of 31 other strains of C. coli and C. jejuni showed that 69% carried a transcribed spacer of either ca. 147 or ca. 37 bp. Comparison of all sequenced Campylobacter transcribed spacers showed that the Campylobacter inserts were related in sequence and percent G+C content. All Campylobacter strains carrying transcribed spacers in their 23S rRNA genes produced fragmented 23S rRNAs. Other strains which produced unfragmented 23S rRNAs did not appear to carry transcribed spacers at this position in their 23S rRNA genes. At the 1850 region (E. coli numbering), Campylobacter 23S rRNA displayed a base pairing signature most like that of the beta and gamma subdivisions of the class Proteobacteria, but in the 270 region, Campylobacter 23S rRNA displayed a helix signature which distinguished it from the alpha, beta, and gamma subdivisions. Phylogenetic analysis comparing C. coli VC167 23S rRNA and a C. jejuni TGH9011 (ATCC 43431) 23S rRNA with 53 other completely sequenced (eu)bacterial 23S rRNAs showed that the two campylobacters form a sister group to the alpha, beta, and gamma proteobacterial 23S rRNAs, a positioning consistent with the idea that the genus Campylobacter belongs to the epsilon subdivision of the class Proteobacteria.
Bacteria↗
Construction of new Campylobacter cloning vectors and a new mutational cat cassette.
We have developed new Campylobacter shuttle vectors which are 6.5-6.8-kb plasmids carrying Campylobacter and Escherichia coli replicons, a multiple cloning site (MCS), the lacZ alpha gene, oriT and either a kanamycin or chloramphenicol resistance-encoding gene (KmR or CmR) from Campylobacter which functions in both hosts. These vectors can be mobilized efficiently from E. coli into C. jejuni or C. coli, and stably maintained in these hosts. Plasmids pRY107 and pRY108 carry a KmR marker and 17 unique cloning sites in two different orientations in lacZ alpha, allowing easy blue/white color selection. Plasmids pRY111 and pRY112 contain a CmR gene and 17 unique sites in both orientations. In addition, MCS are flanked by T7 and T3 late promoters and M13 forward and reverse primer sites, facilitating expression in T7 or T3 expression systems and sequence analysis. A Campylobacter CmR gene cartridge, bracketed by six restriction sites, has been developed for use in site-specific mutagenesis of Campylobacter genes.
Base Sequence↗
Unusual microtubule-dependent endocytosis mechanisms triggered by Campylobacter jejuni and Citrobacter freundii.
Bacterial invasion of six different human epithelial cell lines showed that some strains of the intestinal pathogen Campylobacter jejuni invaded intestinal cell lines at a level 10(2)-10(4) times higher than reported previously for other Campylobacter strains. Separately, urinary tract isolates of Citrobacter freundii triggered a high-efficiency invasion of bladder cells. Use of multiple inhibitors with known effects on eukaryotic cell structures/processes allowed us to define in these genetically distinct bacterial genera unusual bacterial invasion mechanisms that uniquely require microtubules but not microfilaments. Campylobacter jejuni strain 81-176 uptake into 407 intestinal cells and Citrobacter entry into T24 bladder cells was blocked by microtubule depolymerization and inhibitors of coated-pit formation but not by microfilament depolymerization. Inhibitors of endosome acidification had no significant impact on intracellular survival of Campylobacter jejuni or Citrobacter freundii, but monensin markedly reduced Citrobacter uptake. Epithelial cell invasion by both of these bacterial genera was dependent upon de novo bacterial protein synthesis but not upon de novo eukaryotic cell protein synthesis. In contrast to the T24 cell line-specific, strict microtubule-dependent uptake, Citrobacter entry into other cell lines was inhibited by both microtubule- and microfilament-depolymerization, suggesting that these bacteria encode two separate pathways for uptake (i, microtubule-dependent; ii, microfilament-dependent) that are cell line-specific and are recognized perhaps depending on the presence and abundance of appropriate eukaryotic receptors.
Campylobacter jejuni↗
Enteroaggregative Escherichia coli heat-stable enterotoxin 1 represents another subfamily of E. coli heat-stable toxin.
Enteroaggregative Escherichia coli (EAggEC) are associated with persistent diarrhea in young children. Some of these organisms produce a low-molecular-weight, heat-stable, plasmid-encoded enterotoxin that has been named EAggEC heat-stable enterotoxin 1 (EAST1). We have cloned a 4.4-kb DNA fragment from the virulence plasmid of prototype EAggEC strain 17-2, which expresses enterotoxic activity as measured by electrogenic response in Ussing chambers mounted with rabbit ileal tissue. DNA-sequence analysis of this fragment identified an open reading frame (ORF) encoding a cysteine-rich polypeptide of 38 amino acids (M(r), 4100). Insertional and deletional mutations in this ORF resulted in loss of enterotoxic activity. The ORF was cloned into a T7 expression vector, and postinduction culture filtrates exhibited enterotoxic activity and increased ileal tissue cGMP levels. A synthetic peptide consisting of predicted amino acid residues 8-29 also showed enterotoxic activity. These data indicate that this ORF, named astA (EAggEC heat-stable enterotoxin), represents the EAST1 structural gene. EAST1 shows significant homology with the enterotoxic domain of heat-stable enterotoxin a (STa) of enterotoxigenic E. coli and with guanylin, a mammalian analog of STa. Unlike STa, which requires six cysteines and three disulfide linkages for full biological activity, both EAST1 and guanylin contain four cysteine residues. Based on the cGMP data and the sequence homology to STa and guanylin, it is predicted that EAST1 stimulates the particulate form of guanylate cyclase through the same receptor-binding region as STa and guanylin.
Amino Acid Sequence↗
Significance of duplicated flagellin genes in Campylobacter.
The complex flagellum of Campylobacter coli VC167 contains two highly related (98%) flagellin subunit proteins which are produced from two 92% homologous, tandemly orientated genes, flaA and flaB. Mutants expressing only flaA form a full-length flagellar filament that confers slightly less than wild-type motility to the bacterium. However, flagellin mutants expressing only flaB produce extremely short, truncated filaments, and are only slightly motile. We have shown that the presence of two essentially identical genes is advantageous, in that flaAflaB+ mutants become highly motile upon passage by an event which allows the production of a full length simple flagellar filament containing a single FlaA-FlaB chimeric flagellin protein. Furthermore, we have demonstrated that the reassortment of DNA that results in this chimeric protein can occur by two mechanisms: intragenomic recombination and transformation-mediated intergenomic recombination.
Amino Acid Sequence↗
A pyrimido[1,6-a]benzimidazole that enhances DNA cleavage mediated by eukaryotic topoisomerase II: a novel class of topoisomerase II-targeted drugs with cytotoxic potential.
Recently, a number of novel quinolones with potent activity against topoisomerase II and eukaryotic cells have been described. Many of these compounds contain aromatic substituents in their C-7 ring positions. To determine whether pyrimido[1,6-a]benzimidazoles, a class of drugs modeled on quinolones, also display activity toward eukaryotic systems, the effects of Ro 46-7864 and Ro 47-3359 on Drosophila melanogaster topoisomerase II and Kc cells were characterized. While the former drug contains an aliphatic group (4-N-methylpiperazine) at the ring position equivalent to C-7 in quinolones, the latter compound contains an aromatic substituent (2,6-dimethylpyridine). Both pyrimido[1,6-a]benzimidazoles inhibited DNA relaxation catalyzed by the type II enzyme. However, only Ro 47-3359 enhanced topoisomerase II-mediated DNA cleavage and was toxic to Kc cells. At a concentration of 100 microM, this drug approximately doubled the levels of DNA breakage in vitro and killed > 50% of the initial cell population of cultures. These results strongly suggest that selected pyrimido[1,6-a]benzimidazoles may function as topoisomerase II-targeted drugs with cytotoxic potential.
4-Quinolones↗
Distribution and polymorphism of the flagellin genes from isolates of Campylobacter coli and Campylobacter jejuni.
The complex flagellar filaments of the LIO8 serogroup member Campylobacter coli VC167 are composed of two highly related subunit proteins encoded by the flaA and flaB genes which share 92% identity. Using oligonucleotide primers based on the known DNA sequence of both the flaA and flaB genes from C. coli VC167 in the polymerase chain reaction, we have shown conservation of both fla genes among isolates within the LIO8 heat-labile serogroup by digestion of the amplified product with PstI and EcoRI restriction endonucleases. Amplification and subsequent restriction analysis of the flaA flagellin gene from Campylobacter isolates belonging to 13 different LIO serogroups further identified 10 unique polymorphic groups. Within most of the serogroups examined, isolates appeared to contain flaA genes with conserved primary structures. Only in serogroups LIO11 and LIO29 did independent isolates possess flagellin genes with different primary structures. Furthermore, by employing primers specific for the flaB gene of C. coli VC167, all serogroups examined contained a second fla gene corresponding to flaB. In all serogroups except the LIO5 and LIO6 isolates which were identical to each other, the polymorphic pattern of this flaB gene was identical to that of the corresponding flaA gene. These data indicate that the presence of a second highly homologous flagellin gene is widespread throughout Campylobacter isolates and that in most instances, the primary structure of the two fla genes is conserved within isolates belonging to the same heat-labile LIO serogroup. This may represent the presence of clonal evolutionary groups in Campylobacter spp.
Antigens, Bacterial↗
The Campylobacter sigma 54 flaB flagellin promoter is subject to environmental regulation.
The complex flagellum of Campylobacter coli VC167 is encoded by two tandemly oriented flagellin genes which are transcribed as two discrete transcriptional units from two different classes of promoters. The flaB gene, which encodes the minor FlaB filament protein, is controlled by a sigma 54 promoter. A transcriptional fusion between a promoterless chloramphenicol acetyltransferase (CAT) reporter gene cartridge and C. coli VC167 DNA carrying flaB transcription and translation signals, including the typical position -13-to-(-)26 flaB sigma 54 consensus promoter sequence, was constructed. When carried on plasmid pRIC1013, the sigma 54-CAT fusion expressed chloramphenicol resistance in Escherichia coli, and CAT production was affected by the pH of the growth medium, the composition of the growth atmosphere, and the growth temperature, with production being significantly higher at 42 degrees C. A conjugative suicide vector, pRIC1028, containing the sigma 54-CAT fusion was constructed and used to recombine the flaB-CAT fusion back into the C. coli chromosome in the correct position with respect to the flaA gene and its transcription terminator. CAT production from the flaB sigma 54 promoter in the C. coli transconjugant VC167-T2/28-1 was shown to peak at mid-log phase and to be modulated by growth medium pH, growth temperature, and the concentration of certain inorganic salts and divalent cations in the growth medium. Under growth conditions which promoted elevated flaB sigma 54 promoter activity, a flaA flaB+ mutant of C. coli VC167 produced increased amounts of FlaB flagellar protein and displayed increased motility.
Bacterial Proteins↗
Variation in antigenicity and molecular weight of Campylobacter coli VC167 flagellin in different genetic backgrounds.
Campylobacter coli VC167 has been shown to undergo a reversible flagellar antigenic variation between antigenic type 1 (T1) and antigenic type 2 (T2). VC167 contains two flagellin genes, and the products of both genes are incorporated into a complex flagellar filament in both antigenic types. Although there are only minor amino acid changes in the flagellins expressed by T1 and T2 cells, the two antigenic types of flagellins can be distinguished by differences in apparent M(r) on sodium dodecyl sulfate-polyacrylamide gels and by immunoreactivity with T1-specific (LAH1) or T2-specific (LAH2) antiserum. The isolation of stable variants of T1 and T2 has allowed for the transfer via natural transformation of the flagellin structural genes from the T1 background into the T2 background and from the T2 background into the T1 background. In addition, the flagellin genes from VC167 T1 and T2 have been transferred into strains of Campylobacter jejuni. The results indicate that the observed antigenic variations of VC167 flagellins are dependent on the host genetic background and independent of the primary amino acid sequence. These data provide evidence that posttranslational modifications are responsible for the antigenic variation seen in VC167 flagellins.
Antigenic Variation↗
Specific detection of Campylobacter jejuni and Campylobacter coli by using polymerase chain reaction.
Development of a routine detection assay for Campylobacter jejuni and Campylobacter coli in clinical specimens was undertaken by using the polymerase chain reaction (PCR). An oligonucleotide primer pair from a conserved 5' region of the flaA gene of C. coli VC167 was used to amplify a 450-bp region by PCR. The primer pair specifically detected 4 strains of C. coli and 47 strains of C. jejuni; but it did not detect strains of Campylobacter fetus, Campylobacter lari, Campylobacter upsaliensis, Campylobacter cryaerophila, Campylobacter butzleri, Campylobacter hyointestinalis, Wolinella recta, Helicobacter pylori, Escherichia coli, Shigella spp., Salmonella spp., Vibrio cholerae, Citrobacter freundii, or Aeromonas spp. By using a nonradioactively labeled probe internal to the PCR product, the assay could detect as little as 0.0062 pg of purified C. coli DNA, or the equivalent of four bacteria. In stools seeded with C. coli cells, the probe could detect between 30 and 60 bacteria per PCR assay. The assay was also successfully used to detect C. coli in rectal swab specimens from experimentally infected rabbits and C. jejuni in human stool samples.
Animals↗
Role of two flagellin genes in Campylobacter motility.
Campylobacter coli VC167 T2 has two flagellin genes, flaA and flaB, which share 91.9% sequence identity. The flaA gene is transcribed from a o-28 promoter, and the flaB gene from a o-54 promoter. Gene replacement mutagenesis techniques were used to generate flaA+ flaB and flaA flaB+ mutants. Both gene products are capable of assembling independently into functional filaments. A flagellar filament composed exclusively of the flaA gene product is indistinguishable in length from that of the wild type and shows a slight reduction in motility. The flagellar filament composed exclusively of the flaB gene product is severely truncated in length and greatly reduced in motility. Thus, while both flagellins are not necessary for motility, both products are required for a fully active flagellar filament. Although the wild-type flagellar filament is a heteropolymer of the flaA and flaB gene products, immunogold electron microscopy suggests that flaB epitopes are poorly surface exposed along the length of the wild-type filament.
Amino Acid Sequence↗
Analysis of the role of flagella in the heat-labile Lior serotyping scheme of thermophilic Campylobacters by mutant allele exchange.
Flagellin mutations originally constructed in Campylobacter coli VC167 (serotype LIO8) by a gene replacement mutagenesis technique (P. Guerry, S. M. Logan, S. Thornton, and T. J. Trust, J. Bacteriol. 172:1853-1860, 1990) were moved from the original host into Campylobacter strains of a number of other Lior serogroups by a natural transformation procedure. This is the first report of the use of this transformation method to transfer a mutated locus among Campylobacter strains. Flagellin mutants were constructed in a number of heat-labile LIO serotypes and were serotyped and analyzed by immunoelectron microscopy with LIO typing antisera. In six cases, isogenic nonflagellated mutants were able to be serotyped in the same serogroup as their parent, and immunogold electron microscopy confirmed that antibodies in the typing antisera bound to components on the surface of both parent and mutant cells. However, in only one case, a strain belonging to serogroup LIO4, was a nonflagellated mutant untypeable, and immunogold electron microscopy showed that antibodies bound to the flagella filament of the parent but not to the cell surface. Furthermore, after introduction and expression as a flagellar filament of a LIO8 flagellin gene in this mutant, the strain could not be serotyped. These results indicate that a nonflagellar antigen is often the serodeterminant in the heat-labile Lior serotyping scheme.
Alleles↗
Inhibitors of 2,3-oxidosqualene lanosterol-cyclase as potential antifungal agents.
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Animals↗
Polynucleotide sequence relationships among flagellin genes of Campylobacter jejuni and Campylobacter coli.
DNA probes that encode a complete flagellin gene and various internal regions of the Campylobacter coli VC167 flagellin genes were hybridized to 30 strains of C. coli or C. jejuni from 20 different Lior serogroups. The results indicated a high overall degree of homology among all of the strains examined. Although the most variable regions occurred within the middle of the gene, significant DNA homology was observed among many serogroups in this region of the molecule.
Bacterial Proteins↗