A role for lipopolysaccharide in turkey tracheal colonization by Bordetella avium as demonstrated in vivo and in vitro.
ABSTRACT: We isolated two insertion mutants of Bordetella avium that exhibited a peculiar clumped-growth phenotype and found them to be attenuated in turkey tracheal colonization. The mutants contained transposon insertions in homologues of the wlbA and wlbL genes of Bordetella pertussis. The wlb genetic locus of B. pertussis has been previously described as containing 12 genes involved in lipopolysaccharide (LPS) biosynthesis. Polyacrylamide gel analysis of LPS from B. avium wlbA and wlbL insertion mutants confirmed an alteration in the LPS profile. Subsequent cloning and complementation of the wlbA and wlbL mutants in trans with a recombinant plasmid containing the homologous wlb locus from B. avium eliminated the clumped-growth phenotype and restored the LPS profile to that of wild-type B. avium. Also, a parental level of tracheal colonization was restored to both mutants by the recombinant plasmid. Interestingly, complementation of the wlbA and wlbL mutants with a recombinant plasmid containing the heterologous wlb locus from B. pertussis, B. bronchiseptica, or Bordetella parapertussis eliminated the clumped-growth phenotype and resulted in a change in the LPS profile, although not to that of wild-type B. avium. The mutants also acquired resistance to a newly identified B. avium-specific bacteriophage, Ba1. Complementation of both wlbA and wlbL mutants with the homologous wlb locus of B. avium, but not the heterologous B. pertussis locus, restored sensitivity to Ba1. Complementation of the wlbL mutant, but not the wlbA mutant, with the heterologous wlb locus of Bordetella bronchiseptica or B. parapertussis restored partial sensitivity to Ba1. Comparisons of the LPS profile and phage sensitivity of the mutants upon complementation by wlb loci from the heterologous species and by B. avium suggested that phage sensitivity required the presence of O-antigen. At the mechanistic level, both mutants showed a dramatic decrease in serum resistance and a decrease in binding to turkey tracheal rings in vitro. In the case of serum resistance, complementation of both mutants with the homologous wlb locus of B. avium restored serum resistance to wild-type levels. However, in the case of epithelial cell binding, only complementation of the wlbA mutant completely restored binding to wild-type levels (binding was only partially restored in the wlbL mutant). This is the first characterization of LPS mutants of B. avium at the genetic level and the first report of virulence changes by both in vivo and in vitro measurements.
Project description:The products of the bvgAS locus activate expression of a majority of the known Bordetella virulence factors but also exert negative control over a class of genes called vrg genes (bvg-repressed genes). BvgAS negatively controls the production of flagella and the phenotype of motility in Bordetella bronchiseptica. In this study flaA, the flagellin gene, was cloned and characterized to facilitate studies of this negative control pathway. An internal flaA probe detected hybridizing sequences on genomic Southern blots of Bordetella pertussis, Bordetella parapertussis, and Bordetella avium, although B. pertussis and B. parapertussis are nonmotile. FlaA is similar to the FliC flagellins of Salmonella typhimurium and Escherichia coli, and flaA complemented an E. coli flagellin mutant. Insertional inactivation of the chromosomal flaA locus eliminated motility, which was restored by complementation with the wild-type locus. Analysis of flaA mRNA production by Northern (RNA) blotting and primer extension indicated that negative regulation by BvgAS occurs at the level of transcription. The transcriptional start site of flaA mapped near a consensus site for the alternative sigma factor, sigma F, encoded by fliA in E. coli and S. typhimurium. Consistent with a role for a fliA analog in B. bronchiseptica, transcriptional activation of a flaA-lacZ fusion in E. coli required fliA and a flaA-linked locus designated frl.frl also efficiently complemented mutations in the flagellar master regulatory locus, flhDC, of E. coli. Our analysis of the motility phenotype of B. bronchiseptica suggests that the Bordetella virulence control system mediates transcriptional control of flaA through a regulatory hierarchy that includes the frl locus and an alternative sigma factor.
Project description:A DNA locus from Bordetella pertussis capable of reconstituting lipopolysaccharide (LPS) O-antigen biosynthesis in Salmonella typhimurium SL3789 (rfaF511) has been isolated, by using selection with the antibiotic novobiocin. DNA within the locus encodes a protein with amino acid sequence similarity to heptosyltransferase II, encoded by waaF (previously rfaF) in other gram-negative bacteria. Mutation of this gene in B. pertussis, Bordetella parapertussis, and Bordetella bronchiseptica by allelic exchange generated bacteria with deep rough LPS phenotypes consistent with the proposed function of the gene as an inner core heptosyltransferase. These are the first LPS mutants generated in B. parapertussis and B. bronchiseptica and the first deep rough mutants of any of the bordetellae.
Project description:Chromosomal insertions defining Bordetella bronchiseptica siderophore phenotypic complementation group III mutants BRM3 and BRM5 were found to reside approximately 200 to 300 bp apart by restriction mapping of cloned genomic regions associated with the insertion markers. DNA hybridization analysis using B. bronchiseptica genomic DNA sequences flanking the cloned BRM3 insertion marker identified homologous Bordetella pertussis UT25 cosmids that complemented the siderophore biosynthesis defect of the group III B. bronchiseptica mutants. Subcloning and complementation analysis localized the complementing activity to a 2.8-kb B. pertussis genomic DNA region. Nucleotide sequencing identified an open reading frame predicted to encode a polypeptide exhibiting strong similarity at the primary amino acid level with several pyridoxal phosphate-dependent amino acid decarboxylases. Alcaligin production was fully restored to group III mutants by supplementation of iron-depleted culture media with putrescine (1,4-diaminobutane), consistent with defects in an ornithine decarboxylase activity required for alcaligin siderophore biosynthesis. Concordantly, the alcaligin biosynthesis defect of BRM3 was functionally complemented by the heterologous Escherichia coli speC gene encoding an ornithine decarboxylase activity. Enzyme assays confirmed that group III B. bronchiseptica siderophore-deficient mutants lack an ornithine decarboxylase activity required for the biosynthesis of alcaligin. Siderophore production by an analogous mutant of B. pertussis constructed by allelic exchange was undetectable. We propose the designation odc for the gene defined by these mutations that abrogate alcaligin siderophore production. Putrescine is an essential precursor of alcaligin in Bordetella spp.
Project description:We have characterized a new virulence factor in Bordetella pertussis: serum resistance. Compared with Escherichia coli HB101, wild-type B. pertussis was relatively resistant to classical-pathway, complement-dependent killing by normal human serum. However, a mutant of B. pertussis (BPM2041) which is less virulent in mice and which has Tn5 lac inserted in a previously uncharacterized bvg-regulated gene was found to be at least 10-fold more susceptible to serum killing than the wild type. We have named this locus brk, for Bordetella resistance to killing. We have cloned and sequenced the brk locus, and it encodes two divergently transcribed open reading frames (ORFs), termed BrkA and BrkB. Both ORFs are necessary for serum resistance. Within the 300 bases which separate the two ORFs and upstream of each ORF are putative sites for BvgA binding. BrkA shows 29% identity to pertactin and has two RGD motifs in addition to a conserved proteolytic processing site and an outer membrane targeting signal. Like pertactin, BrkA is involved in adherence and invasion. Despite the similarities, a pertactin mutant was found to be not as sensitive to serum killing as the BrkA or BrkB mutants. BrkB is similar to ORFs in E. coli and Mycobacterium leprae and displays domains of homology to various transporters. On the basis of its hydropathy profile, BrkB is predicted to be a cytoplasmic membrane protein. By Southern blot, brk sequences were found in Bordetella bronchiseptica and Bordetella parapertussis but not in Bordetella avium.
Project description:Bordetellosis is an upper respiratory disease of turkeys caused by Bordetella avium in which the bacteria attach specifically to ciliated respiratory epithelial cells. Little is known about the mechanisms of pathogenesis of this disease, which has a negative impact in the commercial turkey industry. In this study, we produced a novel explant organ culture system that was able to successfully reproduce pathogenesis of B. avium in vitro, using tracheal tissue derived from 26 day-old turkey embryos. Treatment of the explants with whole cells of B. avium virulent strain 197N and culture supernatant, but not lipopolysaccharide (LPS) or tracheal cytotoxin (TCT), specifically induced apoptosis in ciliated cells, as shown by annexin V and TUNEL staining. LPS and TCT are known virulence factors of Bordetella pertussis, the causative agent of whooping cough. Treatment with whole cells of B. avium and LPS specifically induced NO response in ciliated cells, shown by uNOS staining and diaphorase activity. The explant system is being used as a model to elucidate specific molecules responsible for the symptoms of bordetellosis.
Project description:Bordetella avium causes an upper respiratory tract disease (bordetellosis) in avian species. Commercially raised turkeys are particularly susceptible. Like other pathogenic members of the genus Bordetella (B. pertussis and B. bronchiseptica) that infect mammals, B. avium binds preferentially to ciliated tracheal epithelial cells and produces similar signs of disease. These similarities prompted us to study bordetellosis in turkeys as a possible nonmammalian model for whooping cough, the exclusively human childhood disease caused by B. pertussis. One impediment to accepting such a host-pathogen model as relevant to the human situation is evidence suggesting that B. avium does not express a number of the factors known to be associated with virulence in the other two Bordetella species. Nevertheless, with signature-tagged mutagenesis, four avirulent mutants that had lesions in genes orthologous to those associated with virulence in B. pertussis and B. bronchiseptica (bvgS, fhaB, fhaC, and fimC) were identified. None of the four B. avium genes had been previously identified as encoding factors associated with virulence, and three of the insertions (in fhaB, bvgS, and fimC) were in genes or gene clusters inferred as being absent or incomplete in B. avium, based upon the lack of DNA sequence similarities in hybridization studies and/or the lack of immunological cross-reactivity of the putative products. We further found that the genotypic arrangements of most of the B. avium orthologues were very similar in all three Bordetella species. In vitro tests, including hemagglutination, tracheal ring binding, and serum sensitivity, helped further define the phenotypes conferred by the mutations. Our findings strengthen the connection between the causative agents and the pathogenesis of bordetellosis in all hosts and may help explain the striking similarities of the histopathologic characteristics of this upper airway disease in avian and mammalian species.
Project description:Transcription of the pertussis toxin operon (ptx) is positively regulated in Bordetella pertussis by the bvgAS locus. However, a ptx-lacZ transcriptional fusion in Escherichia coli cannot be activated by bvgAS in trans. This suggests that an additional factor(s) is required for transcription of ptx. A gene encoding a Bvg accessory factor (Baf) was identified by its ability to activate an E. coli ptx-lacZ fusion in the presence of bvgAS. The expression of ptx-lacZ was decreased by the addition of 40 mM MgSO4, a compound that also modulates ptx expression in B. pertussis. Baf alone did not activate expression of an E. coli fhaB-lacZ fusion, nor did it increase expression of fhaB-lacZ in trans with bvgAS. The gene encoding Baf was localized, sequenced, and found to produce a novel 28-kDa protein. Sequences homologous to B. pertussis baf were identified in Bordetella bronchiseptica and Bordetella parapertussis but not in Bordetella avium. When an additional copy of baf was integrated into the chromosome of BC75, a B. pertussis mutant that produces a low level of pertussis toxin, pertussis toxin production was partially complemented in the cointegrate strain.
Project description:Bordetella pertussis and Bordetella bronchiseptica, which are respiratory mucosal pathogens of mammals, produce and utilize the siderophore alcaligin to acquire iron in response to iron starvation. A predicted permease of the major facilitator superfamily class of membrane efflux pumps, AlcS (synonyms, OrfX and Bcr), was reported to be encoded within the alcaligin gene cluster. In this study, alcS null mutants were found to be defective in growth under iron starvation conditions, in iron source utilization, and in alcaligin export. trans complementation using cloned alcS genes of B. pertussis or B. bronchiseptica restored the wild-type phenotype to the alcS mutants. Although the levels of extracellular alcaligin measured in alcS strain culture fluids were severely reduced compared with the wild-type levels, alcS mutants had elevated levels of cell-associated alcaligin, implicating AlcS in alcaligin export. Interestingly, a deltaalcA mutation that eliminated alcaligin production suppressed the growth defects of alcS mutants. This suppression and the alcaligin production defect were reversed by trans complementation of the deltaalcA mutation in the double-mutant strain, confirming that the growth-defective phenotype of alcS mutants is associated with alcaligin production. In an alcA::mini-Tn5 lacZ1 operon fusion strain background, an alcS null mutation resulted in enhanced AlcR-dependent transcriptional responsiveness to alcaligin inducer; conversely, AlcS overproduction blunted the transcriptional response to alcaligin. These transcription studies indicate that the alcaligin exporter activity of AlcS is required to maintain appropriate intracellular alcaligin levels for normal inducer sensing and responsiveness necessary for positive regulation of alcaligin system gene expression.
Project description:We report the isolation and preliminary phenotypic characterization of manganese-resistant Bordetella bronchiseptica mutants with respect to deregulation of siderophore and iron-regulated protein expression. The fur gene of Bordetella pertussis was cloned by genetic complementation of this deregulated phenotype and confirmed as fur by nucleotide sequence analysis.
Project description:Bordetella pertussis 18323 produces a bvg-regulated 39.1-kDa porin-like protein, OmpQ. OmpQ had 61% similarity to the major porin of B. pertussis and contains conserved regions common to both the neisserial and enteric porin families. The results of Southern blot analysis indicate that strains of Bordetella parapertussis and Bordetella bronchiseptica but not Bordetella avium contain this gene.