Hfr mapping of mutations in Bordetella pertussis that define a genetic locus involved in virulence gene regulation.

We report the development of techniques for the genetic mapping of point mutations in the bacterial pathogen Bordetella pertussis. A plasmid vector which is self-transmissible by conjugation and which, by insertion into the B. pertussis chromosome, can mobilize chromosomal sequences during conjugation with a recipient B. pertussis bacterium has been constructed. This vector is used in conjunction with a set of strains containing kanamycin resistance gene insertions at defined physical locations in the B. pertussis genome. In crosses between these donor strains and a mutant recipient strain, transfer of a chromosomal segment flanking the kanamycin resistance gene insertion is selected for, and the percentage of exconjugants which reacquire the wild-type trait is scored. In this way the linkage of the mutant allele to these markers, and thus the approximate chromosomal position of the mutant allele, is determined. We have used this genetic system to map a newly described locus in B. pertussis involved in the regulation of the virulence genes ptx (pertussis toxin) and cya (adenylate cyclase toxin).     

Bordetella pertussis adenylate cyclase toxin and hemolytic activities require a second gene, cyaC, for activation.

In these studies, the Bordetella pertussis adenylate cyclase toxin-hemolysin homology to the Escherichia coli hemolysin is extended with the finding of cyaC, a homolog to the E. coli hlyC gene, which is required for the production of a functional hemolysin molecule in E. coli. Mutations produced in the chromosome of B. pertussis upstream from the structural gene for the adenylate cyclase toxin revealed a region which was necessary for toxin and hemolytic activities of the molecule. These mutants produced the 216-kDa adenylate cyclase toxin as determined by Western blot (immunoblot) analysis. The adenylate cyclase enzymatic activities of these mutants were equivalent to that of wild type, but toxin activities were less than 1% of that of wild type, and the mutants were nonhemolytic on blood agar plates and in in vitro assays. The upstream region restored hemolytic activity when returned in trans to the mutant strains. This genetic complementation defined a gene which acts in trans to activate the adenylate cyclase toxin posttranslationally. Sequence analysis of the upstream region defined an open reading frame with homology to the E. coli hlyC gene. In contrast to E. coli, this open reading frame is oriented oppositely from the adenylate cyclase toxin structural gene.     

Inhibition of monocyte oxidative responses by Bordetella pertussis adenylate cyclase toxin

Bordetella pertussis and the other Bordetella species produce a novel adenylate cyclase toxin which enters target cells to catalyze the production of supraphysiologic levels of intracellular cyclic adenosine monophosphate (cAMP). In these studies, dialyzed extracts from B. pertussis containing the adenylate cyclase toxin, a partially purified preparation of adenylate cyclase toxin, and extracts from transposon Tn5 mutants of B. pertussis lacking the adenylate cyclase toxin, were used to assess the effects of adenylate cyclase toxin on human peripheral blood monocyte activities. Luminol-enhanced chemiluminescence of monocytes stimulated with opsonized zymosan was inhibited greater than 96% by exposure to adenylate cyclase toxin-containing extract, but not by extracts from adenylate cyclase toxin-deficient mutants. The chemiluminescence responses to particulate (opsonized zymosan, Leishmania donovani, and Staphylococcus aureus) and soluble (phorbol myristate acetate) stimuli were inhibited equivalently. The superoxide anion generation elicited by opsonized zymosan was inhibited 92% whereas that produced by phorbol myristate acetate was inhibited only 32% by B. pertussis extract. Inhibition of oxidative activity was associated with a greater than 500-fold increase in monocyte cAMP levels, but treated monocytes remained viable as assessed by their ability to exclude trypan blue and continued to ingest particulate stimuli. The major role of the adenylate cyclase toxin in the inhibition of monocyte oxidative responses was demonstrated by: 1) little or no inhibition by extracts from B. pertussis mutants lacking adenylate cyclase toxin; 2) high level inhibition with extract from B. parapertussis, a related species lacking pertussis toxin; and 3) a reciprocal relationship between monocyte cAMP levels and inhibition of opsonized zymosan-induced chemiluminescence using both crude extract and partially purified adenylate cyclase toxin. Pertussis toxin, which has been shown to inhibit phagocyte responses to some stimuli by a cAMP-independent mechanism, had only a small (less than 20%) inhibitory effect when added at concentrations up to 100-fold in excess of those present in B. pertussis extract. These data provide strong support for the hypothesis that B. pertussis adenylate cyclase toxin can increase cAMP levels in monocytes without compromising target cell viability or impairing ingestion of particles and that the resultant accumulated cAMP is responsible for the inhibition of oxidative responses to a variety of stimuli.     

Detection and subcellular localization of three Ptl proteins involved in the secretion of pertussis toxin from Bordetella pertussis.

The ptl locus of Bordetella pertussis contains eight open reading frames which are predicted to encode proteins (PtlA to PtlH) that are essential for secretion of pertussis toxin from the bacterium and which are members of a family of transport proteins found in other types of bacteria. We have detected PtlE, PtlF, and PtlG in immunoblots of extracts of B. pertussis by using antibodies raised to fusion proteins consisting of maltose-binding protein and the individual Ptl proteins. These proteins have apparent molecular weights similar to those predicted by DNA sequence analysis. Cell fractionation studies indicated that all three Ptl proteins are associated with the membranes of B. pertussis, suggesting that the Ptl proteins form a gate or channel which facilitates transport of pertussis toxin. Cell extracts of other Bordetella spp. were probed with antibodies to Ptl proteins for the presence of these transport proteins. Neither Bordetella parapertussis nor Bordetella bronchiseptica contained detectable levels of PtlE or PtlF. This lack of detectable Ptl protein may provide an explanation for previous observations which indicated that introduction of the genes encoding pertussis toxin subunits from B. pertussis into other Bordetella spp. results in production of the toxin but not secretion of the toxin.     

Complementation of mutations in Escherichia coli and Bordetella pertussis by B. pertussis DNA cloned in a broad-host-range cosmid vector

A gene library of Bordetella pertussis DNA was constructed in Escherichia coli using the broad-host-range cosmid vector pLAFR1. The average insert size was 24.9 kb. From 500 members of the gene library, clones were identified which complemented trpE, glnA and Thr- mutations in E. coli but none which complemented trpD, trpC, trpB, trpA, proA or Leu- mutations. Four clones were identified which complemented trpE in E. coli. Anthranilate synthase activity was detected in a trpE strain only when it harboured a plasmid from one of these clones; activity was repressed when tryptophan was included in the growth medium. Two clones were identified which complemented glnA of E. coli. A recombinant plasmid from one of these clones also restored some of the nitrogen acquisition functions of glnG and glnL in E. coli. Expression of several B. pertussis virulence-associated products (haemolysin, heat-labile toxin, adenylate cyclase, filamentous haemagglutinin, and the cell-envelope polypeptide of Mr 30,000) was not detected in 500 independent clones. However, by transferring the recombinant plasmids to a mutant of B. pertussis deficient in haemolysin and adenylate cyclase, a plasmid was identified which restored both these activities.     

Serum resistance in bvg-regulated mutants of Bordetella pertussis

Serum resistance, or resistance to killing by antibody dependent pathway of complement, in Bordetella pertussis is bvg-regulated and the Bordetella resistance to killing (brk) locus mediates much of the resistance. Here we examined whether other bvg-regulated proteins contribute to serum resistance. We found that neither pertussis toxin, adenylate cyclase toxin, filamentous hemagglutinin, dermonecrotic toxin, tracheal colonization factor, nor Vag8 mutants were sensitive to serum killing compared to the wild-type. Filamentous hemagglutinin has been reported to bind C4 binding protein, an inhibitor of complement, but this activity does not appear to contribute to serum resistance, as evidenced by the resistant phenotype of FHA mutants. Clinical isolates were serum resistant and wild-type strains possessing an additional copy of the brk locus were 2–5-fold more resistant to serum killing.     

Genetic analysis of a region of the Bordetella pertussis chromosome encoding filamentous hemagglutinin and the pleiotropic regulatory locus vir.

The vir locus of Bordetella pertussis apparently encodes a trans-acting positive regulator that is required for the coordinate expression of genes associated with virulence: pertussis toxin, filamentous hemagglutinin (FHA), hemolysin, and adenylate cyclase toxin. DNA clones of vir and of genes required for the synthesis of some of the factors under vir control were obtained with DNA probes from the chromosomal DNA surrounding sites of Tn5 insertion mutations that inactivated those genes. Two vir clones were found which also contained genes required for the proper expression of FHA in B. pertussis. The plasmids which contained both the fha and vir genes expressed immunologically reactive FHA in Escherichia coli, as detected by colony blots, whereas plasmids which contained only fha or vir were negative in this assay. The regulation of FHA production in E. coli, as in B. pertussis, was temperature dependent and inhibited by high concentrations of either magnesium ions or nicotinic acid, indicating that the sequences cloned in E. coli contained the information required to preserve the physiological responses seen in B. pertussis. Further characterization of the vir-fha clones by Tn5 mutagenesis in E. coli and by the return of cloned sequences to B. pertussis in trans and to the B. pertussis chromosome led to the localization of the vir locus, the structural gene for FHA, and genes that are possibly required for the synthesis and export of FHA.     

Bordetella pertussis adenylate cyclase toxin. Structural and functional independence of the catalytic and hemolytic activities.

The Bordetella pertussis calmodulin-dependent adenylate cyclase (CyaA) is a 1706-residue-long toxin, endowed with hemolytic activity. We have constructed B. pertussis mutant strains producing modified CyaAs devoid of adenylate cyclase activity. Our results show that such modified CyaAs display hemolytic activity identical to the wild-type toxin, thus demonstrating that the hemolytic activity is independent of the adenylate cyclase activity. Furthermore, B. pertussis and Escherichia coli strains producing CyaA lacking the catalytic domain (residues 1-373) were constructed. The truncated protein exhibits hemolytic activity comparable to the wild-type toxin, thus establishing that the carboxyl-terminal 1332 residues alone are endowed with hemolytic activity. Together, these findings show that adenylate cyclase and hemolytic activities are located in two distinct regions of the molecule (respectively, approximately amino acids 1-400 and 401-1706) and that the two regions of CyaA are functionally independent.     

Bordetella pertussis lipopolysaccharide resists the bactericidal effects of pulmonary surfactant protein A

Surfactant protein A (SP-A) plays an important role in the innate immune defense of the respiratory tract. SP-A binds to lipid A of bacterial LPS, induces aggregation, destabilizes bacterial membranes, and promotes phagocytosis by neutrophils and macrophages. In this study, SP-A interaction with wild-type and mutant LPS of Bordetella pertussis, the causative agent of whooping cough, was examined. B. pertussis LPS has a branched core structure with a nonrepeating trisaccharide, rather than a long-chain repeating O-Ag. SP-A did not bind, aggregate, nor permeabilize wild-type B. pertussis. LPS mutants lacking even one of the sugars in the terminal trisaccharide were bound and aggregated by SP-A. SP-A enhanced phagocytosis by human monocytes of LPS mutants that were able to bind SP-A, but not wild-type bacteria. SP-A enhanced phagocytosis by human neutrophils of LPS-mutant strains, but only in the absence of functional adenylate cyclase toxin, a B. pertussis toxin that has been shown to depress neutrophil activity. We conclude that the LPS of wild-type B. pertussis shields the bacteria from SP-A-mediated clearance, possibly by sterically limiting access to the lipid A region.     

The PtlE protein of Bordetella pertussis has peptidoglycanase activity required for Ptl-mediated pertussis toxin secretion

Pertussis toxin of Bordetella pertussis is secreted by a type IV secretion system comprised of the products of the nine ptl (pertussis toxin liberation) genes. These proteins are believed to form a complex spanning both the inner and outer membranes and passing through the peptidoglycan layer. Peptidoglycan acts as a barrier for transport through the periplasm of large folded molecules. Assembled pertussis toxin and the secretion component proteins PtlC through PtlH are too large to diffuse through intact peptidoglycan. Therefore, we hypothesized that the Ptl system contains a peptidoglycanase activity. The PtlE protein was found to exhibit a sequence match to the active site of glycohydrolase enzymes. An N-terminally polyhistidine-tagged PtlE fusion protein, constructed and expressed in Escherichia coli and in B. pertussis, exhibited peptidoglycanase activity on activity gels. A fusion protein with alanine substitutions at the putative active site residues (aspartic acid at position 53 and glutamic acid at position 62) lacked peptidoglycanase activity. B. pertussis strains with the amino acid substitutions were deficient for pertussis toxin secretion. Based on these results, we concluded that PtlE is a peptidoglycanase responsible for the local removal or rearrangement of the peptidoglycan layer during Ptl secretion complex assembly.     

Virulence factors of Bordetella pertussis

Clearly, B. pertussis has evolved very elaborate mechanisms to maintain itself in the human host. Three different proteins (FHA, pertussis toxin and fimbriae) have been implicated in adherence. Furthermore, a number of toxins are produced (pertussis toxin, adenylate cyclase, dermonecrotic toxin, and tracheal cytotoxin) which destroy the clearance mechanisms of the respiratory tract, or suppress the immune response. There is evidence that B. pertussis may survive intracellularly, and the possibility that it is a facultative intracellular parasite should certainly be explored. The availability of a large number of cloned virulence genes, and a system to construct well defined mutants by allelic exchange (Stibbitz et al. 1986) will greatly facilitate the study of Bordetella virulence factors at the molecular level. It opens the possibility to construct avirulent strains, which are still able to colonize and stimulate the local immune response. Such strains may be used as live, oral vaccines, to present (heterologous) antigens to the mucosal immune system of the respiratory tract.     

The calmodulin-sensitive adenylate cyclase of Bordetella pertussis: cloning and expression in Escherichia col

The adenylate cyclase toxin of the prokaryote Bordetella pertussis is stimulated by the eukaryotic regulatory protein, calmodulin. A general strategy, using the adenylate-cyclase-calmodulin interaction as a tool, has permitted cloning and expression of the toxin in Escherichia coli in the absence of any B. pertussis trans-activating factor. We show that the protein is synthesized in a large precursor form composed of 1706 amino acids. The calmodulin-stimulated catalytic activity resides in the amino-terminal 450 amino acids of the adenylate cyclase. The enzyme expressed in E. coli is recognized in Western blots by antibodies directed against purified B. pertussis adenylate cyclase, and its activity is inhibited by these antibodies.     

Distinction between Clostridium botulinum type A strains associated with food-borne botulism and those with infant botulism in Japan in intraintestinal toxin production in infant mice and some other properties

In Bordetella pertussis virulence-associated genes, including adenylate cyclase toxin (Cya), are coordinately regulated in response to environmental signals by proteins coded by the bvg-locus. We have constructed cya-lac fusions in Escherichia coli and have shown that the cya operon is not expressed in E. coli, neither is it activated by bvg, when introduced in trans. The cya-lac fusion is fully active when returned to B. pertussis by homologous recombination and responds to bvg-dependent activation and environmental regulation. These results indicate that in B. pertussis the activation of the cya operon by bvg is indirect.     

Sequential activation and environmental regulation of virulence genes in Bordetella pertussis.

Bacterial pathogens undergo profound physiological changes when they infect their host and require co-ordinated regulation of gene expression in response to the stress encountered during infection. In Bordetella pertussis, the human pathogen which causes whooping cough, virulence factors are synthesized in response to environmental signals under the control of the bvg regulatory locus. Here we demonstrate that the bvg locus is responsible for two events of gene activation. In the first step the bvg locus transactivates its own autoregulated promoter (P1) and the promoter of the adherence factor filamentous haemagglutinin (PFHA). The second step occurs several hours later and consists of the transactivation of adenylate cyclase and pertussis toxin genes. We provide evidence that the second step of transactivation requires overexpression of regulatory proteins. Our results imply that bacterial adhesion and tissue colonization--intoxication are two separate steps at the molecular level.     

Differential response of the bvg virulence regulon of Bordetella pertussis to MgSO4 modulation.

Magnesium sulfate is known to repress the expression of the virulence factors of Bordetella pertussis that are coordinately regulated by the bvg locus. We have tested the time required by MgSO4 to repress the synthesis of several bvg-regulated mRNA species and found that the promoters of the virulence genes (pertussis toxin, adenylate cyclase, and filamentous hemagglutinin) are repressed in 6 min, while the autogenously regulated promoters of the bvg locus (P1, P3, and P4) are repressed only several hours later. These data show a differential behavior between regulated and autoregulated genes of the bvg regulon.     

Actions of cyclic adenosine monophosphate on the cytodifferentiation of ovarian cells: studies in cultured swine granulosa cells using a novel exogenous adenylate cyclase from Bordetella pertussis

The regulation by cAMP of cholesterol side-chain cleavage activity and the synthesis of immunoisolated cytochrome P-450scc and adrenodoxin proteins was investigated in primary cultures of swine ovarian (granulosa) cells. Administration of a novel adenylate cyclase toxin isolated from Bordetella pertussis increased granulosa-cell cAMP accumulation up to 200-fold over basal. These effects were additive with those of FSH, forskolin, and cholera toxin. In contrast, bacterial extracts BP 347 and BP 348 from mutant strains of B. pertussis that lack either all virulent factors or the adenylate cyclase toxin and hemolysin were devoid of effect. Granulosa-cell cAMP accumulation supported by active bacterial adenylate cyclase was accompanied by 2- to 11-fold, time-dependent increases in [35S]methionine incorporation into immunospecific cytochrome P-450scc and adrenodoxin. These increases in the synthesis of cholesterol side-chain cleavage proteins were associated with enhanced pregnenolone production in response to exogenous sterol substrate, 25-hydroxycholesterol, and augmented progesterone secretion both in the absence and presence of exogenous lipoprotein. Moreover, the effects of Bordetella adenylate cyclase toxin on granulosa cell steroidogenesis were functionally integrated with other regulatory responses, since the non-cAMP dependent effector, estradiol 17-beta, interacted synergistically with bacterial adenylate cyclase in stimulating progesterone production. We conclude that exogenous adenylate cyclase isolated from B. pertussis can be functionally integrated into the cAMP-dependent effector pathway of granulosa cells with a resulting increase in intracellular cAMP concentrations, augmented biosynthesis of progesterone and pregnenolone, enhanced synthesis of immunospecific cytochrome P-450scc and adrenodoxin, and synergistic interactions with a non-cAMP-dependent ovarian effector hormone (estradiol).(ABSTRACT TRUNCATED AT 250 WORDS)