Bacterial pathogens undergo profound physiological changes when they infect their host and require coordinated 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.

Regulation of the genes coding for virulence factors in Bordetela pertussis is controlled by the bvg locus, which encodes one putative sensory protein (BvgS) and one positive regulator of transcription (BvgA). We have studied the transcription of the bvg locus and found that this is controlled by a 350-base-pair DNA fragment, which contains five promoters, three of which transcribe the bvg locus, one transcribes an antisense RNA, and one transcribes a virulence-associated gene. Under noninducing conditions, only the promoter P2 is active and this is responsible for the production of low amounts of regulatory proteins. Upon induction, the other four promoters become active and, by a mechanism that may involve transcriptional and translational regulation, cause a 50-fold increase of the transcriptional activator BvgA. A model of the autoregulation of the bvg locus is presented.

The nucleotide sequence of the structural gene for filamentous haemagglutinin (FHA), fhaB, a crucial adherence factor for Bordetella pertussis, has been determined. Its 10774 nucleotides are far more than necessary to encode the 220 kD biologically active, mature polypeptide product, suggesting a role for co- or post-translational processing. Fusion proteins derived from various portions of the fhaB open reading frame (ORF) were used to generate polyclonal antisera. Western immunoblot analysis of purified FHA and Bordetella sp. whole cell extracts with these antisera indicated that the 220 kD product is encoded by the 5' portion of the ORF and that the smaller polypeptide species are breakdown products of this polypeptide. These data, as well as N-terminal amino acid sequencing of the major polypeptide species, suggest a scheme for the proteolytic processing of an FHA precursor polypeptide.

The toxicity of pertussis toxin is mediated by the ADP-ribosyltransferase activity of subunit S1. ADP-ribosylation of the target substrates in eukaryotic cells is a common mechanism of action of many bacterial protein toxins (1-3). The best-studied molecules that adopt this mechanism are diphtheria toxin (4-7), Pseudomonas exotoxin A (8-10), cholera toxin (11-13), and pertussis toxin (14-16), but several other toxins possessing ADP-ribosyltransferase activity have also been described (17,18). The toxins usually contain two functional moieties: A, which is enzymatically active, and B-, which recognizes and binds the receptors on the cell surface facilitating the entry of the enzymatically active subunit into the target cells.The proteins that are ADP-ribosylated by diphtheria, (20)(21)(22).For diphtheria and Pseudomonas toxins, a series of studies, which include nitrosoguanidine mutagenesis of the genes (23), characterization of the mutant genes (5) and their products (24-26), photoaffinity labeling of the toxins with NAD (27, 28), site-directed mutagenesis (29, 30), and crystallographic structure of the Pseudomonas exotoxin A (31) have led to the identification of amino acids essential to the enzymatic activity of the proteins. For example, Glu-148 of diphtheria toxin and Glu-553 of Pseudomonas -exotoxin A, located within the catalytic site of the two enzymes, cannot be replaced even with an aspartic acid without abolishing enzymatic activity (29,30).We used a similar approach to study the structure-function relationship of the S1 subunit ofpertussis toxin. By carboxyland amino-terminal deletion analysis and site-directed mutagenesis, we identified at least three regions of the S1 subunit that are essential for enzyme function. Substitutions within these regions produce enzymatically inactive molecules. MATERIALS AND METHODSConstruction of the Si Deletion Mutants. The S1 subunit of pertussis toxin was expressed in Escherichia coli fused to the 98 amino-terminal amino acids of the MS2 polymerase. This fusion protein (PTE255) contains amino acids 2-235 of the S1 subunit and is enzymatically active (32). The gene coding for the amino acids 2-235 is contained within a BamHI-Xba I fragment flanked by an EcoRI site at the 5' end and a HindIII site at the 3' end. To obtain the plasmids expressing carboxylterminal deletion mutants of the S1 protein, the plasmid pTE255 was digested first with Xba I and then with Nco I, Nru I, Bal I, Sal I, and Sph I, respectively.' The sticky ends generated by the restriction enzymes were then repaired by the large fragment of DNA polymerase, and the plasmids were circularized by DNA ligase. During this process, the natural stop codon of the S1 subunit was lost, and therefore the new proteins contained a few amino acids fused at the carboxyl terminus: NCO, NRU, and SPH proteins had the following carboxyl-terminal unrelated amino acids: Leu-ProArg-Ala-Phe-Arg. BAL and SAL proteins contained, respectively, 50 and 16 amino acids deriving from the sequence of pBR322 (33) fused at th...

Bordetella pertussis produces a number of virulence factors whose expression is coordinately regulated by the bvgA3 locus. Transcription of virulence genes is repressed by environmental factors such as low temperature (25 "C) and chemical stimuli. Temperature shift of bacterial cultures from 25 "C to 37 "C activates two classes of bvg-regulated virulence genes: the early genes, which are activated within 10 min, and late genes, which require 2 4 h for activation. During the interval between the activation of the early and late genes, the intracellular concentration of BvgA increases 50-fold. It has been proposed that this increased concentration may be required for the activation of the late genes. Here we have analysed the response of the bvg locus to intermediate temperatures and to repeated temperature shifts. Temperature shifts of B.pertussis cultures from 22 "C to 28 "C, 32 "C or 35 "C resulted in the synthesis of low, intermediate, and high amounts of BvgA. This implied that the intracellular concentration of BvgA is temperature-dependent. We have also observed that the amount of virulence factors produced correlates with the BvgA concentration. When bacteria grown at 37 "C were shifted to 22 "C, transcription from the adenylate cyclase toxin haemolysis promoter (PAC) was repressed after 30 min, while transcription from the bvg (P,) and filamentous haemagglutinin (PFHA) promoters was repressed after 2 h. During this time, the amount of BvgA did not decrease. A subsequent temperature shift from 22 "C to 37 "C induced transcription from the P, and P, , promoters after 10 min and transcription from the PAC promoter after 20 min. This result shows that in the presence of a high concentration of BvgA, the time lag between temperature shift and late promoter transcription is reduced from 2 4 h to 20 min. The above data support the proposal that the concentration of BvgA plays a role in activating expression of the late genes.

Introduction: Barriers to vaccination and the important role of healthcare professionals (HCPs) in influencing immunization decisions made by parents/patients have been well documented. Little information describes challenges that HCPs face in carrying out their role as vaccinators.Methods: We conducted a focus group study asking HCPs to describe their expectations as frontline vaccinators versus the day-to-day reality they faced. Participants described challenges impacting their ability and motivation as vaccinators, and proposed key solutions to the most important challenges. A total of 75 nurses and physicians (sixteen groups of frontline vaccinators) from the United Kingdom, United States, Germany and India participated in 2 hour focus-group discussions.Results: There was disconnect between how participants viewed their role in preserving population health when they started their career, and the reality of real-world practice today. Challenges experienced and reflected were similar across professional groups and countries. Low patient-level vaccine knowledge, patient miseducation, untimely vaccine information, frequently changing vaccine schedules, time pressures, lack of centralized record systems, pressure to achieve vaccination targets, and in some instances vaccine costs, all impacted the efficiency and enthusiasm of HCPs. Identified solutions by the same providers included improving patient-level information, equipping HCPs with effective information, and practical ways to reduce the vaccination burden by improved administrative processes and centralized recording coupled with delegating vaccinator roles.Conclusion: This focus group gives unique insights into needs of HCPs to fulfill their role as vaccinators. Supporting and equipping vaccinators is critical to the continuing success of vaccination programs and the proposed life-course immunization strategy. (Supplementary Appendix 1)

We report the identification of a protein homologous to a histone H1 in Bordetella pertussis. The B. pertussis histone homologue, BpH1, varies in size in different strains from 182 to 206 amino acids. The variability of the size of the protein is due to gene variability by insertion or deletion of DNA modules. Insertion of a kanamycin cassette into the bpH1 gene generates a BpH1 null mutant with phenotypic properties and growth rate similar to those of the wild-type strain, showing that this gene is dispensable. In vitro, the BpH1 protein prevents chromosomal DNA degradation from DNase I and constrains supercoiled DNA. Transcription of the bpH1 gene is activated during exponential growth of the bacteria, whereas it is repressed during the stationary phase of growth. It is proposed that BpH1 plays a role in chromatin formation and condensation during DNA replication and that repression of transcription depends upon a reduced rate of DNA replication.

The expression of the virulence-associated genes in Bordetella species is co-ordinately regulated by the gene products encoded by the bvg locus. In Bordetella pertussis the expression of this locus is regulated by the P1, P2, P3 and P4 promoters which are located in a 350 bp DNA fragment also containing the PFHA promoter. Here we report the transcriptional regulation of the bvg locus and the fha gene in Bordetella parapertussis and a sequence analysis of the bvg-regulated promoters. The Pp1, Pp2, Pp4 and PpFHA promoters are indistinguishable, both in transcription initiation sites and environmental regulation, from the corresponding promoters of B. pertussis, while the Pp3 promoter is not active. Sequence homologies from nine bvg-regulated promoters show a conserved dinucleotide, 5'-TG-3', at approximately one turn of helix upstream of the -10 5'-A.AaTat-3' region, and a 5'-TTTCC-3' sequence in the -90 region. Since the nucleotide sequence of the inactive Pp3 promoter shows several base substitutions with respect to the found sequence homologies, it is likely that some of these bases play an essential role in promoter activity.