Bordetella: Molecular Microbiology Chapter Abstracts

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The Bordetella genome project generated and analysed the genome sequences of representative strains of B. bronchiseptica, B. pertussis and B. parapertussis. This was the first project to sequence the genomes of several such closely related bacteria at the same time, and with the intention of a comparative analysis. Information from this project is fueling future research into the biology of the Bordetellae and allowing the generation of hypotheses addressing the differing pathogenicities and host ranges of these bacteria, and their evolutionary relationships. The genome sequence of B. avium is complete, and that of B. petrii is continuing, and these data will expand and enhance these analyses.

Chapter 2
Phylogeny, Evolution and Epidemiology of Bordetellae
Frits R. Mooi, Qiushui He and Nicole Guiso

Before the introduction of vaccination, pertussis caused more infant deaths than diphtheria, scarlet fever, and measles combined. Morbidity and mortality due to pertussis were significantly reduced by the introduction of whole cell vaccines in the 1940's. Despite the availability of pertussis vaccines, pertussis remains one of the 10 infectious diseases with the highest morbidity and mortality world-wide. Further, even in countries with a long history of high vaccine uptake, pertussis has remained endemic and in some countries a dramatic resurgence has occurred. One of the hallmarks of the pertussis resurgence is a shift in the incidence towards older age categories, a phenomenon attributed to waning vaccine-induced immunity. Annual infection frequencies in adolescents and adults have been estimated at 1-8%. The persistence of B. pertussis, despite vaccination, is particularly intriguing in view of its extreme low genetic diversity, implicating a limited ability to adapt. However, highly directional changes have been observed in B. pertussis populations, and in many countries antigenic divergence between vaccine strains and clinical isolates has occurred. These changes may have been immune-driven although their effect on the persistence and resurgence of pertussis remains contentious. Recently vaccinated children are well-protected against clinical disease implying a limited effect of strain variation. However, vaccine-induced immunity lasts only for 5 to 10 years, and the relatively small mismatches observed between circulating strains and vaccine strains may be important in hosts with low, waning, immunity. The shift towards older age categories shown in vaccinated population may be the compound effect of waning immunity and pathogen adaptation.

Chapter 3
The Bvg Regulon
Scott Stibitz

The bvgASR locus of Bordetella species controls the expression of a large number of genes in both a positive and a negative manner. This includes those encoding all known protein virulence factors. Control by bvgASR is sensitive to environmental signals via a phenomenon, known historically as antigenic modulation, and currently understood to be in keeping with its membership in the family of two-component regulatory systems. The activity of the regulator BvgA is dependent on phosphorylation by BvgS which is, in turn, responsive to environmental signals. Itself a Bvg-activated gene, bvgR is required for the repression of the reciprocally regulated Bvg-repressed genes. Although virulence gene activation appears to involve no regulatory inputs other than BvgA~P, the promotor architecture of regulated genes dictates differential responsiveness such that genes are activated at either low or high levels of BvgA~P. At genes belonging to the class of intermediate phase genes, BvgA~P acts as an activator at lower concentrations and as a repressor at higher concentrations. These characteristics apparently allow different virulence genes to be expressed in different spatial or temporal patterns during infection.

Chapter 4
The Bordetella Adhesins
Françoise Jacob-Dubuisson and Camille Locht

The virulence of Bordetella relies strongly on the ability of this micro-organism to adhere to various target cells within the infected host. Several different cell types may serve as targets for this pathogen, and both biochemical and more recent, molecular biological studies have revealed that the pathogenic Bordetella species produce an amazing array of different protein adhesins. The structural genes of most of them, as well as the accessory genes involved in their biogenesis, are under the stringent control of the BvgA/S regulatory system. The involvement of the individual adhesins in the different steps of pathogenesis has been evaluated in various in vitro and animal infection models. Several of the adhesins, such as filamentous hemagglutinin, pertactin and the fimbriae, are also potent immunogens and are included in the acellular pertussis vaccines. Antigenic drifts have been observed for some of them over the last decades, which may be due to immune pressure and may serve to immune-evasion of the pathogen. In addition to their adherence capacities, some of the adhesins also express immuno-modulatory activities, able to subvert the immune system to the benefit of the bacterium. This also may represent a mechanism of immune-evasion evolved in pathogenic Bordetella species.

Chapter 5
Bordetella Toxins
Erik L. Hewlett and Gina M. Donato

Bacteria of the species Bordetella express a number of virulence factors that appear to aid in the establishment of infection and production of clinical disease. Based on the actions of pertussis toxin, pertussis was declared a "toxin-mediated" disease in the past. It is now clear, however, that pertussis toxin alone is not responsible for the manifestations of this illness. Pathogenesis is much more complicated, involving multiple toxins, adhesins and other bacterial products. The molecular actions of most of the known toxins have been elucidated and a number of their structures have been solved. The primary deficiency in the body of knowledge about Bordetella toxins is in the role that each plays in pathogenesis and how they work in a concerted manner to cause different diseases in multiple hosts.

Chapter 6
Type III Secretion in Bordetella Subspecies
Natalia A. Kozak, Ekaterina M. Panina and Jeff F. Miller

Type III secretion (TTS) is used by Gram negative bacteria to deliver effector proteins directly into the cytosol of eukaryotic cells, where they alter a wide range of host functions. The hallmark of the B. bronchiseptica TTSS is the efficient induction of death in a remarkably broad range of cells in vitro. In vivo, the B. bronchiseptica TTSS appears to promote persistent colonization, presumably by modulating host immunity. A nearly identical type III secretion system (TTSS) gene cluster is found in the genomes of B. bronchiseptica, B. pertussis and B. parapertussis. Although human adapted Bordetella strains do not readily express TTSS-associated phenotypes in vitro, TTSS genes are intact, transcribed and regulated. Differential regulation of TTS may have played an important role in the evolution of host specificity in Bordetella subspecies that cause respiratory diseases in mammals.

Chapter 7
The Ptl Type IV Secretion System
Amy A. Rambow-Larsen and Alison A. Weiss

Pertussis toxin is secreted by the Ptl Type IV secretion system. Type IV secretion systems were originally associated with conjugation, or the transfer of DNA from the cytoplasm of one cell to another. In contrast, the Ptl Type IV secretion system transfers the multimeric protein, pertussis toxin, from the bacterial periplasm past the outer membrane, releasing the toxin from the cell. The type IV secretion systems differ remarkably in the substrates, origins, and final destinations of their secreted products, however the secretion systems have retained tremendous simarities, and comparitive studies have allowed for rapid advancement in our understanding of these important bacterial processes.

Chapter 8
Primary Metabolism and Physiology of Bordetella Species
Sandra K. Armstrong and Roy Gross

Of the members of the genus Bordetella, the classical pathogens Bordetella bronchiseptica, Bordetella pertussis and Bordetella parapertussis, have been studied for many years. The physiology of B. pertussis has been most extensively analyzed, since this Bordetella species is challenging to grow in vitro and efficient growth of virulent-phase organisms is essential for pertussis vaccine production. The recent availability of the genome sequences of these species has allowed for in silico predictions adding to established knowledge of their metabolic pathways and physiological traits. In this chapter, genomic information is correlated with experimental observations from the literature to further our understanding of the physiology of the pathogenic Bordetellae and a nonpathogenic environmental species, Bordetella petrii. Results of these analyses support the notion of the existence a free-living B. bronchiseptica-like progenitor with a versatile nonfermentative metabolism centered on the oxidation of amino acids and citric acid cycle intermediates. From this ancestor, the pathogenic Bordetellae may have differentially evolved to become specialists that inhabit their present host environments.

Chapter 9
Bordetella Bacteriophage and Diversity-Generating Retroelements
Asher Hodes, Roman Barbalat, Louise Temple and Jeff F. Miller

Numerous Bordetella bacteriophages have been discovered and a few have been characterized. In the course of studies on mechanisms of pathogenesis, we identified a group of temperate bacteriophages that generate diversity in a gene that specifies tropism for receptor molecules on the Bordetella cell surface. This microevolutionary adaptation is produced by a novel genetic element that combines the basic retroelement life cycle of transcription, reverse transcription and integration with site-directed, nucleotide-specific mutagenesis. Central to this process is a reverse transcriptase-mediated exchange between two repeats, one serving as an donor template (TR) and the other as a recipient of variable sequence information (VR). Recent work has focused on the genetic basis of diversity-generation. The directionality of information transfer is determined by the initiation of mutagenic homing (IMH) sequence present at the 3' end of VR. The diversity-generating system targets variability to a subset of bases encoding the ligand-banding surface of a C-type lectin fold. The CLec protein (termed "major tropism determinant" or Mtd) forms highly stable trimers which serve as a rigid scaffolds on which diversified receptor-binding sequences are displayed. This rigidity contrasts with the highly flexible immunoglobulin fold which serves as a diversity-scaffold for the human immune system. Using the Bordetella phage cassette as a signature, we have identified an array of related elements in multiple bacterial genomes, including Vibrio, Bacteroides, Treponema, Bifidobacterium, Trichodesmium and Nostoc species. These comprise a new family of retroelements, designated "Diversity-Generating Retroelements", with the potential to confer powerful selective advantages to their host genomes.

Chapter 10
Biotechnological Applications of the Bordetella pertussis Adenylate cyclase toxin
Marcela Simsova, Gouzel Karimova, Peter Sebo and Daniel Ladant

The remarkable features of the Adenylate cyclase toxin (ACT) of Bordetella pertussis have prompted a number of very exciting biotechnolological applications. The ability of this protein to bind to antigen-presenting cells and to penetrate these cells has been used to develop the molecule as a very efficient vehicle for the presentation of epitopes and antigens to CD4+ and to CD8+ T lymphocytes. This can be used for diagnostics and vaccine development, and strong protective T cell responses have been induced against tumors and viral infections. The activation of ACT by intracellular calmodulin has been exploited as a reporter for intracellular delivery of proteins or peptides fused to the active domain of the toxin. Intramolecular complementation that occurs between two adenylate cyclase fragments when they are physically joined has been utilised to develop powerful screening methods for protein-protein interactions and for protease or protease inhibitor activities. However, in addition to ACT, the properties of other B. pertussis virulence factors have also a potential to be used for biotechnological applications. The immunomodulatory activities of pertussis toxin and the antigen-presenting properties of filamentous haemagglutinin, as well as the efficient secretion machinery of filamentous haemagglutinin to be used for the secretion of heterologous proteins are just some examples.

Chapter 11
Pertussis Vaccines
Jann Storsaeter, Joanne Wolter and Camille Locht

Since their inception during the mid 20th century, pertussis vaccines have prevented millions of deaths in children and have profoundly influenced the epidemiology of pertussis disease in countries where their use has been widespread. Despite their proven effectiveness and safety, public rejection of whole-cell pertussis vaccines stimulated the development of second generation, acellular pertussis vaccines. Molecular biology has contributed in many ways to the development of acellular vaccines, in particular by detailed characterization of the protective antigens and by generating defined, genetically detoxified vaccines. However, despite more acceptable reactogenicity profiles of the new vaccines, many problems, such as limited duration of protection, their inability to prevent sub-clinical disease and transmission to unprotected infants, and rising reactogenicity with booster doses, remain common to pertussis vaccines as a whole. Current priorities to continue global progress in pertussis control are finding the means to prevent severe and potentially fatal pertussis in infants too young to receive currently available vaccines, increasing vaccine coverage amongst adolescents, and increasing vaccine coverage rates in developing countries where 90% of pertussis cases occur annually.

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