Role of the Type III Secretion System in a Hypervirulent Lineage of Bordetella bronchiseptica

Despite the fact that closely related bacteria can cause different levels of disease, the genetic changes that cause some isolates to be more pathogenic than others are generally not well understood. We use a combination of approaches to determine which factors contribute to the increased virulence of a Bordetella bronchiseptica lineage. A strain isolated from a host with B. bronchiseptica-induced disease, strain 1289, was 60-fold more virulent in mice than one isolated from an asymptomatically infected host, strain RB50. Transcriptome analysis and quantitative reverse transcription-PCR showed that the type III secretion system (TTSS) genes were more highly expressed by strain 1289 than strain RB50. Compared to strain RB50, strain 1289 exhibited greater TTSS-mediated cytotoxicity of a mammalian cell line. Additionally, we show that the increase in virulence of strain 1289 compared to that of RB50 was partially attributable to the TTSS. Using multilocus sequence typing, we identified another strain from the same lineage as strain 1289. Similar to strain 1289, we implicate the TTSS in the increased virulence of this strain. Together, our data suggest that the TTSS is involved in the increased virulence of a B. bronchiseptica lineage which appears to be disproportionately associated with disease. These data are consistent with the view that B. bronchiseptica lineages can have different levels of virulence, which may contribute to this species' ability to cause different severities of respiratory disease.

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title: Emergence of a bacterial clone with enhanced virulence by acquisition of a phage encoding a secreted phospholipase A2
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title: Molecular Pathogenesis, Epidemiology, and Clinical Manifestations of Respiratory Infections Due to Bordetella pertussis and Other Bordetella Subspecies
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title: Bordetella pertussis, the Causative Agent of Whooping Cough, Evolved from a Distinct, Human-Associated Lineage of B. bronchiseptica
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title: Genetic diversity and relationships in populations of Bordetella spp
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title: Molecular evolution and host adaptation of Bordetella spp.: phylogenetic analysis using multilocus enzyme electrophoresis and typing with three insertion sequences
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title: bvg Repression of alcaligin synthesis in Bordetella bronchiseptica is associated with phylogenetic lineage
abstract: Recent studies have shown that Bordetella bronchiseptica utilizes a siderophore-mediated transport system for acquisition of iron from the host iron-binding proteins lactoferrin and transferrin. We recently identified the B. bronchiseptica siderophore as alcaligin, which is also produced by B. pertussis. Alcaligin production by B. bronchiseptica is repressed by exogenous iron, a phenotype of other microbes that produce siderophores. In this study, we report that alcaligin production by B. bronchiseptica RB50 and GP1SN was repressed by the Bordetella global virulence regulator, bvg, in addition to being Fe repressed. Modulation of bvg locus expression with 50 mM MgSO4 or inactivation of bvg by deletion allowed strain RB50 to produce alcaligin. In modulated organisms, siderophore production remained Fe repressed. These observations contrasted with our previous data indicating that alcaligin production by B. bronchiseptica MBORD846 and B. pertussis was repressed by Fe but bvg independent. Despite bvg repression of alcaligin production, strain RB50 was still able to acquire Fe from purified alcaligin, suggesting that expression of the bacterial alcaligin receptor was not repressed by bvg. We tested 114 B. bronchiseptica strains and found that bvg repression of alcaligin production was strongly associated with Bordetella phylogenetic lineage and with host species from which the organisms were isolated.

title: Bacteriological variation among Bordetella bronchiseptica isolates from dogs and other species
abstract: Bacteriological properties of 50 isolates of Bordetella bronchiseptica were compared. Phase variation, which involved colonial morphology and its associated characters of hemagglutination, hemolysis, acriflavine agglutination; crystal violet staining, flagellation, and fimbriation, occurred among these isolates. Organisms representing the three observed morphotypes did not have different growth rates, nor were any differences in their bacteriological characteristics observed after repeated subculture on agar. There were also variations in antimicrobial drug susceptibility, especially to sulfonamide-trimethoprim, and in nitrate reduction. The relationships among these variable parameters were not apparent. None of the observed variations could be attributed to differences in the species of origin.

title: Antigenic polymorphism of the lipopolysaccharides from human and animal isolates of Bordetella bronchiseptica.
abstract: Six monoclonal antibodies (mAbs) against lipopolysaccharides (LPS) from Bordetella pertussis (P1P3, 60.5), B. parapertussis (PP2, PP6, PPB) and B. bronchiseptica (BRg1) were used to examine the presence of antigenic determinants of LPS on B. bronchiseptica cells. Forty-eight clinical isolates of this Gram-negative bacterium (4 canine, 3 equine, 6 porcine, 4 rabbit and 31 human) were examined. Significant cross-reactivities with the heterologous anti-pertussis and anti-parapertussis mAbs were observed. The isolates also exhibited marked antigenic polymorphism. The 48 isolates could be classified in six immunogroups. Purified LPS preparations extracted from some isolates were analysed by ELISA, thin-layer chromatography, and tricine-SDS-PAGE. The results show that four main types of antigenic polymorphism of B. bronchiseptica LPSs exist: (a) heterogeneity of the core, (b) presence or absence of O-chains, (c) differences in the hinge region between O-chain and core, and (d) differences in interactions of LPS with other cell-surface constituents. Smooth-type LPS molecules, detectable with mAb PP6, were more frequently observed in animal isolates (94%) than in human isolates (52%). Reverse frequencies were found with mAb 60.5 (48% of human isolates, 18% of animal isolates), which is unable to react with long-chain LPSs. This observation could be due to the general absence of some lectin-like receptor, specific to the O-chain, on human bronchoalveolar tissues.

title: Virulence factors of Bordetella bronchiseptica associated with the production of infectious atrophic rhinitis and pneumonia in experimentally infected neonatal swine
abstract: Four strains of Bordetella bronchiseptica (CSU-P-1, 64-C-0406, 1120-A-83-013, and B205BT) with defined virulence for neonatal swine were examined, and an attempt was made to correlate the presence of certain in vitro phenotypic characteristics with the ability of a particular B. bronchiseptica strain to produce turbinate and lung lesions in piglets. All of the strains except CSU-P-1 colonized the nasal passages of the pigs heavily, and strains 1120-A-83-013 and B205BT produced moderate to severe nasal and lung lesions in experimentally infected piglets. All of the strains attached equally well to porcine tracheal ring explant cultures, and all of the strains except CSU-P-1 produced smooth, hemolytic colonies on Bordet-Gengou medium, agglutinated porcine erythrocytes, and possessed adenylate cyclase activity. Strains 1120-A-83-013 and B205BT produced considerably higher levels of dermonecrotic toxin activity than did strains CSU-P-1 and 64-C-0406. These results indicate that production of nasal and lung lesions in neonatal piglets by the B. bronchiseptica strains tested can be directly correlated with their level of dermonecrotic toxin production.

title: Phosphorelay control of virulence gene expression in Bordetella.
abstract: In Bordetella, the BvgAS phosphorelay comprising the polydomain sensor, BvgS, and response regulator, BvgA, controls the expression of at least three distinct phenotypic phases in response to differences in the concentration of a few chemical compounds. Each phenotypic phase is characterized by maximal expression of some genes and minimal expression of others, and current data support the hypothesis that each plays a distinct and important role in the Bordetella infectious cycle. The ability to control expression of multiple phenotypic states in response to subtle differences in signal intensity could be a characterizing feature of phosphorelays that contain polydomain sensors.

title: The vir locus and phase-variation in Bordetella pertussis.
abstract: By a phenomenon known as phase-variation Bordetella pertussis is capable of changing between a virulent-phase in which multiple virulence-associated determinants are expressed, and an avirulent-phase in which the virulence-associated determinants are not expressed. Mutations in the vir locus of B. pertussis have a similar effect. We have examined the state of the vir locus in each of a series of strains derived one from the other by phase-variation. We have found that a single base-pair change is associated with the change between the virulent and avirulent phases. This single base-pair change corresponds to a frameshift mutation in the vir locus.

title: Role of the Dermonecrotic Toxin of Bordetella bronchiseptica in the Pathogenesis of Respiratory Disease in Swine
abstract: Bordetella bronchiseptica is one of the etiologic agents causing atrophic rhinitis and pneumonia in swine. It produces several purported virulence factors, including the dermonecrotic toxin (DNT), which has been implicated in the turbinate atrophy seen in cases of atrophic rhinitis. The purpose of these experiments was to clarify the role of this toxin in respiratory disease by comparing the pathogenicity in swine of two isogenic dnt mutants to their virulent DNT+ parent strains. Two separate experiments were performed, one with each of the mutant-parent pairs. One-week-old cesarean-derived, colostrum-deprived pigs were inoculated intranasally with the parent strain, the dnt mutant strain, or phosphate-buffered saline. Weekly nasal washes were performed to monitor colonization of the nasal cavity, and the pigs were euthanized 4 weeks after inoculation to determine colonization of tissues and to examine the respiratory tract for pathology. There was evidence that colonization of the upper respiratory tract, but not the lower respiratory tract, was slightly greater for the parent strains than for the dnt mutants. Moderate turbinate atrophy and bronchopneumonia were found in most pigs given the parent strains, while there was no turbinate atrophy or pneumonia in pigs challenged with the dnt mutant strains. Therefore, production of DNT by B. bronchiseptica is necessary to produce the lesions of turbinate atrophy and bronchopneumonia in pigs infected with this organism.

title: Species- and Strain-Specific Control of a Complex, Flexible Regulon by Bordetella BvgAS
abstract: The Bordetella master virulence regulatory system, BvgAS, controls a spectrum of gene expression states, including the virulent Bvg+ phase, the avirulent Bvg− phase, and at least one Bvg-intermediate (Bvgi) phase. We set out to define the species- and strain-specific features of this regulon based on global gene expression profiling. Rather than functioning as a switch, Bvg controls a remarkable continuum of gene expression states, with hundreds of genes maximally expressed in intermediate phases between the Bvg+ and Bvg− poles. Comparative analysis of Bvg regulation in B. pertussis and B. bronchiseptica revealed a relatively conserved Bvg+ phase transcriptional program and identified previously uncharacterized candidate virulence factors. In contrast, control of Bvg−- and Bvgi-phase genes diverged substantially between species; regulation of metabolic, transporter, and motility loci indicated an increased capacity in B. bronchiseptica, compared to B. pertussis, for ex vivo adaptation. Strain comparisons also demonstrated variation in gene expression patterns within species. Among the genes with the greatest variability in patterns of expression, predicted promoter sequences were nearly identical. Our data suggest that the complement of transcriptional regulators is largely responsible for transcriptional diversity. In support of this hypothesis, many putative transcriptional regulators that were Bvg regulated in B. bronchiseptica were deleted, inactivated, or unregulated by BvgAS in B. pertussis. We propose the concept of a “flexible regulon.” This flexible regulon may prove to be important for pathogen evolution and the diversification of host range specificity.

title: Environmental regulation of expression of virulence determinants in Bordetella pertussis
abstract: The trans-activator vir is required for expression of all virulence-associated genes in Bordetella pertussis. The nature of the global regulation of these factors by vir and environmental signals was examined by Northern blot analysis and with beta-galactosidase transcriptional fusions in five vir-regulated genes. Northern blots suggested that vir regulates at the level of transcription since Vir- organisms did not exhibit detectable mRNA from vir-regulated loci. Environmental signals such as high levels of salts, nicotinic acid, and 6-chloronicotinic acid or growth at low temperatures were examined. Of all of the cations and anions examined, only SO4 ions eliminated transcription of vir-regulated genes and reduced transcription of vir itself, suggesting that global regulation is obtained by modifying expression of the essential component, vir. Organisms grown on 6-chloronicotinic acid or quinaldic acid did not have detectable transcription from vir-regulated loci. Modulation by nicotinic acid, on the other hand, was strain dependent, acting at the level of transcription in strain 18-323 but not in Tohama I derivatives. Growth at lower temperatures reduced, but did not eliminate, transcription from vir-regulated loci. At 28 degrees C the ratio of pertussis toxin mRNA to recA mRNA (a non-vir-regulated factor) was equivalent to that at 37 degrees C, suggesting that transcription at low temperatures is reduced in a proportional manner and need not involve vir.

title: Construction and validation of a first-generation Bordetella bronchiseptica long-oligonucleotide microarray by transcriptional profiling the Bvg regulon
abstract: BackgroundBordetella bronchiseptica is a bacterial respiratory pathogen that infects a broad range of mammals, causing chronic and often subclinical infections. Gene expression in Bordetella is regulated by a two-component sensory transduction system, BvgAS, which controls the expression of a spectrum of phenotypic phases transitioning between a virulent (Bvg+) phase and a non-virulent (Bvg-) phase.ResultsBased on the genomic sequence and using the freely available software ArrayOligoSelector, a long oligonucleotide B. bronchiseptica microarray was designed and assembled. This long-oligonucleotide microarray was subsequently tested and validated by comparing changes in the global expression profiles between B. bronchiseptica RB50 and its Bvg- phase-locked derivative, RB54. Data from this microarray analysis revealed 1,668 Bvg-regulated genes, which greatly expands the BvgAS regulon defined in previous reports. For previously reported Bvg-regulated transcripts, the gene expression data presented here is congruent with prior findings. Additionally, quantitative real-time PCR data provided an independent verification of the microarray expression values.ConclusionThe results presented here provide a comprehensive, genome-wide portrait of transcripts encompassing the BvgAS regulon, while also providing data validating the long-oligonucleotide microarray described here for studying gene expression in Bordetella bronchiseptica.

title: Ectopic expression of the flagellar regulon alters development of the bordetella-host interaction
abstract: Signal transduction molecules within the two-component family represent a conserved adaptation for the control of genes involved in pathogenesis. The Bordetella virulence control locus, bvgAS, activates and represses gene expression in response to environmental signals. While infection requires virulence gene activation, the role of gene repression during infection is not understood. By altering regulatory genes and reversing regulatory connections, we found evidence that the BvgAS-repressed genes responsible for motility are neither required nor expressed during colonization of the host. Expression of this Bvg- phase-specific phenotype in the Bvg+ growth phase resulted in a defect in tracheal colonization. Therefore, BvgAS promotes virulence both by activating genes required for colonization and by repressing genes that inhibit the development of infection.

title: BvgAS-mediated signal transduction: analysis of phase-locked regulatory mutants of Bordetella bronchiseptica in a rabbit model
abstract: Members of the Bordetella genus alternate between two distinct phenotypic phases in response to changes in their environment. This switch, termed phenotypic modulation, is mediated by the BvgAS sensory transduction system. We developed an animal model based on the interaction of Bordetella bronchiseptica with one of its natural hosts, the rabbit. To investigate the importance of BvgAS signal transduction, we constructed constitutive (RB53) and Bvg- (RB54) phase-locked derivatives of a wild-type strain, RB50. RB50 and RB53, but not RB54, established respiratory infections in B. bronchiseptica-free rabbits with an intranasal 50% infective dose of less than 200 organisms, and the course of the infection closely resembled that observed with naturally infected rabbits. Bacteria were recovered from the nasal cavity, larynx, trachea, and lungs in similar numbers from RB50- and RB53-infected rabbits, yet no pathology was detected by histological examination of lung and tracheal sections. The antibody responses in rabbits inoculated with RB50 or RB53 were quantitatively and qualitatively indistinguishable; high titers of antibodies were generated primarily against Bvg(+)-phase-specific antigens. No response against flagella, a Bvg- phase factor, was detected. Assessment of bacteria associated with alveolar macrophages indicated that only a small percentage of bacteria, if any, appear to be residing within lung macrophages. We also tested the ability of these strains to survive in a nutrient poor environment, conditions which may be encountered within certain niches in the host or in an environmental reservoir. The Bvg- phase was advantageous for growth under these conditions. Our results indicate the Bvg+ phase is sufficient for establishment of respiratory tract infection in the rabbit and the normal BvgAS-mediated response to environmental signals is not required during initial colonization. The Bvg- phase may play a role at later stages of infection, including persistence, transmission, or survival in the environment.

title: Molecular characterization and assembly of the needle complex of the Salmonella typhimurium type III protein secretion system.
abstract: Many bacterial pathogens of plants and animals have evolved a specialized protein-secretion system termed type III to deliver bacterial proteins into host cells. These proteins stimulate or interfere with host cellular functions for the pathogen's benefit. The Salmonella typhimurium pathogenicity island 1 encodes one of these systems that mediates this bacterium's ability to enter nonphagocytic cells. Several components of this type III secretion system are organized in a supramolecular structure termed the needle complex. This structure is made of discrete substructures including a base that spans both membranes and a needle-like projection that extends outward from the bacterial surface. We demonstrate here that the type III secretion export apparatus is required for the assembly of the needle substructure but is dispensable for the assembly of the base. We show that the length of the needle segment is determined by the type III secretion associated protein InvJ. We report that InvG, PrgH, and PrgK constitute the base and that PrgI is the main component of the needle of the type III secretion complex. PrgI homologs are present in type III secretion systems from bacteria pathogenic for animals but are absent from bacteria pathogenic for plants. We hypothesize that the needle component may establish the specificity of type III secretion systems in delivering proteins into either plant or animal cells.

title: The BvgAS virulence control system regulates type III secretion in Bordetella bronchiseptica
abstract: The BvgAS signal transduction system in Bordetella spp. mediates a transition between infectious (Bvg+) and non‐infectious (Bvg−) phases by sensing environmental conditions and regulating gene expression. Using differential display, arbitrary‐primed polymerase chain reaction (PCR), we identified a gene expressed in the Bvg+ phase of Bordetella bronchiseptica that shows a high degree of sequence similarity to a locus involved in providing energy for type III secretion in pathogenic Gram‐negative bacteria (yscN in Yersinia spp.). We determined that the expression of this homologue in B. bronchiseptica (designated bscN ) is regulated by bvg. Several open reading frames surrounding the bscN locus also show sequence similarity to loci encoding type III secretion apparatus components in other bacteria. An  deletion of bscN in B. bronchiseptica leads to decreased secretion of several proteins, decreased cytotoxicity towards cultured cell lines and a defect in causing tyrosine dephosphorylation of specific proteins in infected cells in vitro. The deletion strain also revealed that bscN‐mediated secretion is required for persistent colonization of the trachea in a rat infection model. Loci encoding type III secretion homologues were identified in four strains of B. pertussis and two strains of B. parapertussis. B. pertussis strain 18323 and an ovine isolate of B. parapertussis show significant transcription of the genes in vitro.

title: Regulation of type III secretion in Bordetella
abstract: The BvgAS virulence control system regulates the expression of type III secretion genes in Bordetella subspecies that infect humans and other mammals. We have identified five open reading frames, btrS, btrU, btrX, btrW and btrV, that are activated by BvgAS and encode regulatory factors that control type III secretion at the levels of transcription, protein expression and secretion. The btrS gene product bears sequence similarity to ECF (extracytoplasmic function) sigma factors and is required for transcription of the bsc locus. btrU, btrW and btrV encode proteins predicted to contain PP2C‐like Ser phosphatase, HPK (His protein kinase)‐like Ser kinase and STAS anti‐sigma factor antagonist domains, respectively, which are characteristic of Gram‐positive partner switching proteins in Bacillus subtilis. BtrU and BtrW are required for secretion of proteins that are exported by the bsc type III secretion system, whereas BtrV is specifically required for protein synthesis and/or stability. Bordetella species have thus evolved a unique cascade to differentially regulate type III secretion that combines a canonical phosphorelay system with an ECF sigma factor and a set of proteins with domain signatures that define partner switchers, which were traditionally thought to function only in Gram‐positive bacteria. The presence of multiple layers and mechanisms of regulation most likely reflects the need to integrate multiple signals in controlling type III secretion. The bsc and btr loci are nearly identical between broad‐host‐range and human‐specific Bordetella. Comparative analysis of Bordetella subspecies revealed that, whereas bsc and btr loci were transcribed in all subspecies, only broad‐host‐range strains expressed a functional type III secretion system in vitro. The block in type III secretion is post‐transcriptional in human‐adapted strains, and signal recognition appears to be a point of divergence between subspecies.

title: BopC Is a Novel Type III Effector Secreted by Bordetella bronchiseptica and Has a Critical Role in Type III-dependent Necrotic Cell Death*
abstract: In Bordetella bronchiseptica, the functional type III secretion system (TTSS) is required for the induction of necrotic cell death in infected mammalian cells. To identify the factor(s) involved in necrotic cell death, type III-secreted proteins from B. bronchiseptica were analyzed using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry and electrospray ionization tandem mass spectrometry. We identified a 69-kDa secreted protein designated BopC. The gene encoding BopC is located outside of the TTSS locus and is also highly conserved in both Bordetella parapertussis and Bordetella pertussis. The results of a lactate dehydrogenase release assay and terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end-labeling assay demonstrated that BopC is required for necrotic cell death. It has been reported that tyrosine-phosphorylated proteins (PY) of host cells are dephosphorylated during B. bronchiseptica infection in a TTSS-dependent manner. We found that BopC is also involved in PY dephosphorylation in infected host cells. It appears that the necrotic cell death triggered by BopC occurs prior to the PY reduction in host cells, because Bordetella-induced cell death was not affected even in the presence of a dephosphorylation inhibitor. Furthermore, a translocation assay showed that the signal sequence for both secretion into culture supernatant and translocation into the host cell is located in 48 amino acid residues of the BopC N terminus. This report reveals for the first time that a novel type III effector, BopC, is required for the induction of necrotic cell death during Bordetella infection.

title: BopB is a type III secreted protein in Bordetella bronchiseptica and is required for cytotoxicity against cultured mammalian cells
abstract: The cytotoxicity of Bordetella bronchiseptica to infected cells is known to be dependent on a B. bronchiseptica type III secretion system. Although the precise mechanism of the type III secretion system is unknown, BopN, BopD and Bsp22 have been identified as type III secreted proteins. In order to identify other proteins secreted via the type III secretion machinery in Bordetella, a type III mutant was generated, and its secretion profile was compared with that of the wild‐type strain. The results showed that the wild‐type strain, but not the type III mutant, secreted a 40‐kDa protein into the culture supernatant. This protein was identified as BopB by the analysis of its N‐terminal amino acid sequence. Severe cytotoxicity such as necrosis was induced in L2 cells by infection with the wild‐type B. bronchiseptica. In contrast, this effect was not observed by the BopB mutant infection. The haemolytic activity of the BopB mutant was greatly impaired compared with that of the wild‐type strain. The results of a digitonin assay strongly suggested that BopB was translocated into HeLa cells infected with the wild‐type strain. Taken together, our results demonstrate that Bordetella secretes BopB via a type III secretion system during infection. BopB may play a role in the formation of pores in the host plasma membrane which serve as a conduit for the translocation of effector proteins into host cells.

title: A genome‐wide screen identifies a Bordetella type III secretion effector and candidate effectors in other species
abstract: Bordetella bronchiseptica utilizes a type III secretion system (TTSS) for induction of non‐apoptotic cytotoxicity in host cells and modulation of host immunity. The identity of Bordetella TTSS effectors, however, has remained elusive. Here we report a genome‐wide screen for TTSS effectors based on shared biophysical and functional characteristics of class I chaperones and their frequent colocalization with TTSS effectors. When applied to B. bronchiseptica, the screen identified the first TTSS chaperone‐effector locus, btcA‐bteA, and we experimentally confirmed its function. Expression of bteA is co‐ordinated with expression of TTSS apparatus genes, BteA is secreted through the TTSS of B. bronchiseptica, it is required for cytotoxicity towards mammalian cells, and it is highly conserved in the human‐adapted subspecies B. pertussis and B. parapertussis. Transfection of bteA into epithlieal cells results in rapid cell death, indicating that BteA alone is sufficient to induce potent cytotoxicity. Finally, an in vitro interaction between BteA and BtcA was demonstrated. The search for TTSS chaperones and effectors was then expanded to other bacterial genomes, including mammalian and insect pathogens, where we identified a large number of novel candidate chaperones and effectors. Although the majority of putative effectors are proteins of unknown function, several have similarities to eukaryotic protein domains or previously identified effectors from other species.

title: The Bordetella bronchiseptica Type III Secretion System Inhibits Gamma Interferon Production That Is Required for Efficient Antibody-Mediated Bacterial Clearance
abstract: Several species of pathogenic microorganisms have developed strategies to survive and persist in vital organs which are normally maintained as sterile by the generation of strong immune responses. Here, we report an immunomodulation involving the Bordetella bronchiseptica type III secretion system (TTSS) which contributes to bacterial survival in the lower respiratory tract of the host. The prolonged persistence of B. bronchiseptica that was observed in gamma interferon (IFN-γ)−/− mice indicates that the efficient clearance of bacteria from the lower respiratory tract requires not only B cells and antibodies but also IFN-γ production. Our data also suggest that interleukin-10 (IL-10)-producing splenocytes are generated early during infection and that IL-10 inhibits IFN-γ-producing cells and delays the clearance of B. bronchiseptica from the lungs. The TTSS of B. bronchiseptica inhibits the generation of IFN-γ-producing splenocytes and is required for long-term bacterial persistence in the lower respiratory tract in wild-type mice. This suggests that a mechanism involving the modulation of IFN-γ production by the TTSS facilitates B. bronchiseptica survival in the lower respiratory tract.

title: Modulation of host immune responses, induction of apoptosis and inhibition of NF‐κB activation by the Bordetella type III secretion system
abstract: Bordetella bronchiseptica establishes respiratory tract infections in laboratory animals with high efficiency. Colonization persists for the life of the animal and infection is usually asymptomatic in immunocompetent hosts. We hypothesize that this reflects a balance between immunostimulatory events associated with infection and immunomodulatory events mediated by the bacteria. We have identified 15 loci that are part of a type III secretion apparatus in B. bronchiseptica and three secreted proteins. The functions of the type III secretion system were investigated by comparing the phenotypes of wild‐type bacteria with two strains that are defective in type III secretion using in vivo and in vitro infection models. Type III secretion mutants were defective in long‐term colonization of the trachea in immunocompetent mice. The mutants also elicited higher titres of anti‐Bordetella antibodies upon infection compared with wild‐type bacteria. Type III secretion mutants also showed increased lethal virulence in immunodeficient SCID‐beige mice. These observations suggest that type III‐secreted products of B. bronchiseptica interact with components of both innate and adaptive immune systems of the host. B. bronchiseptica induced apoptosis in macrophages in vitro and inflammatory cells in vivo and type III secretion was required for this process. Infection of an epithelial cell line with high numbers of wild type, but not type III deficient B. bronchiseptica resulted in rapid aggregation of NF‐κB into large complexes in the cytoplasm. NF‐κB aggregation was dependent on type III secretion and aggregated NF‐κB did not respond to TNFα activation, suggesting B. bronchiseptica may modulate host immunity by inactivating NF‐κB. Based on these in vivo and in vitro results, we hypothesize that the Bordetella type III secretion system functions to modulate host immune responses during infection.

title: Adenylate cyclase activity of a 68,000-molecular-weight protein isolated from the outer membrane of Bordetella bronchiseptica
abstract: A method was developed which is suitable for the isolation of substantial quantities of outer membrane proteins of Bordetella species in a water-soluble form. The extracted material may then be further fractionated in the absence of detergents by ion-exchange chromatography and preparative flat-bed isoelectrofocusing. These procedures facilitated the isolation of one of the proteins, of molecular weight 68,000, for which the antibody titer correlated with the degree of protection against nasal changes induced in specific-pathogen-free piglets by Bordetella bronchiseptica infection (P. Novotny, M. Kobisch, K. Cownley, A. P. Chubb, and J. A. Montaraz, . This protein, which banded between 7.0 and 7.6 pH in preparative isoelectrofocusing, may be further purified with a monoclonal immunosorbent. Immunopurified protein showed adenylate cyclase activity. The enzymatic activity was found to be unstable during processing; i.e., although the crude extract showed up to 150 nmol of cyclic AMP per mg/min, the immunopurified protein showed a maximum of only 200 nmol of cyclic AMP per mg/min. Two strains of B. bronchiseptica, isolated from herds of healthy pigs showing no signs of atrophic rhinitis, did not produce the 68,000-molecular-weight protein and were negative for adenylate cyclase. However, it is not known whether the 68,000-molecular-weight protein is a component of adenylate cyclase or whether it is an unrelated protein associated with this enzyme in some unknown way. Adenylate cyclase activity from culture supernatants of B. bronchiseptica, B. pertussis, and B. parapertussis can be absorbed equally to the same monoclonal immunosorbent.

title: Multiple Roles for BordetellaLipopolysaccharide Molecules during Respiratory Tract Infection
abstract: Bordetella pertussis, Bordetella parapertussis, and Bordetella bronchiseptica are closely related subspecies that cause respiratory tract infections in humans and other mammals and express many similar virulence factors. Their lipopolysaccharide (LPS) molecules differ, containing either a complex trisaccharide (B. pertussis), a trisaccharide plus an O-antigen-like repeat (B. bronchiseptica), or an altered trisaccharide plus an O-antigen-like repeat (B. parapertussis). Deletion of the wlb locus results in the loss of membrane-distal polysaccharide domains in the three subspecies of bordetellae, leaving LPS molecules consisting of lipid A and core oligosaccharide. We have used wlb deletion (Δwlb) mutants to investigate the roles of distal LPS structures in respiratory tract infection by bordetellae. Each mutant was defective compared to its parent strain in colonization of the respiratory tracts of BALB/c mice, but the location in the respiratory tract and the time point at which defects were observed differed significantly. Although the Δwlb mutants were much more sensitive to complement-mediated killing in vitro, they displayed similar defects in respiratory tract colonization in C5−/− mice compared with wild-type (wt) mice, indicating that increased sensitivity to complement-mediated lysis is not sufficient to explain the in vivo defects. B. pertussis andB. parapertussis Δwlb mutants were also defective compared to wt strains in colonization of SCID-beige mice, indicating that the defects were not limited to interactions with adaptive immunity. Interestingly, the B. bronchiseptica Δwlbstrain was defective, compared to the wt strain, in colonization of the respiratory tracts of BALB/c mice beginning 1 week postinoculation but did not differ from the wt strain in its ability to colonize the respiratory tracts of B-cell- and T-cell-deficient mice, suggesting that wlb-dependent LPS modifications in B. bronchiseptica modulate interactions with adaptive immunity. These data show that biosynthesis of a full-length LPS molecule by these three bordetellae is essential for the expression of full virulence for mice. In addition, the data indicate that the different distal structures modifying the LPS molecules on these three closely related subspecies serve different purposes in respiratory tract infection, highlighting the diversity of functions attributable to LPS of gram-negative bacteria.

title: The Complex Mechanism of Antibody-Mediated Clearance of Bordetella from the Lungs Requires TLR41
abstract: Although the antibacterial effects of Abs are well studied in in vitro systems, the in vivo effects of Abs cannot always be accurately predicted. Complicated cross-talk between different effector functions of Abs and various arms of the immune system can affect their activities in vivo. Using the mouse respiratory pathogen Bordetella bronchiseptica, we examined the mechanisms of Ab-mediated clearance of bacteria from the respiratory tract. Interestingly, although TLR4 was not necessary for protective immunity following infection, it was required for rapid bacterial clearance in mice that were vaccinated or adoptively transferred Abs. TLR4 was important for the rapid recruitment of neutrophils that are necessary for Ab-mediated bacterial clearance via a mechanism that requires both FcγR and CR3. These data are consistent with a model in which TLR4-mediated inflammatory responses aid in the recruitment of neutrophils, which phagocytose Ab- and complement-opsonized bacteria via FcγRs and CR3. Although pattern recognition receptors are known to be involved in innate immunity and the generation of adaptive immunity, their contributions to specific adaptive immune functions should be considered in ongoing efforts to improve vaccine-induced protective immunity.

title: Phase variation in Bordetella pertussis by frameshift mutation in a gene for a novel two-component system
abstract: BORDETELLA pertussis, the aetiological agent of whooping cough, coordinately regulates the expression of many virulence-associated determinants, including filamentous haemagglutinin, pertussis toxin, adenylyl cyclase toxin, dermonecrotic toxin and haemolysin. The coordinate regulation is apparent in the repression of synthesis of these determinants in response to environmental stimuli1,2; a phenomenon known as antigenic or phenotypic modulation. B. pertussis also varies between metastable genetic states, or phases. There is a virulent phase in which virulence-associated determinants are synthesized, and an avirulent phase in which they are not3. Previous studies4,5 have shown that a genetic locus, vir, is required for expression from many virulence-associated loci, and that replacing the cloned vir locus in trans can restore the virulent phase phenotype to spontaneously occurring avirulent phase strains. Here, we show that phase variation in one series of strains is due to a frameshift mutation within an open reading frame that is predicted to code for a Vir protein product. The deduced protein sequence is similar to both components of the 'two-component' regulatory systems which control gene expression in response to environmental stimuli in a range of bacterial species.

title: Toll-Like Receptor 4-Dependent Early Elicited Tumor Necrosis Factor Alpha Expression Is Critical for Innate Host Defense against Bordetella bronchiseptica
abstract: Toll-like receptor 4 (TLR4) mediates the response to lipopolysaccharide, and its activation induces the expression of a large number of inflammatory genes, many of which are also induced by other pathogen-associated molecular patterns. Interestingly, the subset of genes that are dependent on TLR4 for optimal expression during gram-negative bacterial infection has not been determined. We have previously shown that TLR4-deficient mice rapidly develop acute pneumonia after inoculation with Bordetella bronchiseptica, suggesting that TLR4 is required for expression of early elicited gene products in this model. Microarray analysis with macrophages derived from wild-type and TLR4-deficient mice was used to identify genes whose expression, within 1 h of bacterial exposure, is dependent on TLR4. The results of this investigation suggest that TLR4 is not required for the majority of the transcriptional response to B. bronchiseptica. However, early tumor necrosis factor alpha (TNF-α) mRNA expression is primarily dependent on TLR4 and in vitro and in vivo protein levels substantiate this finding. TLR4-deficient mice and TNF-α−/− mice are similarly susceptible to infection with relatively low doses of B. bronchiseptica and in vivo neutralization studies indicate that it is the TLR4-dependent early elicited TNF-α response that is critical for preventing severe pneumonia and limiting bacterial growth. These results suggest that one critical role for TLR4 is the generation of a robust but transient TNF-α response that is critical to innate host defense during acute gram-negative respiratory infection.

title: CD11b is required for the resolution of inflammation induced by Bordetella bronchiseptica respiratory infection
abstract: CD11b is a cell surface receptor that contributes to many cellular processes which are involved in the generation of a protective immune response against pathogenic organisms. In this work, the natural host‐pathogen model of murine Bordetella bronchiseptica infection was used to explore the role of CD11b in respiratory immunity. Following intranasal inoculation, CD11b–/– mice rapidly succumb to B. bronchiseptica respiratory infection, highlighting the prominent role of CD11b in the generation of a protective immune response in this model. CD11b appears to be required for both the control of bacterial numbers and the regulation of cellular responses in the lungs. An increased accumulation of neutrophils in the lungs of CD11b–/– mice as compared with wild‐type mice suggests that CD11b contributes to the regulation of cellular responses to respiratory infection. This accumulation may be explained by a decrease in apoptosis that is observed in the absence of CD11b following cellular interactions with B. bronchiseptica. Interestingly, this role for CD11b in the regulation of cellular accumulation appears to be critically important for the resolution of damage associated with the type III secretion system (TTSS) of B. bronchiseptica. These data provide new insight into the key role CD11b plays in the resolution of damage in the lower respiratory tract, as well as the B. bronchiseptica virulence determinant that induces it.

title: Pertussis toxin inhibits neutrophil recruitment to delay antibody-mediated clearance of Bordetella pertussis.
abstract: Whooping cough is considered a childhood disease, although there is growing evidence that children are infected by adult carriers. Additionally, increasing numbers of vaccinated adults are being diagnosed with Bordetella pertussis disease. Thus it is critical to understand how B. pertussis remains endemic even in highly vaccinated or immune populations. Here we used the mouse model to examine the nature of sterilizing immunity to B. pertussis. Antibodies were necessary to control infection but did not rapidly clear B. pertussis from the lungs. However, antibodies affected B. pertussis after a delay of at least a week by a mechanism that involved neutrophils and Fc receptors, suggesting that neutrophils phagocytose and clear antibody-opsonized bacteria via Fc receptors. B. pertussis blocked migration of neutrophils and inhibited their recruitment to the lungs during the first week of infection by a pertussis toxin-dependent (PTx-dependent) mechanism; a PTx mutant of B. pertussis induced rapid neutrophil recruitment and was rapidly cleared from the lungs by adoptively transferred antibodies. Depletion of neutrophils abrogated the defects of the PTx mutant. Together these results indicate that PTx inhibits neutrophil recruitment, which consequently allows B. pertussis to avoid rapid antibody-mediated clearance and therefore successfully infect immune hosts.

title: Significance analysis of microarrays applied to the ionizing radiation response
abstract: Microarrays can measure the expression of thousands of genes to identify changes in expression between different biological states. Methods are needed to determine the significance of these changes while accounting for the enormous number of genes. We describe a method, Significance Analysis of Microarrays (SAM), that assigns a score to each gene on the basis of change in gene expression relative to the standard deviation of repeated measurements. For genes with scores greater than an adjustable threshold, SAM uses permutations of the repeated measurements to estimate the percentage of genes identified by chance, the false discovery rate (FDR). When the transcriptional response of human cells to ionizing radiation was measured by microarrays, SAM identified 34 genes that changed at least 1.5-fold with an estimated FDR of 12%, compared with FDRs of 60 and 84% by using conventional methods of analysis. Of the 34 genes, 19 were involved in cell cycle regulation and 3 in apoptosis. Surprisingly, four nucleotide excision repair genes were induced, suggesting that this repair pathway for UV-damaged DNA might play a previously unrecognized role in repairing DNA damaged by ionizing radiation.

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

title: mlstdbNet – distributed multi-locus sequence typing (MLST) databases
abstract: BackgroundMulti-locus sequence typing (MLST) is a method of typing that facilitates the discrimination of microbial isolates by comparing the sequences of housekeeping gene fragments. The mlstdbNet software enables the implementation of distributed web-accessible MLST databases that can be linked widely over the Internet.ResultsThe software enables multiple isolate databases to query a single profiles database that contains allelic profile and sequence definitions. This separation enables isolate databases to be established by individual laboratories, each customised to the needs of the particular project and with appropriate access restrictions, while maintaining the benefits of a single definitive source of profile and sequence information. Databases are described by an XML file that is parsed by a Perl CGI script. The software offers a large number of ways to query the databases and to further break down and export the results generated. Additional features can be enabled by installing third-party (freely available) tools.ConclusionDevelopment of a distributed structure for MLST databases offers scalability and flexibility, allowing participating centres to maintain ownership of their own data, without introducing duplication and data integrity issues.

title: Bordetella pertussis Expresses a Functional Type III Secretion System That Subverts Protective Innate and Adaptive Immune Responses
abstract: Certain bacteria use a type III secretion system (TTSS) to deliver effector proteins that interfere with cell function into host cells. While transcription of genes encoding TTSS components has been demonstrated, studies to date have failed to identify TTSS effector proteins in Bordetella pertussis. Here we present the first evidence of a functionally active TTSS in B. pertussis. Three known TTSS effectors, Bsp22, BopN, and BopD, were identified as TTSS substrates in B. pertussis 12743. We found expression of Bsp22 in a significant proportion of clinical isolates but not in common laboratory-adapted strains of B. pertussis. We generated a TTSS mutant of B. pertussis 12743 and showed that it induced significantly lower respiratory tract colonization in mice than the wild-type bacteria. Respiratory infection of mice with the mutant bacteria induced significantly greater innate proinflammatory cytokine production in the lungs soon after challenge, and this correlated with significantly higher antigen-specific interleukin-17, gamma interferon, and immunoglobulin G responses later in infection. Our findings suggest that the TTSS subverts innate and adaptive immune responses during infection of the lungs and may be a functionally important virulence factor for B. pertussis infection of humans.

title: Probing the Function of Bordetella bronchiseptica Adenylate Cyclase Toxin by Manipulating Host Immunity
abstract: We have examined the role of adenylate cyclase-hemolysin (CyaA) by constructing an in-frame deletion in the Bordetella bronchiseptica cyaA structural gene and comparing wild-type andcyaA deletion strains in natural host infection models. Both the wild-type strain RB50 and its adenylate cyclase toxin deletion (ΔcyaA) derivative efficiently establish persistent infections in rabbits, rats, and mice following low-dose inoculation. In contrast, an inoculation protocol that seeds the lower respiratory tract revealed significant differences in bacterial numbers and in polymorphonuclear neutrophil recruitment in the lungs from days 5 to 12 postinoculation. We next explored the effects of disarming specific aspects of the immune system on the relative phenotypes of wild-type and ΔcyaA bacteria. SCID, SCID-beige, or RAG-1−/− mice succumbed to lethal systemic infection following high- or low-dose intranasal inoculation with the wild-type strain but not the ΔcyaA mutant. Mice rendered neutropenic by treatment with cyclophosphamide or by knockout mutation in the granulocyte colony-stimulating factor locus were highly susceptible to lethal infection by either wild-type or ΔcyaA strains. These results reveal the significant role played by neutrophils early in B. bronchiseptica infection and by acquired immunity at later time points and suggest that phagocytic cells are a primary in vivo target of theBordetella adenylate cyclase toxin.

title: Intraspecies variation in bacterial genomes: the need for a species genome concept.
abstract: Bacterial populations are clonal. Their evolution involves not only divergence between orthologous genes but also gain of genes from other clones or species, which has only recently been widely appreciated through macrorestriction mapping, genomic subtraction and complete genome sequencing. Genes can also be lost in response to selection or by random mutation after becoming redundant. The bacterial genome is a dynamic structure and intraspecies variation needs to be included in genome analysis if we are to gain insight into the full species genome.

title: Hypervirulence and pathogen fitness.
abstract: Historically, pathogenesis research has focused on the identification and characterization of virulence factors. More recently, 'anti-virulence' genes have been discovered. Mutations in these loci result in a hypervirulent phenotype, as measured by a lower lethal dose, a colonization advantage, reduced clearance or decreased survival time of the host. If these genes function to reduce pathogen virulence, why have they been retained? Multiple hypotheses have been offered to explain this phenomenon.

title: Coevolution of hosts and parasites.
abstract: The coevolution of parasites and their hosts has both general biological interest and practical implications in agricultural, veterinary and medical fields. Surprisingly, most medical, parasitological and ecological texts dismiss the subject with unsupported statements to the effect that ‘successful’ parasite species evolve to be harmless to their hosts. Recently, however, several people have explored theoretical aspects of the population genetics of host-parasite associations; these authors conclude that such associations may be responsible for much of the genetic diversity found within natural populations, from blood group polymorphisms (Haldane, 1949) to protein polymorphisms in general (Clarke, 1975, 1976) and to histocompatibility systems (Duncan, Wakeland & Klein, 1980). It has also been argued that pathogens may constitute the selective force responsible for the evolution and maintenance of sexual reproduction in animal and plant species (Jaenike, 1978; Hamilton, 1980, 1981, 1982; Bremermann, 1980).

title: The evolution of parasitic diseases.
abstract: Parasites are characterized by their fitness-reducing effect on their hosts. Studying the evolution of parasitic diseases is an attempt to understand these negative effects as an adaptation of the parasite, the host, both or neither. Dieter Ebert and E. Allen Herre here discuss how the underlying concepts are general and are applicable for all types of disease-producing organisms, broadly defined here as parasites. The evolutionary processes that lead to the maintenance of the harmful effects are believed to be characterized by genetic correlations with other fitness components of the parasite. Depending on the shape of these correlations, any level of virulence can evolve.