Adaptation of Bordetella pertussis to the Respiratory Tract.

There is a lack of insight into the basic mechanisms by which Bordetella pertussis adapts to the local host environment during infection. We analyzed B. pertussis gene expression in the upper and lower airways of mice and compared this to SO4-induced in vitro Bvg-regulated gene transcription. Approximately 30% of all genes were differentially expressed between in vitro and in vivo conditions. This included several novel potential vaccine antigens that were exclusively expressed in vivo. Significant differences in expression profile and metabolic pathways were identified between the upper versus the lower airways, suggesting distinct antigenic profiles. We found high-level expression of several Bvg-repressed genes during infection, and mouse vaccination experiments using purified protein fractions from both Bvg- and Bvg+ cultures demonstrated protection against intranasal B. pertussis challenge. This study provides novel insights into the in vivo adaptation of B. pertussis and may facilitate the improvement of pertussis vaccines.

Bordetella pertussis Isolates from Argentinean Whooping Cough Patients Display Enhanced Biofilm Formation Capacity Compared to Tohama I Reference Strain.

Pertussis is a highly contagious disease mainly caused by Bordetella pertussis. Despite the massive use of vaccines, since the 1950s the disease has become re-emergent in 2000 with a shift in incidence from infants to adolescents and adults. Clearly, the efficacy of current cellular or acellular vaccines, formulated from bacteria grown in stirred bioreactors is limited, presenting a challenge for future vaccine development. For gaining insights into the role of B. pertussis biofilm development for host colonization and persistence within the host, we examined the biofilm forming capacity of eight argentinean clinical isolates recovered from 2001 to 2007. All clinical isolates showed an enhanced potential for biofilm formation compared to the reference strain Tohama I. We further selected the clinical isolate B. pertussis 2723, exhibiting the highest biofilm biomass production, for quantitative proteomic profiling by means of two-dimensional fluorescence difference gel electrophoresis (2D-DIGE) coupled with mass spectrometry, which was accompanied by targeted transcriptional analysis. Results revealed an elevated expression of several virulence factors, including adhesins involved in biofilm development. In addition, we observed a higher expression of energy metabolism enzymes in the clinical isolate compared to the Tohama I strain. Furthermore, all clinical isolates carried a polymorphism in the bvgS gene. This mutation was associated to an increased sensitivity to modulation and a faster rate of adhesion to abiotic surfaces. Thus, the phenotypic biofilm characteristics shown by the clinical isolates might represent an important, hitherto underestimated, adaptive strategy for host colonization and long time persistence within the host.

Proteomics-identified Bvg-activated autotransporters protect against bordetella pertussis in a mouse model.

Pertussis is a highly infectious respiratory disease of humans caused by the bacterium Bordetella pertussis. Despite high vaccination coverage, pertussis has re-emerged globally. Causes for the re-emergence of pertussis include limited duration of protection conferred by acellular pertussis vaccines (aP) and pathogen adaptation. Pathogen adaptations involve antigenic divergence with vaccine strains, the emergence of strains which show enhanced in vitro expression of a number of virulence-associated genes and of strains that do not express pertactin, an important aP component. Clearly, the identification of more effective B. pertussis vaccine antigens is of utmost importance. To identify novel antigens, we used proteomics to identify B. pertussis proteins regulated by the master virulence regulatory system BvgAS in vitro. Five candidates proteins were selected and it was confirmed that they were also expressed in the lungs of naïve mice seven days after infection. The five proteins were expressed in recombinant form, adjuvanted with alum and used to immunize mice as stand-alone antigens. Subsequent respiratory challenge showed that immunization with the autotransporters Vag8 and SphB1 significantly reduced bacterial load in the lungs. Whilst these antigens induced strong opsonizing antibody responses, we found that none of the tested alum-adjuvanted vaccines - including a three-component aP - reduced bacterial load in the nasopharynx, suggesting that alternative immunological responses may be required for efficient bacterial clearance from the nasopharynx.

Differentially expressed genes in Bordetella pertussis strains belonging to a lineage which recently spread globally.

Pertussis is a highly contagious, acute respiratory disease in humans caused by the Gram-negative pathogen Bordetella pertussis. Pertussis has resurged in the face of intensive vaccination and this has coincided with the emergence of strains carrying a particular allele for the pertussis toxin promoter, ptxP3, which is associated with higher levels of pertussis toxin (Ptx) production. Within 10 to 20 years, ptxP3 strains have nearly completely replaced the previously dominant ptxP1 strains resulting in a worldwide selective sweep. In order to identify B. pertussis genes associated with the selective sweep, we compared the expression of genes in ptxP1 and ptxP3 strains that are under control of the Bordetella master virulence regulatory locus (bvgASR). The BvgAS proteins comprise a two component sensory transduction system which is regulated by temperature, nicotinic acid and sulfate. By increasing the sulfate concentration, it is possible to change the phase of B. pertussis from virulent to avirulent. Until recently, the only distinctive phenotype of ptxP3 strains was a higher Ptx production. Here we identify additional phenotypic differences between ptxP1 and ptxP3 strains which may have contributed to its global spread by comparing global transcriptional responses under sulfate-modulating conditions. We show that ptxP3 strains are less sensitive to sulfate-mediated gene suppression, resulting in an increased production of the vaccine antigens pertactin (Prn) and Ptx and a number of other virulence genes, including a type III secretion toxin, Vag8, a protein involved in complement resistance, and lpxE involved in lipid A modification. Furthermore, enhanced expression of the vaccine antigens Ptx and Prn by ptxP3 strains was confirmed at the protein level. Identification of genes differentially expressed between ptxP1 and ptxP3 strains may elucidate how B. pertussis has adapted to vaccination and allow the improvement of pertussis vaccines by identifying novel vaccine candidates.

The vaccine potential of Bordetella pertussis biofilm-derived membrane proteins.

Pertussis is an infectious respiratory disease of humans caused by the gram-negative pathogen Bordetella pertussis. The use of acellular pertussis vaccines (aPs) which induce immunity of relative short duration and the emergence of vaccine-adapted strains are thought to have contributed to the recent resurgence of pertussis in industrialized countries despite high vaccination coverage. Current pertussis vaccines consist of antigens derived from planktonic bacterial cultures. However, recent studies have shown that biofilm formation represents an important aspect of B. pertussis infection, and antigens expressed during this stage may therefore be potential targets for vaccination. Here we provide evidence that vaccination of mice with B. pertussis biofilm-derived membrane proteins protects against infection. Subsequent proteomic analysis of the protein content of biofilm and planktonic cultures yielded 11 proteins which were ≥three-fold more abundant in biofilms, of which Bordetella intermediate protein A (BipA) was the most abundant, surface-exposed protein. As proof of concept, mice were vaccinated with recombinantly produced BipA. Immunization significantly reduced colonization of the lungs and antibodies to BipA were found to efficiently opsonize bacteria. Finally, we confirmed that bipA is expressed during respiratory tract infection of mice, and that anti-BipA antibodies are present in the serum of convalescent whooping cough patients. Together, these data suggest that biofilm proteins and in particular BipA may be of interest for inclusion into future pertussis vaccines.

Gene expression profiling of pituitary melanotrope cells during their physiological activation.

The pituitary melanotrope cells of the amphibian Xenopus laevis are responsible for the production of the pigment-dispersing peptide α-melanophore-stimulating hormone, which allows the animal to adapt its skin color to its environment. During adaptation to a dark background the melanotrope cells undergo remarkable changes characterized by dramatic increases in cell size and secretory activity. In this study we performed microarray mRNA expression profiling to identify genes important to melanotrope activation and growth. We show a strong increase in the expression of the immediate early gene (IEG) c-Fos and of the brain-derived neurotrophic factor gene (BDNF). Furthermore, we demonstrate the involvement of another IEG in the adaptation process, Nur77, and conclude from in vitro experiments that the expression of both c-Fos and Nur77 are partially regulated by the adenylyl cyclase system and calcium ions. In addition, we found a steady up-regulation of Ras-like product during the adaptation process, possibly evoked by BDNF/TrkB signaling. Finally, the gene encoding the 105-kDa heat shock protein HSPh1 was transiently up-regulated in the course of black-background adaptation and a gene product homologous to ferritin (ferritin-like product) was >100-fold up-regulated in fully black-adapted animals. We suggest that these latter two genes are induced in response to cellular stress and that they may be involved in changing the mode of mRNA translation required to meet the increased demand for de novo protein synthesis. Together, our results show that microarray analysis is a valuable approach to identify the genes responsible for generating coordinated responses in physiologically activated cells.

ERK-regulated double cortin-like kinase (DCLK)-short phosphorylation and nuclear translocation stimulate POMC gene expression in endocrine melanotrope cells.

We tested whether double cortin-like kinase-short (DCLK-short), a microtubule-associated Ser/Thr kinase predominantly expressed in the brain, is downstream of the ERK signaling pathway and is involved in proopiomelanocortin gene (POMC) expression in endocrine pituitary melanotrope cells of Xenopus laevis. Melanotropes form a well-established model to study physiological aspects of neuroendocrine plasticity. The amphibian X. laevis adapts its skin color to the background light intensity by the release of α-MSH from the melanotrope cell. In frogs on a white background, melanotropes are inactive but they are activated during adaptation to a black background. Our results show that melanotrope activation is associated with an increase in DCLK-short mRNA and with phosphorylation of DCLK-short at serine at position 30 (Ser-30). Upon cell activation phosphorylated Ser-30-DCLK-short was translocated from the cytoplasm into the nucleus, and the ERK blocker U0126 inhibited this process. The mutation of Ser-30 to alanine also inhibited the translocation and reduced POMC expression, whereas overexpression stimulated POMC expression. This is the first demonstration of DCLK-short in a native endocrine cell. We conclude that DCLK-short is physiologically regulated at both the level of its gene expression and protein phosphorylation and that the kinase is effectively regulating POMC gene expression upon its ERK-mediated phosphorylation.

Pertussis: a matter of immune modulation.

Pertussis, or whooping cough, is a highly contagious, acute respiratory disease of humans that is caused by the Gram-negative bacterial pathogen Bordetella pertussis. In the face of extensive global vaccination, this extremely monomorphic pathogen has persisted and re-emerged, causing approximately 300,000 deaths each year. In this review, we discuss the interaction of B. pertussis with the host mucosal epithelium and immune system. Using a large number of virulence factors, B. pertussis is able to create a niche for colonization in the human respiratory tract. The successful persistence of this pathogen is mainly due to its ability to interfere with almost every aspect of the immune system, from the inhibition of complement- and phagocyte-mediated killing to the suppression of T- and B-cell responses. Based on these insights, we delineate ideas for the rational design of improved vaccines that can target the 'weak spots' in the pathogenesis of this highly successful pathogen.