Project description:Bordetella pertussis is the etiological agent of whooping cough, a bacterial infection of especially children, which may be fatal without treatment. In frame of studies to investigate putative effects of vaccination on host-pathogen interaction and clonal distribution of strains, in addition to Corynebacterium diphtheriae and Clostridium tetani toxoid vaccines, also whole-cell and acellular pertussis vaccines were analyzed by mass spectrometry.
Project description:Filamentous hemagglutinin (FHA), Bordetella pertussis' key adhesin, is both present at the cell surface and released in the extracellular milieu. To comprehend the role free FHA plays in the pathogenic process, we analyzed the global transcriptional changes in human peripheral blood mononuclear cells (PBMC) after exposure to that purified virulence factor. This analysis was undertaken with four different FHA preparations (FHA-1, FHA-2, FHA-3, and FHA-4), purified from four different B. pertussis strains. Cultures comprising 3x106 PBMC were stimulated for 0.5, 2, 4, and 6 hours with 5 ug/ml of each purified B. pertussis FHA. Control PBMC cultures were simultaneously treated with similar volumes of elution buffers (Mock-1 and Mock-2), and sampled at the same time points. The array experiment was composed of 27 samples. Most of the transcripts known to be part of the "common host-transcriptional response" that mediates inflammation were among the 1,235 array elements (representing 683 known unique genes) showing a greater than 3-fold change in transcript abundance between FHA-treated and untreated cells. We also identified an FHA-specific signature not observed in cells stimulated with B. pertussis LPS or heat-killed B. pertussis. This response, mounted by 13 genes, largely (69%, 9/13 genes) interferon (IFN)-regulated genes. Further analysis revealed that 18.3% (125 out of 683) of the FHA-responsive genes were in fact regulated by IFN. These included several major players of the antiviral IFN type I response, as well as three key components of the ISGylation pathway. Induction of the ubiquitin-like protein ISG15 and its specific protease USP18 was confirmed at the mRNA level by relative real-time polymerase chain reaction (RT-PCR). Western-blot analysis demonstrated the presence of both free ISG15 and several ISGylated conjugates in FHA-stimulated PBMC cell lysate. Intracellular FACS analysis provided further evidence that monocytes and a natural-killer (NK)-enriched cell population are the primary producers of ISG15 after FHA stimulation. Our results revealed new activities of free B. pertussis FHA: activation of the host IFN and ISGylation pathways. The consequences of such activation are yet to be resolved, but we hypothesize that FHA utilizes the ISGylation pathway or USP18 to regulate the IFN response, which in turn sensitizes the host to the infection and induces cell death. Groups of assays that are related as part of a time series. Infection: treatment with purified B. pertussis filamentous hemagglutinin (FHA) at 0.005 mg/mL Time: Samples were collected after various treatment time (hours) Keywords: time_series_design Computed
Project description:Bordetella pertussis, the etiological agent of whooping cough, regulates the expression of its virulence factors by the well-defined BvgA/S two-component regulatory system. Phosphorylated BvgA activates the virulence activated genes (vags) and represses via the activation of the bvgR gene the expression of the virulence repressed genes (vrgs). In the presence of MgSO4, known as modulating condition, the BvgA/S system is inactive, and the vrgs are expressed. Here, we show that, except for 6 genes of unknown function, the expression of all the vrgs depends on RisA, another transcriptional two-component regulator. In addition to the vrgs, RisA also regulates the expression of a many other genes. Whereas the expression of most vags was not affected by the deletion of risA, some vags, including the genes coding for pertactin, filamentous hemagglutinin, fimbriae and adenylate cyclase toxin, were surprisingly no longer modulated by MgSO4 in the risA- background. Yet another set of genes, mostly of unknown function, were modulated only in the risA- background. Finally, a large group of genes were up-regulated under modulating conditions only in the risA- background. They comprise genes of the chemotaxis and flagellar operons, iron-regulated genes, some of which are under the control of BvgA/S, while others are not. We identified RisK as the cognate RisA kinase, and found that most of the RisA-dependent genes also required RisK for expression, whereas a few genes were independent of RisK. These results were confirmed by the replacement of RisA with the phosphoablative RisAD60N analog and the phosphomimetic RisAD60E analog. Thus, according to its phosphorylation state, RisA regulates the expression of many genes, which may or may not also be regulated by the BvgA/S system, adding a new layer of complexity of B. pertussis gene regulation. Comparison of mutant strains with their corresponding parental wild type strains using 2 to 4 Biological replicates.