In Bordetella pertussis, expression of virulence factors is controlled by the Bvg proteins, which comprise a sensor-regulator two-component signal transduction system. Previously, we described a mutant strain of B.pertussis that had reduced transcription of pertussis toxin and adenylate cyclase toxin genes, while other virulence factors were relatively unaffected. We obtained a B. pertussis clone that repaired the defect in both this strain and an independent mutant strain with a similar phenotype when introduced onto the chromosome by allelic exchange. Further analysis revealed that the mutations were just upstream of the translational start site of the rpoA gene encoding the a subunit of RNA polymerase. We confirmed that these mutations were responsible for the mutant phenotype by site-directed mutagenesis. Our hypothesis that these mutations cause an overexpression of rpoA was confirmed by Western immunoblotting and translational fusion analysis. Corroboration of this effect was obtained by overexpressing rpoA on a plasmid in wild-type B. pertussis, which caused the same phenotype as the mutants showed. Conclusions in regard to the identity of the transcription activator of the toxin genes are discussed.

A CD8+ cytolytic T-lymphocyte (CTL) response to antigen-presenting cells generally requires intracellular delivery or synthesis of antigens in order to access the major histocompatibility complex (MHC) class I processing and presentation pathway. To test the ability of pertussis toxin (PT) to deliver peptides to the class I pathway for CTL recognition, we constructed fusions of CTL epitope peptides with a genetically detoxified derivative of PT (PT9K/129G). Two sites on the A (S1) subunit of PT9K/129G tolerated the insertion of peptides, allowing efficient assembly and secretion of the holotoxin fusion by Bordetella pertussis. Target cells incubated with these fusion proteins were specifically lysed by CTLs in vitro, and this activity was shown to be MHC class I restricted. The activity was inhibited by brefeldin A, suggesting a dependence on intracellular trafficking events, but was not inhibited by the proteasome inhibitors lactacystin andN-acetyl-l-leucyl-l-leucyl-l-norleucinal (LLnL). Furthermore, the activity was present in mutant antigen-presenting cells lacking the transporter associated with antigen processing, which transports peptides from the cytosol to the endoplasmic reticulum for association with MHC class I molecules. PT may therefore bypass the proteasome-dependent cytosolic pathway for antigen presentation and deliver epitopes to class I molecules via an alternative route.

Overexpression of the RNA polymerase alpha subunit in Bordetella pertussis reduces expression of the virulence factor pertussis toxin. Here we show that this reduction is at the level of transcription, is reversed by overexpression of the transcriptional activator BvgA, and is dependent on the C-terminal domain of alpha.In Bordetella pertussis, expression of virulence factors is regulated by the Bvg two-component signal transduction system, comprising the sensor BvgS and the transcriptional activator BvgA (1, 11). The Bvg system is modulated (with loss of virulence factor expression) by reduced temperature (Ͻ30°C) or the presence of sulfate ions or nicotinic acid in the growth medium (11). Previously we showed that mutant B. pertussis strains with reduced expression of pertussis toxin (Ptx) and adenylate cyclase/hemolysin toxin, but not of other Bvg-regulated virulence factors such as filamentous hemagglutinin (Fha), had mutations upstream of the rpoA gene, which encodes the alpha subunit of RNA polymerase (RNAP) (3). These mutations caused a two-to threefold overexpression of alpha through an increase in translation of the rpoA gene (3). We also showed that inducible overexpression of alpha from a recombinant plasmid in B. pertussis had the same effect (3). The alpha subunit is a common site of interaction of RNAP with transcription activator proteins (6). We therefore hypothesized that the observed effect on virulence factor expression was due to interaction of the excess alpha with BvgA, effectively reducing the level of BvgA present in cells for functional interactions with RNAP. To obtain further evidence that the excess alpha affects BvgA-dependent transcription activation, we first assessed the effect of overexpressing alpha on transcription of both the ptx and fha genes.Overexpression of alpha reduces transcription of both ptx and fha. We introduced a ptx-lac transcriptional fusion (8) into the chromosome of wild-type (Tohama I) and mutant (alphaoverexpressing strains BC75 and RPV3 and the bvg knockout strain Tohama I ⌬bvg) B. pertussis strains as previously described (8). We also introduced a fha-lac transcriptional fusion into the chromosome of the same set of strains, by allelic exchange from the plasmid pSS1581 (kindly provided by Scott Stibitz). The fusion strains were grown at 37°C in SS medium (9) to mid-log phase (nonmodulating conditions that allow full Bvg activity), and then ␤-galactosidase assays (8) were performed on the cultures to determine the level of ptx and fha transcription. As seen in Fig. 1, the level of ptx transcription is significantly reduced in both alpha-overexpressing mutants (approximately twofold in RPV3 and threefold in BC75), but the level of fha transcription is not significantly reduced in these strains. Since there is only a modest (two-to threefold) overexpression of alpha in RPV3 and BC75 (3), we introduced the plasmid pNMD120 (encoding IPTG [isopropyl-␤-D-thiogalactopyranoside]-inducible expression of B. pertussis rpoA) (3), as well as the vector control plasmid pNM...