Analysis of BvgA Activation of the Pertactin Gene Promoter in Bordetella pertussis

Abstract

Bordetella pertussis, the causative agent of whooping cough, regulates expression of its virulence factors via a two-component signal transduction system encoded by the bvgregulatory locus. It has been shown by activation kinetics that several of the virulence factors are differentially regulated. fhais transcribed at 10 min following an inducing signal, whileptx is not transcribed until 2 to 4 h after the inducing signal. We present data indicating that prn is transcribed at 1 h, an intermediate time compared to those offha and ptx. We have identifiedcis-acting sequences necessary for expression ofprn in B. pertussis by usingprn-lac fusions containing alterations in the sequence upstream of the prn open reading frame. In vitro transcription and DNase I footprinting analyses provided evidence to support our hypothesis that BvgA binds to this sequence upstream ofprn to activate transcription from the promoter. Our genetic data indicate that the region critical for prnactivation extends upstream to position −84. However, these data do not support the location of the prn transcription start site as previously published. We used a number of methods, includingprn-lac fusions, reverse transcriptase PCR, and 5′ rapid amplification of cDNA ends, to localize and identify thebvg-dependent 5′ end of the prn transcript to the cytosine at −125 with respect to the published start site.

Demonstration of differential virulence gene promoter activation in vivo in Bordetella pertussis using RIVET

“…In this study we have shown that differential regulation of bvg-regulated promoters occurs in vivo. We have also documented the temporal regulation of the fha, prn and cya promoters in vivo and shown that the general pattern is the same as in vitro, when analysed using the RIVET system or when compared to patterns established using other methods (Scarlato et al, 1991;Kinnear et al, 1999;Stibitz, 1998). This indicates that the numerous in vitro studies conducted analysing Bvg-mediated regulation are relevant to events that occur in vivo during infection.…”

“…Adenylate cyclase toxin production is controlled by cya, a late promoter. Pertactin, also an adhesin, is governed by prn, which evidence indicates is a middle promoter (Kinnear et al, 1999). Finally, ptx, another late promoter (Scarlato et al, 1991), is responsible for pertussis toxin expression.…”

“…As shown in Fig. 4, the percentage of Kan-sensitive colonies (hereafter called the per cent resolution) increased over time in a manner showing differential expression similar to that observed in vitro using other methods ( (Scarlato et al, 1991;Stibitz, 1998;Kinnear et al, 1999). Given that the highest standard deviation for a resolution time point is 15.6%, for comparison between experiments we chose 20% resolution (indicated by the heavy black line) as a reliable indicator of a promoter beginning to show full expression.…”

“…The 'late' promoters, which include cya and ptx , take longer to be expressed after being shifted to non-modulating conditions and require lower concentrations of modulator to maintain the modulated state (Scarlato et al ., 1991;Stibitz, 1998). In addition, prn has been described as an 'intermediate' promoter, since following release from modulation, it is expressed after the early but before the late class of bvg -activated promoters (Kinnear et al ., 1999). This expression pattern is not to be confused with the third class of bvg -regulated genes, which has been termed the 'intermediate phase' or Bvg i (Cotter and Miller, 1997); hence intermediate bvg -activated promoters such as prn will be referred to as 'middle' promoters throughout the remainder of this work for clarity.…”

Pertussis: a matter of immune modulation

“…Late genes, which include those encoding adenylate cyclase toxin (ACT) and pertussis toxin (Ptx), show relatively slow induction kinetics and require higher concentrations of BvgA-P for expression (Scarlato et al, 1991). The gene encoding pertactin (Prn) has been suggested to belong to the class of intermediate genes (Kinnear et al, 1999). The BvgASR locus is autogenously regulated and belongs to the early genes, providing an additional level of regulation for Bvg-induced class-switching (Roy et al, 1990).…”