Modulation of Pertussis and Adenylate Cyclase Toxins by Sigma Factor RpoE in Bordetella pertussis

Abstract

Bordetella pertussis is a human pathogen that can infect the respiratory tract and cause the disease known as whooping cough. B. pertussis uses pertussis toxin (PT) and adenylate cyclase toxin (ACT) to kill and modulate host cells to allow the pathogen to survive and persist. B. pertussis encodes many uncharacterized transcription factors, and very little is known about their functions. RpoE is a sigma factor which, in other bacteria, responds to oxidative, heat, and other environmental stresses. RseA is a negative regulator of RpoE that sequesters the sigma factor to regulate gene expression based on conditions. In B. pertussis, deletion of the rseA gene results in high transcriptional activity of RpoE and large amounts of secretion of ACT. By comparing parental B. pertussis to an rseA gene deletion mutant (PM18), we sought to characterize the roles of RpoE in virulence and determine the regulon of genes controlled by RpoE. Despite high expression of ACT, the rseA mutant strain did not infect the murine airway as efficiently as the parental strain and PM18 was killed more readily when inside phagocytes. RNA sequencing analysis was performed and 263 genes were differentially regulated by RpoE, and surprisingly, the rseA mutant strain where RpoE activity was elevated expressed very little pertussis toxin. Western blots and proteomic analysis corroborated the inverse relationship of PT to ACT expression in the high-RpoE-activity rseA deletion strain. Our data suggest that RpoE can modulate PT and ACT expression indirectly through unidentified mechanisms in response to conditions.

Development of RNAseq methodologies to profile thein vivotranscriptome ofBordetella pertussisduring murine lung infection

“…FhaB is an adhesin that allows B. pertussis to adhere to the airway during infection. In previous experiments we observed that fhaB is one of the most abundant transcripts in the transcriptome (10). We also detected both pertussis and adenylate cyclase toxin transcripts in the infected lung tissue (Table 2).…”

“…Overall, all of the samples were observed to have similar RIN numbers. For comparison with in vitro grown B. pertussis , we used samples from SSM in vitro grown UT25 extracted and analyzed by RNAseq using an identical procedure previously published by our laboratory (10). These In vitro grown B. pertussis UT25 RNA samples had RIN numbers of 9–10.…”

“…(14): cyaF(CGAGGCGGTCAAGGTGAT), cyaR(GCGGAAGTTGGACAGATGC), ptxAF(CCAGAACGGATTCACGGC), ptxAR (CTGCTGCTGGTGGAGACGA), bvgAF (AGGTCATCAATGCCGCCA), bvgAR (GCAGGACGGTCAGTTCGC), fhaBF (CAAGGGCGGCAAGGTGA), fhaBR (ACAGGATGGCGAACAGGCT), rpoBF (GCTGGGACCCGAGGAAAT), rpoBR (CGCCAATGTAGACGATGCC). BP2497 were designed in a previous study: BP2497F (TCGGATCGCACCAATTACTTC) and BP2497R (CCTTGGCGATCAGCGAGTT) (10). The following primers were used for Gapdh: gapdhf (CATGGCCTTCCGTGTTCCT) and gapdhr (GCGGCACGTCAGATCCA).…”

Peptidoglycan Recognition Protein 4 Suppresses Early Inflammatory Responses to Bordetella pertussis and Contributes to Sphingosine-1-Phosphate Receptor Agonist-Mediated Disease Attenuation

“…B. pertussis stress responses are poorly understood. Recent work showed altered virulence factor secretion following induction of the two-component stress response operon Rpo/Rse, with increased secretion of ACT and decreased production of PT (49). Our future work will seek to determine the nature of the PGLYRP4-TCT interaction, whether PGLYRP4 also mediates its anti-inflammatory activity through effects on other virulence factors, whether PGLYRP4 induces B. pertussis stress responses or direct bacterial killing, and the impact of these responses on infection.…”

Albumin, in the Presence of Calcium, Elicits a Massive Increase in Extracellular Bordetella Adenylate Cyclase Toxin

“…We now know that there are other pathways by which virulence is controlled in this genus (57)(58)(59)(60)(61), but how these pathways relate to one another and to Bvg is still to be fully determined. The Bvg system controls production of many bacterial proteins-importantly ACT and other virulence factorsand expression is downregulated in response to sulfate, nicotinic acid, and a shift to lower temperature (25°C).…”