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.

Keywords: Bordetella pertussis; RpoE; adenylate cyclase toxin; pertussis toxin.

FIG 1

Colonization of the murine airway or survival in J774A.1 and neutrophils by B. pertussis strain UT25 and the isogenic PM18 (ΔrseA) strains. CD1 mice were infected with 2 × 10⁷ CFU of the B. pertussis strains UT25 and PM18. At 24, 48, and 144 h postinfection, the bacterial burdens of the nasal wash (A), trachea (B), and lungs (C) were determined. Four to five mice were used for each time point, and all of the values obtained are indicated. At 144 h, the level of PM18 measured by the nasal wash (1 ml) was significantly lower than that of the parental UT25. In the lungs, PM18 was present in a larger amount at 24 h but then decreased as UT25 increased (*, P < 0.05; **, P < 0.01). J774A.1 cells were allowed to phagocytose UT25 or PM18. (D) Less viable PM18 organisms were observed at both 2 and 24 h postinfection. Bone marrow-derived neutrophils were isolated and allowed to phagocytose UT25 or PM18, and similar to J774A.1 cells, PM18 was more efficiently killed intracellularly (E) but not extracellularly (F).

FIG 2

RNA-seq analysis of PM18 compared to UT25 reveals the genes controlled by RpoE. (A) A volcano plot of genes statistically significantly repressed (138; blue) or activated (125; red) are indicated relative to the observed fold changes and their respective P values. (B) The predicted or known localizations of each of the genes that were differentially regulated are shown.

FIG 3

String analysis reveals the systems of genes that are repressed or activated by RpoE. The 138 repressed genes (A) and the 125 activated genes (B) are grouped with the systems or families to which they correspond. Notably, pertussis toxin and metabolism/stress genes are repressed, and most of the activated genes do not belong to known systems except for iron acquisition and branched-chain amino acid uptake.

FIG 4

Biofilm formation, ACT, and cyaABDEX expression in relation to RpoE in B. pertussis. (A) The biofilm-forming capabilities of each strain are shown. Strain UT25 biofilm formation can be suppressed by exogenous ACT. (B) PM18 formed biofilms to the same magnitude as UT25 with exogenous ACT, which correlates with the large amount of ACT secreted by PM18. s, supernatant; p, pelleted bacteria. (C) The relative read coverage of one of the RNA-seq replicates with respect to the cyaC-cyaABDEX operons. Only the type one secretion system-encoding genes cyaB and cyaD were statistically activated in PM18.

FIG 5

Analysis of the UT25 and PM18 proteomes. (A) The breakdown of activated and repressed proteins with respect to their predicted subcellular localizations. (B) The RNA-seq and proteomics were correlated and Venn diagrams are shown. (C) The NSAF-normalized fold change of the protein found in both proteomes is shown and is plotted by location on the chromosome. Proteins that correlated with RNA-seq analysis are highlighted in black halos.

FIG 6

qRT-PCR analysis of selected genes and Western blot measurement of pertussis toxin. UT25 and PM18 were grown up to an OD₆₀₀ of 0.4 and 0.8, and three biological replicates were analyzed with three technical replicates each (*, P < 0.05, **, P < 0.01; ***P < 0.001). (B) A representative Western blot of pertussis toxin is shown for strains UT25 and PM18 where supernatants or cell pellets were used. UT25 has more pertussis toxin expression than PM18, which corroborates RNA-seq, proteomics, and qRT-PCR analyses.

FIG 7

Hypothetical schematic of RpoE-RseA system proteins. The B. pertussis RseA proteases BP1776 (AlgW), BP1426 (MucP), and ClpXP (BP1776/BP1775) are indicated. RpoE would be tethered to the inner membrane by RseA. AlgW, MucP, and then ClpXP would sequentially cleave RseA to release RpoE to drive expression at target promoters. It is possible that this system can be activated during stress conditions to modulate pertussis and adenylate cyclase toxin expression in B. pertussis.