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

Abstract

Pertussis (whooping cough), caused by Bordetella pertussis, is resurging in the United States and worldwide. Adenylate cyclase toxin (ACT) is a critical factor in establishing infection with B. pertussis and acts by specifically inhibiting the response of myeloid leukocytes to the pathogen. We report here that serum components, as discovered during growth in fetal bovine serum (FBS), elicit a robust increase in the amount of ACT, and ≥90% of this ACT is localized to the supernatant, unlike growth without FBS, in which ≥90% is associated with the bacterium. We have found that albumin, in the presence of physiological concentrations of calcium, acts specifically to enhance the amount of ACT and its localization to the supernatant. Respiratory secretions, which contain albumin, promote an increase in amount and localization of active ACT that is comparable to that elicited by serum and albumin. The response to albumin is not mediated through regulation of ACT at the transcriptional level or activation of the Bvg two-component system. As further illustration of the specificity of this phenomenon, serum collected from mice that lack albumin does not stimulate an increase in ACT. These data, demonstrating that albumin and calcium act synergistically in the host environment to increase production and release of ACT, strongly suggest that this phenomenon reflects a novel host-pathogen interaction that is central to infection with B. pertussis and other Bordetella species.

Keywords: Bordetella pertussis; RTX toxins; adenylate cyclase toxin; albumin; calcium.

FIG 1

Serum elicits a massive increase in the amounts of total and extracellular B. pertussis ACT. B. pertussis UT25 was grown in SSM ± 10% FBS for 8 h. The total fraction includes both culture supernatant and bacterial cells. The supernatant was collected after centrifugation. (A) AC enzyme activity was measured as described in Materials and Methods and normalized by OD₆₀₀. Data represent the mean ± standard deviation (SD) from 9 independent experiments. A Student's t test was used to determine statistical significance. ***, P ≤ 0.0001. (B) Western blot analysis using a rabbit polyclonal anti-ACT antibody detecting the 200-kDa ACT protein.

FIG 2

The response to serum is rapid and peaks at 24 h of growth. B. pertussis UT25 was grown in SSM ± 10% FBS, and samples were taken at the indicated time points. The total fraction includes both culture supernatant and bacterial cells. The supernatant was collected after centrifugation. AC enzyme activity was measured as described in Materials and Methods. (A) The data represent the mean ± SD from 3 independent experiments. Comparisons between enzyme activity ± FBS at all time points are statistically significant as determined by an unpaired t test (P ≤ 0.05). Comparisons between bacterial density ± FBS were statistically significant (P ≤ 0.05) at 8, 16, 24, and 32 h. (B) Data represent the mean ± SD from 2 independent experiments done in duplicate.

FIG 3

Albumin and calcium act synergistically to increase ACT production and secretion. B. pertussis UT25 was grown in SSM ± 2 mg/ml BSA, 2 mM CaCl₂, and/or 10% FBS (as indicated) for 8 h. The total fraction includes both culture supernatant and bacterial cells. The supernatant was collected after centrifugation. AC enzyme activity was measured as described in Materials and Methods and normalized by OD₆₀₀. Data represent the mean ± SD from 3 independent experiments done in duplicate. Statistical significance was assessed using a 2-way analysis of variance (ANOVA). **, P ≤ 0.01, and ***, P ≤ 0.001, compared to growth in SSM.

FIG 4

Albumin and calcium-stripped albumin have equivalent effects on ACT amount and release. B. pertussis UT25 was grown in SSM ± 2 mg/ml BSA or EGTA-treated BSA for 8 h. The total fraction includes both culture supernatant and bacterial cells. The supernatant was collected after centrifugation. AC enzyme activity was measured as described in Materials and Methods and normalized by OD₆₀₀. Data represent the mean ± SD from 3 independent experiments done in duplicate. Statistical significance was assessed using a 2-way ANOVA, and comparisons between SSM plus BSA and SSM plus EGTA-treated BSA were not significantly different.

FIG 5

The maximum effect on ACT amount and release requires a minimum of 0.5 mM calcium. B. pertussis UT25 was grown in SSM with 2 mg/ml BSA with the indicated concentrations of total calcium for 8 h. The total fraction includes both culture supernatant and bacterial cells. The supernatant was collected after centrifugation. AC enzyme activity was measured as described in Materials and Methods and normalized by OD₆₀₀. The data presented are the mean ± SD from a single experiment done in duplicate but representative of 4 similar experiments.

FIG 6

Serum collected from analbuminemic mice lacks albumin. Wild-type (WT) C56BL/6 mouse serum or ALB^−/− mouse serum were analyzed by SDS-PAGE and Coomassie staining to detect protein profiles. The band corresponding to albumin in the wild-type sample is indicated with an arrow.

FIG 7

Albumin is required for an increased amount of ACT during growth in the presence of mouse serum. (A) B. pertussis UT25 was grown in SSM ± wild-type C56BL/6 mouse serum or ALB^−/− mouse serum ± 2 mg/ml BSA, all with 2 mM CaCl₂, for 8 h. The total fraction includes both culture supernatant and bacterial cells. The supernatant was collected after centrifugation. AC enzyme activity was measured as described in Materials and Methods and normalized by OD₆₀₀. Data represent the mean ± SD from 3 independent experiments done in duplicate. Statistical significance was assessed using a 2-way ANOVA. ***, P ≤ 0.001 compared to growth in SSM. (B) Western blot analysis using a rabbit polyclonal anti-ACT antibody detecting the 200-kDa ACT protein. Lanes: S, supernatant; T, total.

FIG 8

Human serum and human serum albumin increase the amount of ACT and shift localization of ACT to the supernatant. B. pertussis UT25 was grown in SSM ± HS or HSA (as indicated), all with 2 mM CaCl₂, for 8 h. The total fraction includes both culture supernatant and bacterial cells. AC enzyme activity was measured as described in Materials and Methods and normalized by OD₆₀₀. The data represent the mean ± SD from 2 independent experiments done in duplicate.

FIG 9

Regulation of cyaA during growth ± HSA is not occurring at the level of transcription through the Bvg two-component system. (A) RNA was isolated from B. pertussis UT25 grown in SSM ± 2 mg/ml HSA and/or 40 mM MgSO₄ with 2 mM calcium for 4 h. Expression of target genes was determined using the relative quantification method. Statistical significance was analyzed by an unpaired t test. Data represent the mean ± SD from 3 independent experiments. ***, P ≤ 0.001 compared to growth in SSM. (B) B. pertussis UT25 was grown in SSM with calcium ± 2 mg/ml HSA and/or 40 mM MgSO₄ for 4 h. AC enzyme activity was measured in the total fraction as described in Materials and Methods and normalized by OD₆₀₀. Data represent the mean ± SD from 3 independent experiments done in duplicate. ***, P ≤ 0.001 compared to growth in SSM; ###, P ≤ 0.001 compared to growth in SSM plus MgSO₄.

FIG 10

Transcription of genes encoded in the cya operon is not altered ± HSA. (A) RNA was isolated from B. pertussis UT25 grown in SSM ± 2 mg/ml HSA and/or 40 mM MgSO₄ with 2 mM calcium for 4 h. Expression of target genes was determined using the relative quantification method. Statistical significance was analyzed by an unpaired t test. Data represent the mean ± SD from 3 independent experiments. **, P ≤ 0.01, and ***, P ≤ 0.001, compared to growth in SSM.

FIG 11

Respiratory secretions, isolated by bronchoalveolar lavage (BAL), contain albumin. SSM plus BAL fluid, SSM plus 2.3 mg/ml HSA, and SSM plus 5% HS samples were analyzed by Western blotting with an anti-BSA antibody as well as SDS-PAGE and Coomassie staining to detect protein profiles and confirm the presence of albumin. Molecular masses of known components of serum and respiratory secretions are as follows: albumin, 66 kDa; gammaglobulin heavy chains, 55 to 60 kDa; and gammaglobulin light chains, 25 to 28 kDa.

FIG 12

Respiratory secretions increase the total and extracellular amounts of ACT. (A) B. pertussis UT25 was grown in SSM ± saline, BAL fluid, 2 mg/ml HSA, or 5% HS, all with 2 mM CaCl₂, for 8 h. The total fraction includes both culture supernatant and bacterial cells. The supernatant was collected after centrifugation. AC enzyme activity was measured as described in Materials and Methods and normalized by OD₆₀₀. Data represent the mean ± SD from 2 independent experiments done in duplicate. Statistical significance was assessed using a 2-way ANOVA. ***, P ≤ 0.001 compared to growth in SSM. (B) Western blot analysis using a rabbit polyclonal anti-ACT antibody detecting the 200-kDa ACT protein. Lanes: S, supernatant; T, total.

FIG 13

Extracellular ACT produced during growth with respiratory secretions is a functional toxin. J774 cells were incubated with the supernatant from growth of B. pertussis UT25 in SSM plus BAL fluid or rACT (normalized to equivalent enzyme activity) for 3 h at 37°C. The number of viable cells was calculated using the CCK8 assay (see Materials and Methods). The data represent the mean ± SD from a single experiment done in triplicate, which is representative of three additional experiments.