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. 2017 May 23;85(6):e00937-16.
doi: 10.1128/IAI.00937-16. Print 2017 Jun.

Cyclic AMP-Elevating Capacity of Adenylate Cyclase Toxin-Hemolysin Is Sufficient for Lung Infection but Not for Full Virulence of Bordetella pertussis

Karolina Skopova  1 , Barbora Tomalova  1 , Ivan Kanchev  2 , Pavel Rossmann  1 , Martina Svedova  1 , Irena Adkins  1 , Ilona Bibova  1 , Jakub Tomala  1 , Jiri Masin  1 , Nicole Guiso  3   4 , Radim Osicka  1 , Radislav Sedlacek  2 , Marek Kovar  1 , Peter Sebo  5
Affiliations

Cyclic AMP-Elevating Capacity of Adenylate Cyclase Toxin-Hemolysin Is Sufficient for Lung Infection but Not for Full Virulence of Bordetella pertussis

Karolina Skopova et al. Infect Immun. .

Abstract

The adenylate cyclase toxin-hemolysin (CyaA, ACT, or AC-Hly) of Bordetella pertussis targets phagocytic cells expressing the complement receptor 3 (CR3, Mac-1, αMβ2 integrin, or CD11b/CD18). CyaA delivers into cells an N-terminal adenylyl cyclase (AC) enzyme domain that is activated by cytosolic calmodulin and catalyzes unregulated conversion of cellular ATP into cyclic AMP (cAMP), a key second messenger subverting bactericidal activities of phagocytes. In parallel, the hemolysin (Hly) moiety of CyaA forms cation-selective hemolytic pores that permeabilize target cell membranes. We constructed the first B. pertussis mutant secreting a CyaA toxin having an intact capacity to deliver the AC enzyme into CD11b-expressing (CD11b+) host phagocytes but impaired in formation of cell-permeabilizing pores and defective in cAMP elevation in CD11b- cells. The nonhemolytic AC+ Hly- bacteria inhibited the antigen-presenting capacities of coincubated mouse dendritic cells in vitro and skewed their Toll-like receptor (TLR)-triggered maturation toward a tolerogenic phenotype. The AC+ Hly- mutant also infected mouse lungs as efficiently as the parental AC+ Hly+ strain. Hence, elevation of cAMP in CD11b- cells and/or the pore-forming capacity of CyaA were not required for infection of mouse airways. The latter activities were, however, involved in bacterial penetration across the epithelial layer, enhanced neutrophil influx into lung parenchyma during sublethal infections, and the exacerbated lung pathology and lethality of B. pertussis infections at higher inoculation doses (>107 CFU/mouse). The pore-forming activity of CyaA further synergized with the cAMP-elevating activity in downregulation of major histocompatibility complex class II (MHC-II) molecules on infiltrating myeloid cells, likely contributing to immune subversion of host defenses by the whooping cough agent.

Keywords: Bordetella pertussis; adenylate cyclase toxin-hemolysin; cAMP intoxication; lung colonization; pore-forming activity; virulence.

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Figures

FIG 1
FIG 1
The AC+ Hly− variant of CyaA is produced at normal levels and preserves the DC-binding and cAMP-elevating activity of intact CyaA. (A) Parental AC+ Hly+ B. pertussis, the nonhemolytic mutant expressing the CyaA-E570Q+K860R toxin (AC+ Hly−), and the AC− Hly+ mutant producing an enzymatically inactive CyaA-AC− toxoid were grown on Bordet-Gengou agar with 15% defibrinated sheep blood for 5 days at 37°C. (B) Bacteria were grown in liquid Stainer-Scholte (SS) medium for 18 h at 37°C, and CyaA toxin was detected on Western blots of bacterial lysates using the anti-RTX 9D4 antibody. Values below the blot indicate the total AC enzyme activities of CyaA associated with the outer surface of bacterial cells grown in SS medium with 0.13 mM Ca2+ ions. (C) CD11b− sheep erythrocytes (5 × 108/ml) were incubated at 37°C with bacterial lysates (50 ng CyaA/ml). After 30 min, aliquots were taken for determination of the cell-associated AC activity (binding) and of the AC activity internalized into erythrocytes and protected against digestion by externally added trypsin (invasive AC). Activities are expressed as percentages of intact CyaA activity and represent average values ± standard deviations from two independent determinations performed in duplicate. (D) AC domain translocation was assessed by determining the intracellular concentration of cAMP generated in cells following incubation with diluted bacterial lysates (final CyaA concentration of 7.5 ng/ml). The results represent the average of values obtained in at least two independent experiments performed in duplicate.
FIG 2
FIG 2
cAMP-elevating activity alone is sufficient for immunomodulatory shaping of the dendritic cell phenotype by B. pertussis-secreted CyaA. Bone marrow-derived DCs were treated with a suspension of heat-killed parental B. pertussis (HI-Bp; 70°C, 30 min) or infected with the different B. pertussis mutants at an MOI of 100:1. After 1 h of incubation, kanamycin (100 μg/ml) was added to kill the bacteria, and DCs were further incubated for 3 or 23 h. (A) DC survival after 4 h was determined by flow cytometry with TMRE and Hoechst 33258 staining. The viability (TMRE+, Hoechst 33258−) of untreated DCs (medium) was set to 100%. (B) Expression of H-2Kb, I-A/I-E, CD80, CD86, CD40, and CD54 in living CD11c+ DCs was determined by flow cytometry, and (C) the secretion of TNF-α, IL-10, and IL-12p70 at 24 h was determined in DC culture supernatants by ELISA. Expression of maturation-associated molecules and production of cytokines by DCs treated with heat-killed bacteria was set as 100%. Values represent the means ± standard errors of the mean (SEM) of n = 3 (*, P < 0.05). (D) DCs treated as indicated above were coincubated with 0.1 μM OVA protein for presentation on MHC class II molecules to OT-II CD4+ T cells or with 0.2 μM OVA for presentation on MHC class I molecules to OT-I CD8+ T cells. After 4 h of coincubation, the DCs were washed and 2 × 105 naïve OVA-specific CD8+ or CD4+ T cells were added. Production of IL-17, IFN-γ, and IL-10 in cell culture supernatants after 3 days was determined by ELISA. (E) The numbers of Foxp3+ CD4+ CD25+ T regulatory cells were determined by flow cytometry after 3 days of coculture. All experiments were reproduced at least 3 times, and representative dot plots are shown. The graph values represent means ± SEM (*, P < 0.05).
FIG 2
FIG 2
cAMP-elevating activity alone is sufficient for immunomodulatory shaping of the dendritic cell phenotype by B. pertussis-secreted CyaA. Bone marrow-derived DCs were treated with a suspension of heat-killed parental B. pertussis (HI-Bp; 70°C, 30 min) or infected with the different B. pertussis mutants at an MOI of 100:1. After 1 h of incubation, kanamycin (100 μg/ml) was added to kill the bacteria, and DCs were further incubated for 3 or 23 h. (A) DC survival after 4 h was determined by flow cytometry with TMRE and Hoechst 33258 staining. The viability (TMRE+, Hoechst 33258−) of untreated DCs (medium) was set to 100%. (B) Expression of H-2Kb, I-A/I-E, CD80, CD86, CD40, and CD54 in living CD11c+ DCs was determined by flow cytometry, and (C) the secretion of TNF-α, IL-10, and IL-12p70 at 24 h was determined in DC culture supernatants by ELISA. Expression of maturation-associated molecules and production of cytokines by DCs treated with heat-killed bacteria was set as 100%. Values represent the means ± standard errors of the mean (SEM) of n = 3 (*, P < 0.05). (D) DCs treated as indicated above were coincubated with 0.1 μM OVA protein for presentation on MHC class II molecules to OT-II CD4+ T cells or with 0.2 μM OVA for presentation on MHC class I molecules to OT-I CD8+ T cells. After 4 h of coincubation, the DCs were washed and 2 × 105 naïve OVA-specific CD8+ or CD4+ T cells were added. Production of IL-17, IFN-γ, and IL-10 in cell culture supernatants after 3 days was determined by ELISA. (E) The numbers of Foxp3+ CD4+ CD25+ T regulatory cells were determined by flow cytometry after 3 days of coculture. All experiments were reproduced at least 3 times, and representative dot plots are shown. The graph values represent means ± SEM (*, P < 0.05).
FIG 3
FIG 3
AC enzyme activity of CyaA alone supports lung infection by B. pertussis. (A) Infection of the mouse lungs. Four-week-old Swiss CD-1 mice were challenged intranasally with 105 CFU of the parental AC+ Hly+ strain or the AC− Hly+ and AC+ Hly− mutants. The mean values and standard deviations from six mice per time point were plotted (except for day 12, which is a mean value from only three mice). The results represent the average of values obtained in two experiments. (B) Survival rates of 4-week-old mice infected with 1 × 108 CFU of the WT and mutant B. pertussis strains. (C) Survival rates at the increased inoculation dose of 6 × 108 CFU. Mortality was monitored for 10 days postinfection. The results were reproduced in at least 3 experiments using 6 to 8 mice per challenged group per challenge dose.
FIG 4
FIG 4
The B. pertussis AC+ Hly− mutant elicits significantly milder inflammation of infected lungs than the parental strain. Four BALB/c mice per group were infected intranasally with 1.5 × 105 CFU in 50 μl of suspensions of the indicated B. pertussis strains. The animals were sacrificed on day 6, and lungs were processed for staining with hematoxylin and eosin (see Materials and Methods for details) and scanned. Control mice received SS medium only. (A) Longitudinal sections of the left lobes at the original magnification of 1,25× that are representative of 3 serial sections per lung lobe. The right panels show enlargements of representative images of the bronchi and peribronchial parenchyma at magnification 20×. Lungs of animals infected by the AC+ Hly+ strain exhibited bronchopneumonia affecting the regions primarily around the large lobar bronchi. Significantly milder inflammation is observed in the lungs of animals infected by the AC+ Hly− and AC− Hly+ strains. (B) Inflamed parenchyma regions were manually delimited on three consecutive lung sections for 4 infected animals per group (12 sections analyzed in total) and scanned using an AxioScan.Z1 automated slide scanner, and the ratios of inflamed (infl.) to total parenchyma areas were calculated using the ZEN software. ** and *** represent P values of <0.01 and <0.001, respectively.
FIG 5
FIG 5
The AC+ Hly− mutant does not penetrate into lung parenchyma. Lungs of BALB/c mice infected with 105 CFU of the indicated strains in 50 μl of suspensions were examined on day 6 upon immunohistochemical (IHC) staining for B. pertussis antigens with a polyclonal rabbit serum. The parental AC+ Hly+ B. pertussis infected the bronchial tree and invaded the lung alveoli and parenchyma, resulting in massive inflammatory response and pneumonic foci with cellular infiltrate (right panel). The AC+ Hly− mutant infected the bronchial tree and was found attached to the ciliated epithelial cells of bronchi without invading the parenchyma. IHC magnifications: left panels, 1.25×; right panels, 40×.
FIG 6
FIG 6
The cell-permeabilizing activity of CyaA triggers infiltration of neutrophils into B. pertussis-infected lungs. (A and B) BALB/c mice infected intranasally with 105 CFU of the indicated strains were sacrificed on day 6, and the sections of unperfused lung tissue were examined upon NASDCL histochemical and F4/80 immunohistochemical staining for neutrophils and macrophages, respectively. Representative sections documenting the main inflammatory cell components from the B. pertussis-induced lesions are shown. Neutrophil infiltration was significantly reduced in both AC+ Hly− and AC− Hly+ strain-infected lungs, while macrophage counts were reduced only in sections of lungs infected by the AC− Hly+ strain. Magnification, 20×. (B) The numbers of neutrophils and macrophages per surface unit were quantified by analysis of all inflamed parenchyma regions that were manually delimited on three consecutive lung sections for 4 infected animals per group (12 sections analyzed in total). (C to E) Distribution of cell subsets in infected mouse lungs. BALB/c mice were infected with 1.5 × 105 CFU of the various B. pertussis strains (AC+ Hly+, AC− Hly+, AC+ Hly−), using medium as a control, and suspensions of unperfused lungs were analyzed by flow cytometry. (C) Dot plots of cell subtypes in one representative mouse lung suspension per experimental group. (D) Total counts of indicated cell subsets in the infected lungs (n = 5) that were not perfused prior to homogenization. (E) Relative distribution of myeloid cell subsets in suspensions of nonperfused lungs (n = 5). Groups were compared using ANOVA followed by Tukey test for pairwise comparison of subgroups. *, **, and *** represent P values of <0.05, <0.01, and <0.001, respectively. The experiment was repeated twice with similar results. EOS, eosinophils; Mϕ, macrophages; MONO, monocytes; NEU, neutrophils; cDC, conventional dendritic cells; pDC, plasmacytoid dendritic cells.
FIG 6
FIG 6
The cell-permeabilizing activity of CyaA triggers infiltration of neutrophils into B. pertussis-infected lungs. (A and B) BALB/c mice infected intranasally with 105 CFU of the indicated strains were sacrificed on day 6, and the sections of unperfused lung tissue were examined upon NASDCL histochemical and F4/80 immunohistochemical staining for neutrophils and macrophages, respectively. Representative sections documenting the main inflammatory cell components from the B. pertussis-induced lesions are shown. Neutrophil infiltration was significantly reduced in both AC+ Hly− and AC− Hly+ strain-infected lungs, while macrophage counts were reduced only in sections of lungs infected by the AC− Hly+ strain. Magnification, 20×. (B) The numbers of neutrophils and macrophages per surface unit were quantified by analysis of all inflamed parenchyma regions that were manually delimited on three consecutive lung sections for 4 infected animals per group (12 sections analyzed in total). (C to E) Distribution of cell subsets in infected mouse lungs. BALB/c mice were infected with 1.5 × 105 CFU of the various B. pertussis strains (AC+ Hly+, AC− Hly+, AC+ Hly−), using medium as a control, and suspensions of unperfused lungs were analyzed by flow cytometry. (C) Dot plots of cell subtypes in one representative mouse lung suspension per experimental group. (D) Total counts of indicated cell subsets in the infected lungs (n = 5) that were not perfused prior to homogenization. (E) Relative distribution of myeloid cell subsets in suspensions of nonperfused lungs (n = 5). Groups were compared using ANOVA followed by Tukey test for pairwise comparison of subgroups. *, **, and *** represent P values of <0.05, <0.01, and <0.001, respectively. The experiment was repeated twice with similar results. EOS, eosinophils; Mϕ, macrophages; MONO, monocytes; NEU, neutrophils; cDC, conventional dendritic cells; pDC, plasmacytoid dendritic cells.
FIG 7
FIG 7
The cell-permeabilizing and AC enzyme activities of CyaA synergize in provoking reduction of MHC-II molecule expression on myeloid cells in infected lungs. Expression of the MHC-II molecule on selected cell subsets in lungs of BALB/c mice infected with B. pertussis 6 days after infection with 1.5 × 105 CFU of the various strains or with medium (Control). (A) Histograms for one representative mouse lung suspension per experimental group. (B) Total counts of MHC-II-expressing cells per million lung cells (n = 5). (C) Mean level (MFI) of MHC-II molecules detected on the surface of selected cell subsets in the infected lungs (n = 5). Groups were compared using ANOVA followed by Tukey test for pairwise comparison of subgroups. *, **, and *** represent P values of <0.05, <0.01, and <0.001, respectively. The experiment was repeated twice with similar results. Mϕ, macrophages; MONO, monocytes; cDC, conventional dendritic cells; pDC, plasmacytoid dendritic cells.
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