The immunomodulatory and cytotoxic effects of different forms of recombinant adenylate cyclase toxin

This thesis describes the small- and large-scale purification of four different recombinant forms of CyaA, namely; fully functional enzymically-active, acylated and invasive CyaA; an acylated and invasive CyaA form lacking adenylate cyclase (AC) enzymic activity (CyaA*); and the non-acylated, poorly-invasive forms of these toxins, proCyaA and proCyaA*, respectively. Only proCyaA and CyaA showed AC enzymic activity. Only CyaA and CyaA* were haemolytic towards sheep erythrocytes and cytotoxic towards mouse J774.2 macrophage-like cells. Both haemolysis and cytotoxicity by CyaA and CyaA* only occurred in the presence of calcium. CyaA was cytotoxic towards J774.2 cells at low concentrations (50% killing at 0.1 ?g protein/ml) and this paralleled low levels of caspase 3/7 activity, a measure of apoptotic activity. At higher toxin concentrations of CyaA (> 0.5 ?g protein/ml), caspase 3/7 activity declined despite high levels of cell killing measured by the cytotoxicity assay. This may have been due to the onset of necrotic killing at higher concentrations. CyaA* could only kill J774.2 cells at a toxin concentration greater than 0.5 ?g protein/ml and the effect was more calcium-dependent than that of CyaA. Cell killing by CyaA* also occurred in the absence of apoptosis. These data suggested that CyaA killed J774.2 cells by two different mechanisms, involving apoptosis at low concentrations (which was cAMP dependent) and necrosis by pore-formation at higher concentrations. ProCyaA and proCyaA* showed no apoptotic, cytotoxic or haemolytic activities. CyaA inhibited the zymosan stimulated oxidative burst of J774.2 cells. ProCyaA and CyaA* were ~500-fold less active at inhibiting the zymosan-stimulated oxidative burst of J774.2 cells compared with native CyaA. Thus, inhibition of the zymosan-stimulated oxidative burst by CyaA was a result of both invasive and AC enzymic activities. This also indicated that proCyaA was able to invade cells, albeit at a very low level, but that any increase in intracellular cAMP levels created by intracellular proCyaA was unable to induce apoptosis. Circular dichroism (CD) was used to assess any differences in the secondary structure of the four CyaA forms. There were similar changes in tertiary structure of all the CyaA forms as shown by intrinsic tryptophan fluorescence and near UV CD studies in the absence and presence of 1 mM CaCl2. Similar conformational changes in protein secondary structure of all four CyaA forms, at 0.5 mg protein/ml, were observed by far UV CD in the absence and presence of 1 mM CaCl2. However, at 0.05 mg protein/ml, the spectral amplitude of CyaA was decreased by 2-fold suggesting that CyaA was able to adopt two stochiometric forms. Preliminary analytical ultracentrifugation (AUC) studies indicated that CyaA did not exist as a single molecular species in solution. At 3 mg protein/ml, different oligomeric forms of CyaA (monomers, dimers and trimers) as well as the presence of degradation products (20 - 80 kDa) were present. Further optimisation of AUC studies is required to overcome the problem of non-ideality (caused by either a repulsion between charged molecules (proteins) that is not shielded by solvent counter-ions or by size-exclusion effects arising from extended/elongated conformations) which may have interfered with the analyses. (Abstract shortened by ProQuest.)

Bordetella pertussis commits human dendritic cells to promote a Th1/Th17 response through the activity of adenylate cyclase toxin and MAPK-pathways.

The complex pathology of B. pertussis infection is due to multiple virulence factors having disparate effects on different cell types. We focused our investigation on the ability of B. pertussis to modulate host immunity, in particular on the role played by adenylate cyclase toxin (CyaA), an important virulence factor of B. pertussis. As a tool, we used human monocyte derived dendritic cells (MDDC), an ex vivo model useful for the evaluation of the regulatory potential of DC on T cell immune responses. The work compared MDDC functions after encounter with wild-type B. pertussis (BpWT) or a mutant lacking CyaA (BpCyaA-), or the BpCyaA- strain supplemented with either the fully functional CyaA or a derivative, CyaA*, lacking adenylate cyclase activity. As a first step, MDDC maturation, cytokine production, and modulation of T helper cell polarization were evaluated. As a second step, engagement of Toll-like receptors (TLR) 2 and TLR4 by B. pertussis and the signaling events connected to this were analyzed. These approaches allowed us to demonstrate that CyaA expressed by B. pertussis strongly interferes with DC functions, by reducing the expression of phenotypic markers and immunomodulatory cytokines, and blocking IL-12p70 production. B. pertussis-treated MDDC promoted a mixed Th1/Th17 polarization, and the activity of CyaA altered the Th1/Th17 balance, enhancing Th17 and limiting Th1 expansion. We also demonstrated that Th1 effectors are induced by B. pertussis-MDDC in the absence of IL-12p70 through an ERK1/2 dependent mechanism, and that p38 MAPK is essential for MDDC-driven Th17 expansion. The data suggest that CyaA mediates an escape strategy for the bacterium, since it reduces Th1 immunity and increases Th17 responses thought to be responsible, when the response is exacerbated, for enhanced lung inflammation and injury

TLR4 and TLR2 activation.

<p>Triggering of TLR4 and TLR2 in transfected HEK293 cells. HEK293/TLR4/p-Nifty2-SEAP and HEK293/TLR2/p-Nifty2-SEAP cells were either untreated (none) or treated with BpWT or BpCyaA− for 16 h. Positive control (PC) for TLR4 stimulation was <i>E. coli</i> LPS (0.1 µg/ml) and positive control for TLR2 stimulation was Pam2CSK4 (0.1 µg/ml). SEAP activity in supernatants of cell cultures was measured. Data are reported as fold increase of SEAP activity over untreated values. Mean expression ± SE of ten independent experiments is indicated. * <i>p</i><0.05 <i>vs</i> none; ° <i>p</i><0.05 <i>vs</i> PC.</p

MDDC phenotypical modulation by CyaA toxin after treatment with <i>B. pertussis</i> strains^a.

a<p>MDDC were treated with indicated stimuli for 48 h and analyzed for indicated surface markers associated with the mature phenotype. Isotype-matched antibodies were used as negative controls and the values are shown in brackets.</p>b<p>Mean value ± SE of 23 experiments is indicated.</p>c<p>p<0.05 <i>vs</i> none;</p>d<p>p<0.05 <i>vs</i> BpWT;</p>e<p>p<0.05 <i>vs</i> BpCyaA−+CyaA.</p

Cytokine production.

<p>MDDC were either untreated (none) or treated with BpWT or BpCyaA−, the latter in the presence or absence of CyaA and CyaA* (50 ng/ml ) for 48 h. IL-12p70, IL-23, IL-1β, IL-6 and IL-10 were assessed by ELISA. Values are expressed as mean ± SE from nineteen (IL-12p70), eleven (IL-10), six (IL-23, IL-1β and IL-6) independent experiments performed with MDDC obtained from different donors, and expressed as pg/ml of cytokine released. * <i>p</i><0.05 <i>vs</i> none; °<i>p</i><0.05 <i>vs</i> BpWT; ^‡<i>p</i><0.05 <i>vs</i> BpCyaA−; ^§<i>p</i><0.05 <i>vs</i> BpCyaA−+CyaA*.</p

Analysis of MyD88 -dependent pathway induction.

<p><b>A</b> MDDC were either untreated or treated with BpWT or BpCyaA−. Phosphorylation of p38, ERK1/2, SAP/JNK and IκB-α was determined at the indicated time-points by Western blot. A single gel was run and blotted to detect phosphorylated proteins and β tubulin to normalize the results. Data are from one representative out of four independent experiments performed with MDDC obtained from different donors. <b>B</b> MDDC were treated as in panel A, either in the absence or presence of p38 inhibitor (SB203580), ERK1/2 inhibitor (PD98059) or PI3K inhibitor (LY294002) for 48 h. Results of seven independent experiments performed with MDDC obtained from different donors are expressed as the percent of change of maturation markers (CD80, CD83, CD38) with respect to the corresponding stimulus in the absence of inhibitors. Mean ± SE of marker expression in MDDC not treated with inhibitors was for BpWT: CD80 (MFI) = 48±6; CD83 (%) = 23±6; CD38 (MFI) = 18±4; for BpCyaA−: CD80 (MFI) = 72±11; CD83 (%) = 36±7; CD38 (MFI) = 46±5. * <i>p</i><0.05 <i>vs</i> control, calculated from the raw data. <b>C</b> MDDC were treated as in panels A and B. Results of ten (IL-12p70), eight (IL-23 and IL-10) and 5 (IL-1β) independent experiments performed with MDDC obtained from different donors, measured by ELISA, are expressed as the percent of change of cytokines with respect to the corresponding stimulus in the absence of inhibitors. Mean ± SE of cytokine production (pg/ml) in MDDC not treated with inhibitors was for BpWT: IL-12p70 = 0±0; IL-23 = 310±70; IL-1β  = 330±85; IL-10 = 2034±605; for BpCyaA−: IL-12p70 = 374±43; IL-23 = 672±140; IL-1β = 600±87; IL-10 = 2477±593. * <i>p</i><0.05 <i>vs</i> control, calculated from the raw data.</p

Effects of kinase inhibition on Th polarization.

<p><b>A</b> MDDC, either untreated or treated with BpWT or BpCyaA− in the absence or presence of p38 inhibitor (SB203580), ERK1/2 inhibitor (PD98059) or PI3K inhibitor (LY294002) for 48 h, were co-cultured with purified T cells. On day 12, supernatants were collected and secreted cytokines were measured by ELISA. Results of four independent experiments performed with MDDC and T cells obtained from different donors are expressed as the percent of change of cytokines (IFNγ, IL-17 and IL-5) with respect to the corresponding stimulus in the absence of inhibitors. Mean ± SE of cytokine production (pg/ml) in MDDC not treated with inhibitors was for BpWT: IFNγ = 9013±1828; IL-17 = 900±68; IL-5 = 71±19; for BpCyaA−: IFNγ = 9834±1225; IL-17 = 423±94; IL-5 = 21±5. * <i>p</i><0.05 <i>vs</i> control, calculated from the raw data.</p

Analysis of MyD88-independent pathway induction.

<p>MDDC were either untreated (none) or treated with BpWT or BpCyaA−. Phosphorylation of IRF3 was determined at the indicated time-points by Western blot. Data are from one representative out of three independent experiments performed with MDDC obtained from different donors.</p