Probing of Actinobacillus pleuropneumoniae ApxIIIA toxin-dependent cytotoxicity towards mammalian peripheral blood mononucleated cells

Abstract

Background: Actinobacillus pleuropneumoniae, the causative bacterial agent of porcine pleuropneumonia, produces Apx toxins which belong to RTX toxin family and are recognized as the major virulence factors. So far, their target receptor(s) has not been identified and the disease cytopathogenesis remains poorly understood. Production of an active Apx toxin and characterization of its toxic activity constitute the premises necessary to the description of its interaction with a potential receptor. From this point of view, we produced an active recombinant ApxIIIA toxin in order to characterize its toxicity on peripheral blood mononucleated cells (PBMCs) isolated from several species.

Findings: Toxin preparation exercises a strong cytotoxic action on porcine PBMCs which is directly related to recombinant ApxIIIA since preincubation with polymyxin B does not modify the cytotoxicity rate while preincubation with a monospecific polyclonal antiserum directed against ApxIIIA does. The cell death process triggered by ApxIIIA is extremely fast, the maximum rate of toxicity being already reached after 20 minutes of incubation. Moreover, ApxIIIA cytotoxicity is species-specific because llama, human, dog, rat and mouse PBMCs are resistant. Interestingly, bovine and caprine PBMCs are slightly sensitive to ApxIIIA toxin too. Finally, ApxIIIA cytotoxicity is cell type-specific as porcine epithelial cells are resistant.

Conclusion: We have produced an active recombinant ApxIIIA toxin and characterized its specific cytotoxicity on porcine PBMCs which will allow us to get new insights on porcine pleuropneumonia pathogenesis in the future.

Figure 1

A. Spreading of porcine PBMCs elements in SSC/FSC dot plot after rApxIIIA addition. P₁ (red) and P₂ (blue) populations represent mock- (RPMI-1640) and rApxIIIA-exposed porcine PBMCs respectively. X-axis and Y-axis represent FSC and SSC values respectively. B. Surface expression of LFA-1 by mock- (P₁, red) and rApxIIIA (P₂, blue) exposed porcine PBMCs. Surface labeling was made with anti-PoCD18 mAb MCA1972. Labeling with an isotype-matched nonpertinent murine mAb was used as negative control (black). X-axis shows fluorescence intensity (Alexa 488) and the Y-axis represents cell count. The percentage of Alexa 488-positive cells is indicated within the panels. C. Spreading of porcine PBMCs elements in PI/FSC dot plot after rApxIIIA addition. P₁ (red) and P₂ (blue) populations represent mock- and rApxIIIA-exposed porcine PBMCs respectively. Two subpopulations can be observed for P₂: the first shows an intermediate labeling (P₂A) while the second (P₂B) is very strongly tagged, corresponding to intermediate and final stages of cell death respectively. The bar represents the positivity threshold. X-axis and Y-axis represent FSC and PI-fluorescence values. D. Distribution of PI labeling among mock- (red) and rApxIIIA-exposed (blue) porcine PBMCs. The P₂A and P₂B subpopulations are readily detected. X-axis shows fluorescence intensity (PI) and the Y-axis represents cell count. Percentage of PI-positive cells is indicated within the panels.

Figure 2

Kinetics of porcine PBMCs death upon exposition to rApxIIIA crude toxin preparation. Kinetics of the cytotoxic action was characterized by incubating porcine PBMCs with 0.31 μg rApxIIIA crude toxin preparation and stopping the exposure after 1, 10, 20, 30, 45, 60, 90 and 120 minutes. Horizontal axis, duration of exposition; vertical axis, fraction of PI-positive PBMCs. Basal cell death rate was measured from mock-exposed (RPMI-1640) PBMCs for comparison. Values are means ± SDs from three representative experiments.

Figure 3

Dose-dependent cytotoxicity of rApxIIIA on porcine PBMCs. The fraction of PI-positive cells was measured one hour after exposition to serial dilutions of the crude toxin preparation. Values are means ± SDs from three representative experiments. C-, mock-exposed (RPMI-1640) porcine PBMCs incubated for one hour (negative control); C+, paraformaldehyde-exposed PBMCs (positive control).

Figure 4

A. Effect of polymyxin B incorporation on rApxIIIA-containing crude toxin preparation cytotoxicity towards porcine PBMCs. The fraction of PI-positive cells was measured by flow cytometry after a two-hour exposition (i) to 50 μl RPMI-1640 (C-), (ii) to 50 μg/ml or 100 μg/ml polymyxin B alone (Pol50 and Pol100 respectively), (iii) to 50 μg/ml or 100 μg/ml polymyxin B supplemented with 5 μg rApxIIIA during the second hour (Pol50/Apx and Pol100/Apx), or after a one-hour exposition either (iii) to 5 μg rApxIIIA alone (Apx) or (iv) to paraformaldehyde 10% (C+). Values are means ± SDs from three representative experiments. Asterisks (1): PI-positive cell densities significantly higher than that recorded in C-, Pol50 and Pol100 sets of experiments (P < 0.0001). PI-positive cell densities retrieved from Pol50/Apx, Pol100/Apx and rApxIIIA groups were not statistically different from each other (P > 0.5). B. Effect of anti-ApxIIIA antibodies incorporation on rApxIIIA-containing crude toxin preparation cytotoxicity towards porcine PBMCs. The fraction of PI-positive cells was measured by flow cytometry after a one-hour exposition (i) to 50 μl RPMI-1640 (C-), (ii) to a monospecific anti-ApxIIIA polyclonal antiserum diluted 1/1,000 (Ab-Apx), (iii) to a mix of rApxIIIA (0.31 or 0.16 μg) and the anti-ApxIIIA polyclonal antiserum diluted 1/1,000 (Ab-Apx + Apx), and (iv) to rApxIIIA alone (Apx) (0.31 or 0.16 μg). Values are means ± SDs from three representative experiments. Asteriks (2): positive cell densities significantly higher than that recorded in Ab-Apx + Apx sets of experiments (P < 0.01).

Figure 5

Cytotoxicity of rApxIIIA-containing crude toxin preparation towards PBMCs from a set of mammalian species. The fraction of PI-positive cells was measured by flow cytometry after a one-hour exposition to 50 μl RPMI-1640 (mock), 5 μg rApxIIIA or PAF 10%. Values are means ± SDs from three representative experiments. Asterisks: species-specific values significantly different from corresponding mock-exposed value (P < 0.05). Po, Sus scrofa domesticus Piétrain; Wb (wild boar), Sus scrofa scrofa; Bo, Bos taurus; Cap, Capra hircus; Llama, Lama pacos; Hu, Homo sapiens; Dog, Canis familiaris; Mu, Mus musculus; Rat, Rattus norvegicus.