Pet serine protease from enteroaggregative Escherichia coli stimulates the inflammatory response activating human macrophages

Abstract

Background: Pet is a toxin from the family of Serine Protease Autotransporters of Enterobacteriaceae which was initially identified in Enteroaggregative Escherichia coli strains. This protease exhibits enterotoxin properties, damages the cell cytoskeleton and induces intestinal epithelium alterations, which are associated with a severe inflammatory process. An in-vitro study was conducted to evaluate the effect of Pet on the migration of human peripheral blood monocytes-derived macrophages and its participation in the activation of the early inflammatory response and cytokine expression.

Results: In the macrophage migration activation assay, Pet produced a similar effect to that induced by opsonized zymosan (ZAS). Regarding the cytokine expression, an increase of IL-8, TNF-α (pro-inflammatory) and IL-10 (anti-inflammatory) was identified. In addition to the above results, the nuclear translocation of NF-kB pp65 was also identified. These events are probably related to the inflammatory response identified in the histological examination of intestine rat samples inoculated with Pet during a ligated loop assay.

Conclusion: The results showed that Pet participates as an immunostimulant molecule for macrophages, which activates both their mobility and cytokine expression. These observations suggest that the toxin participates in the inflammatory process that is observed during the host infection by EAEC Pet producing.

Keywords: Cytokines; Escherichia coli; Innate immune response; Serin protease.

Fig. 1

Purification of Pet protein by FPLC. Pet protein was purified from supernatant of the minimal clone HB101(pCEFN1).a Culture supernatant ammonium sulfateprecipitated was passed through a Q-Sepharose and FPLC columns. b The toxin specificity was analyzed by Western blot with anti-pet antibody (1:500) and the reaction visualized with rabbit anti-IgG antibody conjugated with alkaline phosphatase (1:5000). The reaction was evidenced adding BCIP/NBT. Lines: MW markers; 1 Pet toxin; 2 Entamoeba histolytica HMI-IMSS antigen

Fig. 2

Histological Study of fractions obtained from rat ileal loops. Rat ileal loops inoculated with: a live O42EAEC bacteria (1.5 × 10⁸ UFC/mL); b 100 μg of purified Pet toxin and c PBS. The intestine preparations were Hematoxilin-Eosin stained and observed in light microscope (400X). Presence of hemorrhage (arrow), necrosis (arrow), ulceration and fibrinopurulent exudates (arrow) of the intestinal epithelium a and b. Non histological changes were observed in c

Fig. 3

Cytokines production of MDM stimulated with Pet toxin from EAEC. Supernatants of MDM stimulated with Pet (1 to 20 μg/ml) were analyzed by ELISA test to determine the cytokines expression. a IL-8, b TNF-α and c IL-10. The MDM cells were treated with E. coli. 0111:B4 LPS (0.1 μg/ml); Polymyxin B (PMB), LPS and Polymyxin B (LPS/PMB), Pet (20 μg/ml) and Polymyxin B (Pet20/PMB), and untreated cells (s/e). The cytokine concentrations weredeterminedat 6(□) and 24 h (■) of stimulation. The results are the means ± of the standard deviation representative of four independent experiments. Significant P < 0.05 (*), as compared between 6 and 24 h of stimulation

Fig. 4

Immunofluorescence analysis of NF-kB activation. a Human macrophages were stimulated during 30 min with Pet (10 μg/ml) or LPS (Positive control) and non-stimulated. The nuclear translocation is determined (arrows) by the blue fluorescence (Hoechst staining) and the NF-kB p65 subunit (arrows) by its green fluorescence (Dylight 488). b Percentage of nucleic positive cells to p65. The results are representative of three independent experiments