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. 1998 Aug;66(8):3825-31.
doi: 10.1128/IAI.66.8.3825-3831.1998.

Activation of the complement classical pathway (C1q binding) by mesophilic Aeromonas hydrophila outer membrane protein

S Merino  1 , M M Nogueras, A Aguilar, X Rubires, S Albertí, V J Benedí, J M Tomás
Affiliations

Activation of the complement classical pathway (C1q binding) by mesophilic Aeromonas hydrophila outer membrane protein

S Merino et al. Infect Immun. 1998 Aug.

Abstract

The mechanism of killing of Aeromonas hydrophila serum-sensitive strains in nonimmune serum by the complement classical pathway has been studied. The bacterial cell surface component that binds C1q more efficiently was identified as a major outer membrane protein of 39 kDa, presumably the porin II described by D. Jeanteur, N. Gletsu, F. Pattus, and J. T. Buckley (Mol. Microbiol. 6:3355-3363, 1992), of these microorganisms. We have demonstrated that the purified form of porin II binds C1q and activates the classical pathway in an antibody-independent manner, with the subsequent consumption of C4 and reduction of the serum total hemolytic activity. Activation of the classical pathway has been observed in human nonimmune serum and agammaglobulinemic serum (both depleted of factor D). Binding of C1q to other components of the bacterial outer membrane, in particular to rough lipopolysaccharide, could not be demonstrated. Activation of the classical pathway by this lipopolysaccharide was also much less efficient than activation by the outer membrane protein. The strains possessing O-antigen lipopolysaccharide bind less C1q than the serum-sensitive strains, because the outer membrane protein is less accessible, and are resistant to complement-mediated killing. Finally, a similar or identical outer membrane protein (presumably porin II) that binds C1q was shown to be present in strains from the most common mesophilic Aeromonas O serogroups.

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Figures

FIG. 1
FIG. 1
Survival of A. hydrophila AH-53 (serum sensitive [20]) in nonimmune serum (•), heat-inactivated (30 min, 56°C) nonimmune serum (○), C1q-deficient serum (□), factor B-deficient serum (▵), and C1q-deficient serum reconstituted with purified C1q (■). The results are the averages of at least three independent experiments (values are means ± standard deviations).
FIG. 2
FIG. 2
Binding of 125I-labeled C1q to A. hydrophila whole cells as described in Materials and Methods of wild-type strains AH-3 (□) and TF7 (○) (serum resistant [20, 22]) and their LPS mutants AH-53 (■) and AH-26 (•) (serum sensitive [20, 22]), respectively. The results are the averages of at least three independent experiments (values are means ± standard deviations). (Insert) Binding of biotinylated C1q to A. hydrophila whole cells as described in Materials and Methods. Cells were incubated with biotinylated C1q and avidin-colloidal gold spheres (A and C) or with avidin-colloidal gold spheres alone (B). Cells in panels A and B correspond to the A. hydrophila LPS mutant AH-26 (serum sensitive [22]), and that in panel C corresponds to wild-type strain TF7 (serum resistant [22]). Bars, 0.4 μm.
FIG. 3
FIG. 3
Isolation of C1q-binding OMP from A. hydrophila AH-53 (serum sensitive [20]). (A) SDS-PAGE and Coomassie blue staining. Lanes: 0, molecular mass standards (in kilodaltons); 1, OMPs of wild-type strain AH-3 (O:34); 2, OMPs of strain AH-53; 3, the purified C1q-binding OMP (probably porin II) boiled in sample buffer for 10 min; 4, as in lane 3 without boiling. (B) Western blot analysis with biotinylated C1q of OMPs and LPS as described in Materials and Methods. Lanes: 0, molecular mass standards (20, 31, 43, 62, and 97 kDa, respectively); 1, 2, and 3, as in panel A; 4, 25 μg of purified BSA; 5 and 6, purified LPS from strains AH-53 and AH-26, respectively. (C) SDS-PAGE and silver staining of purified LPS from different strains (20, 22). Lanes: 1, AH-3 (O:34); 2, AH-53; 3, TF7 (O:11); 4, AH-26.
FIG. 4
FIG. 4
Dot blot analysis with purified C1q labeled with biotin. Purified 39-kDa OMP (probably OM porin II) (row A) and rough LPS from strain AH-53 (row B) and BSA (row C) were dot blotted starting at 0.5 μg (row A), 2 μg (row B), and 1 μg (row C) (column 1) and then with twofold dilutions (columns 2 through 7). Binding of biotinylated C1q was detected with alkaline phosphatase-labeled avidin.
FIG. 5
FIG. 5
Activation of the CPC by the purified 39-kDa OMP (probably porin II). (A) NHS depleted of C1q and factor D and reconstituted with purified C1q was incubated with different amounts (0 to 15 μg) of the porin. After 45 min at 37°C, the remaining C4 (white bars) and CH50 (cross-hatched bars) hemolytic activities were determined as described in Materials and Methods. (B) NHS, NHS depleted of C1q and factor D and reconstituted with purified C1q (RQD+Q), and agammaglobulinemic (Agamma) serum were incubated for 45 min at 37°C with 15 μg of the porin. After incubation, the remaining C4 (white bars) and CH50 (cross-hatched bars) hemolytic activities were assayed as described above for panel A. All the results are the averages of at least three independent experiments (values are means ± standard deviations).
FIG. 6
FIG. 6
Antibody-independent activation of the CPC by OM components. An agammaglobulinemic serum depleted of C1q and factor D and reconstituted with purified C1q was incubated with different amounts of the purified 39-kDa OMP (probably porin II) (•) or purified LPS from serum-sensitive strains AH-26 (■) and AH-53 (▴). After 45 min at 37°C, the remaining CH50 was determined as described in Materials and Methods. The results are the averages of at least three independent experiments (values are means ± standard deviations).
FIG. 7
FIG. 7
OMPs obtained as described in Materials and Methods from different clinical mesophilic Aeromonas strains of O serogroups 2, 3, 6, 11, 12, 14, 16, 17, 18, 23, 29, 33, 34, and 43 (the most common around the world). (A) OMP profiles obtained by SDS-PAGE and Coomassie blue staining. Lanes: 0, molecular mass standards (14, 21, 30, 41, 66, and 92 kDa); 1, serogroup O:2; 2, serogroup O:3; 3, serogroup O:6; 4, serogroup O:11; 5, serogroup O:12; 6, serogroup O:14; 7, serogroup O:16; 8, serogroup O:17; 9, serogroup O:18; 10, serogroup O:23; 11, serogroup O:29; 12, serogroup O:33; 13, serogroup O:34; 14, serogroup O:43. (B) Western blot analysis of the same OMPs incubated with biotinylated C1q as described in Materials and Methods. Lanes 1 to 14 as in panel A.
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