Molecular and virulence characteristics of an outer membrane-associated RTX exoprotein in Pasteurella pneumotropica

Abstract

Background: Pasteurella pneumotropica is a ubiquitous bacterium that is frequently isolated from laboratory rodents and causes various clinical symptoms in immunodeficient animals. Currently two RTX toxins, PnxIA and PnxIIA, which are similar to hemolysin-like high-molecular-weight exoproteins are known in this species. In this study, we identified and analyzed a further RTX toxin named PnxIIIA and the corresponding type I secretion system.

Results: The RTX exoprotein, PnxIIIA, contains only a few copies of the RTX repeat-like sequence and 3 large repeat sequences that are partially similar to the outer membrane protein found in several prokaryotes. Recombinant PnxIIIA protein (rPnxIIIA) was cytotoxic toward J774A.1 mouse macrophage cells, whereas cytotoxicity was attenuated by the addition of anti-CD11a monoclonal antibody. rPnxIIIA could bind to extracellular matrices (ECMs) and cause hemagglutination of sheep erythrocytes. Binding was dependent on the 3 large repeat sequences in PnxIIIA. Protein interaction analyses indicated that PnxIIIA is mainly localized in the outer membrane of P. pneumotropica ATCC 35149 in a self-assembled oligomeric form. PnxIIIA is less cytotoxic to J774A.1 cells than PnxIA and PnxIIA.

Conclusions: The results implicate that PnxIIIA is located on the cell surface and participates in adhesion to ECMs and enhanced hemagglutination in the rodent pathogen P. pneumotropica.

Figure 1

Genetic organization and the predicted primary structure of PnxIIIA in P. pneumotropica ATCC 35149. (A) Schematic representation of the pnxIII operon genetic map and the functions of each gene. Circles represent potential transcriptional termination loops. Predicted functions determined by the protein database are indicated below the gray boxes. (B) Schematic representation of probable domains that were identified by comparing with the HMM database. The numbers represent the regions containing a large repeat sequence. Arrowheads below the number box represent the position of sequence alignment in Additional file 1.

Figure 2

Changes in the cytotoxicity of the rPnxIIIA toward J774A.1 mouse macrophage cells. The cytotoxicity was determined by the release of LDH from J774A.1 cells with or without treatment with anti-CD11a monoclonal antibody cultured with rPnxIIIA.

Figure 3

The binding ability and hemagglutination activity of the rPnxIIIA. The binding ability of rPnxIIIA to the ECMs as determined by ELISA (A to D) and hemagglutination activity of the rPnxIIIA with sheep erythrocytes (E). Changes in the ability of different concentrations of rPnxIIIA to bind to the rat collagen type I (A), rat collagen type II (B), mouse collagen type IV (C), and mouse laminin (D), as determined by measuring the A620. Changes in the hemagglutination activity of different concentration of rPnxIIIA with sheep erythrocytes (E).

Figure 4

Localization of PnxIIIA and the protein interaction analysis of rPnxIIIA. (A) Western blotting analysis of the cell fraction prepared from P. pneumotropica ATCC 35149 cells and culture by using anti-rPnxIIIA IgG. Lanes: 1, SC fraction; 2, IM fraction; 3, OM fraction; 4, UC fraction. (B) Western blotting analysis of the cell fraction prepared from P. pneumotropica ATCC 35149 cells and culture by using anti-rPnxIIIE IgG. Lanes: 1, SC fraction; 2, IM fraction; 3, OM fraction; 4, UC fraction. (C) Western blotting analysis of rPnxIIIA by using anti-rPnxIIIA IgG after cross-linking with only rPnxIIIA or the rPnxIIIE protein and IP with anti-rPnxIIIA IgG. Lanes: 1, rPnxIIIA without cross-linking; 2, 20 μg of rPnxIIIA alone cross-linked with 50 mM BS³ for 60 min and immunoprecipitated; 3, mixture of both rPnxIIIA and rPnxIIIE proteins without cross-linking; 4, 20 μg of both rPnxIIIA and rPnxIIIE proteins cross-linked with 50 mM BS³ for 60 min and immunoprecipitated. (D) Western blotting analysis of rPnxIIIA by using anti-rPnxIIIA IgG after different treatment times with rPnxIIIA alone cross-linked with 50 mM BS³ and immunoprecipitated with anti-rPnxIIIA IgG. Lanes: 1, rPnxIIIA without cross-linking; 2, rPnxIIIA with cross-linking for 5 min; 3, rPnxIIIA with cross-linking for 10 min; 4, rPnxIIIA with cross-linking for 30 min; 5, rPnxIIIA with cross-linking for 60 min. Arrows indicate the position of the bands that appeared.

Figure 5

Transmission electron micrographs of P. pneumotropica ATCC 35149 cells by immunoelectron microscopy with anti-rPnxIIIA IgG. Transmission electron micrographs of the P. pneumotropica ATCC 35149 cells that were first reacted with anti-rPnxIIIA IgG and then labeled with gold particles (10-nm) conjugated with rabbit IgG antibody. Arrows indicate the areas where gold labeling appeared on the cell surface. Left panel, cross-section of the bacterial cell. Right panel, longitudinal section of the bacterial cell. Bar = 0.2 μm.

Figure 6

Presence of PnxIIIA, binding ability, hemagglutination activity, and cytotoxicity of reference strains of P. pneumotropica. (A) Western blotting analysis of cell lysates (5 μg of total protein) of the reference strains by using anti-rPnxIIIA IgG. (B) The binding ability of the reference strains against to the rat collagen type I. A 1-way ANOVA determined that there were significant differences between the strains (P < 0.05). The mean value of A490 of strain ATCC 35149 (numbered as 1) or CCUG 26453 (5) is significantly different from that of the other strains by determination of Duncan's multiple-range test (P < 0.05). (C) Changes in hemagglutination activity of the reference strains with sheep erythrocytes. (D) Percentage of cytotoxicity determined by LDH release from the supernatant of J774A.1 cells cultured with reference strains of P. pneumotropica. A 1-way ANOVA determined that there were significant differences between the strains (P < 0.05). The mean values of cytotoxicity (%) of strain ATCC 35149 (numbered as 1) or ATCC 12555 (2) and CCUG 36632 (6) are significantly different from that of the other strains by determination of Duncan's multiple-range test (P < 0.05). All sections of numbers are represented as follows: 1, ATCC 35149; 2, ATCC 12555; 3, CCUG 26450; 4, CCUG 26451; 5, CCUG 26453; 6, CCUG 36632.