Role of YadA, Ail, and Lipopolysaccharide in Serum Resistance of Yersinia enterocolitica Serotype O:3

Abstract

Complement attack is a host strategy leading to elimination of pathogens. Yersinia enterocolitica expresses several potential complement resistance factors: the outer membrane proteins YadA and Ail as well as lipopolysaccharide (LPS). To study the contribution of these factors to the survival of Y. enterocolitica serotype O:3 in nonimmune human serum, we constructed 23 mutant strains of Y. enterocolitica O:3 expressing different combinations of YadA, Ail, LPS O antigen, and LPS outer core. Survival of bacteria was analyzed in normal serum (with functional classical, lectin, and alternative complement activation pathways) and EGTA-Mg-treated serum (only alternative pathway functional). Kinetic killing tests revealed that the most potent single-serum resistance factor needed for long-term survival was YadA; Ail was also indispensable, but it provided short-term survival and delayed the bacterial killing. On the contrary, the LPS O antigen and outer core, when in combination with YadA, Ail, or both, had a minor and often negative effect on serum resistance. Bacteria in the exponential phase of growth were more resistant to serum killing than stationary-phase bacteria. After exposing bacteria to EGTA-Mg-treated serum, O antigen could prevent deposition of covalently bound C3b on bacteria at 3 min of incubation, even as a single factor. At later time points (15 and 30 min) it had to be accompanied by YadA, Ail, and outer core. In normal serum, the bacteria were less resistant to C3b deposition. However, no direct correlation between the C3 deposition pattern and bacterial resistance was observed.

FIG. 1.

Detection and analysis of YadA, O-ag, OC, and Ail expression in different Y. enterocolitica O:3 strains. (A) Immunoblot analysis using monoclonal antibodies specific for YadA (top), O-ag (middle), and OC (bottom). (B) Silver-stained SDS-PAGE gel showing outer membrane proteins isolated from Ail-expressing and -nonexpressing Y. enterocolitica O:3 and E. coli C600 strains. The arrows point to the migration level of the Ail band. The presence or absence of the factors is indicated below the panels.

FIG. 2.

Survival percentages of YadA-positive Y. enterocolitica O:3 strains (grown to stationary phase at 37°C) in normal and EGTA-Mg-treated sera after 30 or 120 min of incubation. Survival in HIS was set to 100%. In each panel, the left-hand columns show the 30- and 120-min survival percentages in EGTA-Mg-serum, and the right-hand columns show the 30- and 120-min survival percentages in NHS. Standard deviations are indicated by bars. The construction lines of the strains are indicated by arrows between the panels. The inactivated factor in each construction step is indicated by the branching curved arrow, and the manner of this inactivation is marked with the following symbols: ^, KmGB insertion; *, knock out (for example, OC*, OC gene cluster knockout; YadA*, pYV-cured strain). No symbol was ascribed to LPS factors (O-ag and OC) inactivated by spontaneous mutations and isolated using bacteriophage or enterocoliticin selection.

FIG. 3.

Survival percentages of YadA-negative Y. enterocolitica O:3 strains grown at 37°C in normal and in EGTA-Mg-treated sera after 30 or 120 min of incubation. See the legend to Fig. 2 for further explanations.

FIG. 4.

Effect of growth phase on the survival of the wt strain YeO3 in normal and in EGTA-Mg treated sera. Y. enterocolitica O:3 bacteria, grown at RT and at 37°C to stationary or to exponential phase, were exposed to sera for 0.5 and 2 h.

FIG. 5.

Effect of growth phase on the survival of strains YeO3-c-OCR (Ail positive) and YeO3-Ail-OCR (YadA positive) grown at 37°C and incubated in normal and in EGTA-Mg-treated sera.

FIG. 6.

C3 deposition and degradation on different Y. enterocolitica O:3 mutant bacteria. Bacteria were incubated in EGTA-Mg-treated serum and NHS for 3, 15, and 30 min. After being washed, C3 and its derivatives deposited on bacteria were identified by immunoblotting using monoclonal antibody specific for the α-chain of C3. Shown in frames are the depositions of the C3 derivatives on different types of bacteria at different time points. The bacteria were grown at 37°C unless otherwise indicated. For each bacterial sample, its phenotype with respect to expression of YadA, O-ag, Ail, and OC is given below the strain names. For each bacterial sample, C3 deposition in EGTA-Mg-treated serum (a) and in NHS (c) is indicated at the bottom of the figure.