Identification of a domain in Yersinia virulence factor YadA that is crucial for extracellular matrix-specific cell adhesion and uptake

Abstract

For many pathogens, cell adhesion factors are critical virulence determinants. Enteropathogenic Yersinia species express the afimbrial adhesin YadA, the prototype of a class of homotrimeric outer membrane adhesins, which mediates adherence to host cells by binding to extracellular matrix components. In this study, we demonstrate that different pathogenic functions are attributable to highly homologous YadA proteins. YadA of Yersinia pseudotuberculosis (YadA(pstb)) and Yersinia enterocolitica (YadA(ent)) exhibit fundamental differences in their specificity of extracellular matrix substrate binding, they cause dissimilar bacterial aggregation behaviors, and YadA(pstb), but not YadA(ent), promotes efficient uptake into human cells. Evidence is presented here that a unique N-terminal amino acid sequence of YadA(pstb), which is absent in YadA(ent), acts as an "uptake domain" by mediating tight binding to fibronectin bound on alpha(5)beta(1) integrin receptors, which are crucial for initiating the entry process. Deleting this motif in YadA(pstb) generated all features of the YadA(ent) protein, i.e., the molecule lost its adhesiveness to fibronectin and its invasiveness, but gained adhesion potential to collagen and laminin. Loss of the "uptake region" also attenuated host tissue colonization by Y. pseudotuberculosis during oral infections of mice, demonstrating that this motif plays a crucial role in defining pathogen-host cell interaction and pathogenesis. We conclude that even small variations in adhesion factors can provoke major differences in the virulence properties of related pathogens.

Fig. 1.

Outer membrane incorporation, adhesion, and invasion properties of the YadA proteins. (A) Immunoblot analysis demonstrates that comparable amounts of multimeric YadA derivatives (180–200 kDa) are expressed in the outer membrane of Yersinia strain YP31. V, empty vector. Cell adhesion (B) and cell invasion (C) of YadA-expressing E. coli and Y. pseudotuberculosis YP31 recombinant strains are documented. Approximately 10⁶ bacteria were used to challenge 5 × 10⁴ HEp-2 cells. Total numbers of adherent and intracellular bacteria are expressed relative to the adhesion and invasion rate of E. coli and Y. pseudotuberculosis YP31 harboring yadA_pstb, defined as 1.0. Each value represents the mean of at least three independent assays done in triplicate. Data were analyzed by the Student t test. ∗, significantly different from YadA_pstb-expressing bacteria (P < 0.05).

Fig. 2.

clustalw alignment of the head domain of YadA of Y. enterocolitica Serotype O:3 (YadA_ent O:3), O:9 (YadA_ent O:9) and O:8 (YadA_ent O:8), and Y. pseudotuberculosis Type III (YadA_pstb). Asterisks indicate amino acid identity, and dots indicate amino acid similarity. The additional sequence in YadA of Y. pseudotuberculosis, deleted in the YadA_pstbΔ53–83 protein, is indicated with two adjacent black boxes, which also illustrate the portions of the smaller deletions in YadA_pstbΔ53–67 and YadA_pstbΔ68–83. Exchanged amino acids A58E, K59A in the head domain of the YadA_pstb mutant derivatives are indicated by arrows.

Fig. 3.

Different binding properties of YadA-expressing E. coli and Y. pseudotuberculosis YP31 strains to ECM molecules. Plastic wells were coated with collagen type I (A) or laminin (B). Bound bacteria were stained, and absorbance was measured at 595 nm. Expression of YadA_pstb results in a moderate interaction, whereas bacteria expressing YadA_ent and YadA_pstbΔ53–83 exhibit a strong binding capacity to collagen and laminin. Data represent the mean ± SD of three independent experiments done in duplicate. For quantitative analysis of adherence to fibronectin (C), glass coverslips were coated with cellular fibronectin and bound bacteria were counted in a defined area of a microscopic field of ≈4 × 10³ μm². The means and SD of 30 calculated fields are shown. Data were analyzed by the Student t test. ∗, significantly different from YadA_pstb-expressing bacteria (P < 0.05).

Fig. 4.

Inhibition of YadA-promoted invasion by blockage of fibronectin and the fibronectin-binding class of β₁ integrins. HEp-2 cells were blocked with different antibodies directed against ECM molecules (Col, collagen; Lm, Laminin; Fn, fibronectin), GRGDSP/GRGESP peptides, and with antibodies to different α-subunits of β₁ integrins before infection with YadA-expressing bacteria. Approximately 10⁶ bacteria were used to challenge 5 × 10⁴ cells, which were incubated for 1 h at 37°C to determine cell uptake efficiency. Only blockage of fibronectin and α₅β₁ integrins reduced YadA uptake. The proportionate reduction in invasion is expressed relative to the invasion rate promoted by the YadA_pstb WT protein, defined as 1.0. Each value represents the mean of three independent assays done in triplicate. Data were analyzed by the Student t test. ∗, P < 0.05 relative to YadA_pstb without antibodies/peptides (−).