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.