Urinary tract infections (UTIs) caused by uropathogenic Escherichia coli (UPEC) are amongst the most common bacterial infectious diseases in the developed world. The urovirulence of UPEC is mainly associated with the surface-exposed fimbrial adhesins and adhesins of the autotransporter (AT) family. The best studied of these proteins is antigen Ag43 mediating cell aggregation, adhesion and biofilm development as the causes of chronic UTIs. The E. coli IH11128 Dr + (dra + ) strain of the Dr/Afa + family of adhesins possesses two major surfaceexposed virulence factors: Dr fimbrial polyadhesin and DraD protein (fimbrial tip subunit or protein component of the adhesive sheath). Here, we identified for the first time, to our knowledge, the agn43 gene encoding Ag43 in the WT clinical isolate of UPEC Dr + as a new virulence factor not yet tested. We also found that Dr fimbrial expression, which like Ag43 is under the control of a phase-variable mechanism, did not exclude Ag43 surface presentation. However, the presence of Dr fimbriae supported by other structures on the cell surface caused a physical neutralization of Ag43-mediated autoaggregation during in vitro growth. The fimbrial bundling further increased the distance between the adjacent Ag43 + cells, thus preventing head-to-tail association between surface-exposed Ag43 subunits and their interactions with the host cells. The investigations showed that Ag43 did not act as a specific adhesin and invasin, conversely to the major virulence factors of E. coli Dr + , but played significant roles in the viability and metabolic activity of bacterial cells forming biofilm, and in the survival of bacteria within invaded epithelial cells.