Functional Analysis of Antigen 43 in UropathogenicEscherichia coliReveals a Role in Long-Term Persistence in the Urinary Tract

Abstract

Escherichia coli is the primary cause of urinary tract infection (UTI) in the developed world. The major factors associated with the virulence of uropathogenic E. coli (UPEC) are fimbrial adhesins, which mediate specific attachment to host receptors and trigger innate host responses. Another group of adhesins is represented by the autotransporter subgroup of proteins. The best characterized of these proteins, antigen 43 (Ag43), is a self-recognizing adhesin that is associated with cell aggregation and biofilm formation in E. coli K-12. The sequenced genome of prototype UPEC strain CFT073 contains two variant Ag43-encoding genes located on pathogenicity islands. The biological significance of both of these genes and their role in UPEC pathogenesis have not been investigated previously. Here we performed a detailed molecular characterization analysis of Ag43a (c3655) and Ag43b (c1273) from UPEC CFT073. Expression of Ag43a and Ag43b in a K-12 background revealed that they possess different functional properties. Ag43a produced a strong aggregation phenotype and promoted significant biofilm growth. Deletion mutants and strains constitutively expressing Ag43a and Ag43b were also constructed using CFT073. When these mutants were analyzed in a mouse model of UTI, Ag43a (but not Ag43b) promoted long-term persistence in the urinary bladder. Our findings demonstrate that Ag43a contributes to UPEC disease pathogenesis and reveal that there are pathogenicity-adapted variants of Ag43 with distinct virulence-related functions.

The antigen 43 structure reveals a molecular Velcro-like mechanism of autotransporter-mediated bacterial clumping

“…S5). Despite their high sequence similarity, these proteins mediate different degrees of cell aggregation; whereas Ag43a promotes strong aggregation, the aggregation mediated by Ag43b is less pronounced (10). Given the similarity between these two proteins, they are likely to present a very similar overall structure.…”

“…UPEC strain CFT073 encodes two nonidentical copies of Ag43, Ag43a (c3655) and Ag43b (c1273) (10). These proteins share 90% sequence identity overall, with their predicted α-domains sharing 85% sequence identity (SI Appendix, Fig.…”

“…Sequence variation within the α 43 domain of Ag43 paralogues is associated with different functional properties. For example, the reference UPEC strain CFT073 contains two Ag43 variants (Ag43a and Ag43b) that share 85% sequence identity in the functional α-domain; only Ag43a mediates strong aggregation and biofilm formation and long-term colonization of the mouse urinary tract (10). Ag43 from UPEC strain UTI89 is also associated with the formation of Significance Many persistent and chronic bacterial infections are associated with the formation of large cell aggregates and biofilms that are difficult to treat.…”

“…Ag43 is found in most E. coli pathotypes, including uropathogenic E. coli (UPEC) (10). Many UPEC strains also possess multiple copies of the Ag43-encoding agn43 (or flu) gene (10,11).…”

“…Many UPEC strains also possess multiple copies of the Ag43-encoding agn43 (or flu) gene (10,11). Well-characterized functions of Ag43 include cell aggregation and biofilm formation (12)(13)(14).…”