Uropathogenic Escherichia coli (UPEC) strains are among the most prevalent causative agents of urinary tract infections. To establish infection, UPEC must overcome the bactericidal action of host antimicrobial peptides. Previously, the enterohaemorrhagic E. coli outer membrane protease, OmpT, was shown to degrade and inactivate the human antimicrobial peptide LL-37. This study aims to investigate the involvement of UPEC OmpT in LL-37 degradation. An ompT deletion mutant was generated in the prototypical UPEC strain CFT073. Western blot analysis showed that the OmpT protein level is moderate in CFT073. In agreement, OmpT was shown to partially cleave LL-37. However, no difference in the minimum inhibitory concentration of LL-37 was observed between CFT073 and the ompT mutant. Plasmid complementation of ompT, which led to increased OmpT levels, resulted in complete cleavage of LL-37 and a fourfold increase in the minimum inhibitory concentration. The analysis of other UPEC isolates showed similar OmpT activity levels as CFT073. Although UPEC OmpT can cleave LL-37, we conclude that the low level of OmpT limits its contribution to LL-37 resistance. Collectively, these data suggest that UPEC OmpT is likely accompanied by other LL-37 resistance mechanisms.

Bacterial colonization of the urogenital tract is limited by innate defenses, including the production of antimicrobial peptides (AMPs). Uropathogenic Escherichia coli (UPEC) resist AMP‐killing to cause a range of urinary tract infections (UTIs) including asymptomatic bacteriuria, cystitis, pyelonephritis, and sepsis. UPEC strains have high genomic diversity and encode numerous virulence factors that differentiate them from non‐UTI‐causing strains, including ompT. As OmpT homologs cleave and inactivate AMPs, we hypothesized that UPEC strains from patients with symptomatic UTIs have high OmpT protease activity. Therefore, we measured OmpT activity in 58 clinical E. coli isolates. While heterogeneous OmpT activities were observed, OmpT activity was significantly greater in UPEC strains isolated from patients with symptomatic infections. Unexpectedly, UPEC strains exhibiting the greatest protease activities harbored an additional ompT‐like gene called arlC (ompTp). The presence of two OmpT‐like proteases in some UPEC isolates led us to compare the substrate specificities of OmpT‐like proteases found in E. coli. While all three cleaved AMPs, cleavage efficiency varied on the basis of AMP size and secondary structure. Our findings suggest the presence of ArlC and OmpT in the same UPEC isolate may confer a fitness advantage by expanding the range of target substrates.

28Bacterial colonization of the urogenital tract is limited by innate defenses, including the 29 production of antimicrobial peptides (AMPs). Uropathogenic Escherichia coli (UPEC) resist 30 AMP-killing to cause a range of urinary tract infections (UTIs) including asymptomatic 31 bacteriuria, cystitis, pyelonephritis, and sepsis. UPEC strains have high genomic diversity and 32 encode numerous virulence factors that differentiate them from non-UTI causing strains, 33including ompT. As OmpT homologues cleave and inactivate AMPs, we hypothesized that 34 high OmpT protease activity-levels contribute to UPEC colonization during symptomatic 35UTIs. Therefore, we measured OmpT activity in 58 UPEC clinical isolates. While 36 heterogeneous OmpT activities were observed, OmpT activity was significantly greater in 37 UPEC strains isolated from patients with symptomatic infections. Unexpectedly, UPEC 38 strains exhibiting the greatest protease activities harboured an additional ompT-like gene 39 called arlC (ompTp). The presence of two OmpT-like proteases in some UPEC isolates led us 40 to compare the substrate specificities of OmpT-like proteases found in E. coli. While all three 41 cleaved AMPs, cleavage efficiency varied on the basis of AMP size and secondary structure. 42Our findings suggest the presence ArlC and OmpT in the same UPEC isolate may confer a 43 fitness advantage by expanding the range of target substrates. 44 3