Identification of Sat, an autotransporter toxin produced by uropathogenic Escherichia coli

Urinary tract infection (UTI) is a very common extraintestinal infection, a nd Escherichia coil is by far the most common causative organism. Uropathog enic E. coil possess traits that distinguish them from commensal strains of E, coli, such as secretion systems that allow virulence factors to be targ eted to extracytoplasmic compartments. One of at least five characterized s ecretion mechanisms is the autotransporter system, which involves transloca tion of a protein across the inner membrane, presumably via the sec system, and across the outer membrane through a beta-barrel porin structure formed by the carboxy-terminus autotransporter domain. We identified a 107 kDa pr otein that was expressed significantly more often by E. coli strains associ ated with the clinical syndrome of acute pyelonephritis than by faecal stra ins (P = 0.029). We isolated the protein from E. coli CFT073, a strain cult ured from the blood and urine of a patient with acute pyelonephritis. The N -terminal amino acid sequence showed highest similarity to two known SPATE (serine protease autotransporters of Enterobacteriaceae) proteins, Pet and EspC, Using a 509 bp probe from the 5' region of pet, 10 cosmid clones of a n E. coli CFT073 gene library were positive for hybridization. From one cos mid clone, a 7.5 kb EcoRI restriction fragment, which reacted strongly with the probe, was shown to include the entire 3885 bp gene. The predicted 142 kDa protein product possesses the three domains that are typical of SPATE autotransporters: an unusually long signal sequence of 49 amino acids; a 10 7 kDa passenger domain containing a consensus serine protease active site ( GDSGSG); and a C-terminal autotransporter domain of 30 kDa. The protein exh ibited serine protease activity and displayed cytopathic activity on VERO p rimary kidney, HK-2 bladder and HEp-2 cell lines; the name Sat (Secreted au totransporter toxin) was derived from these properties. In addition, Sat an tibodies were present in the serum of mice infected with E. coli CFT073, Ba sed upon its association with pathogenic isolates, its cytopathic phenotype and its ability to elicit a strong antibody response after infection, we p ostulate that Sat represents a novel Virulence determinant of uropathogenic E, coli.