Abstract
Secreted autotransporter toxin (Sat) is a 107-kDa serine protease autotransporter of Enterobacteriaceae (SPATE) presenting cytotoxic activity in renal and bladder cells. Further studies have detected the Sat-encoding gene (sat) in enteroaggregative Escherichia coli (EAEC) and in E. coli strains isolated from neonatal septicemia and meningitis. Here, we investigated the role of Sat as a cytotoxin of EAEC. Sat was purified from a strain of E. coli harboring sat (DEC/Sat+, O126:H2) and used to raise antibodies in rabbit. The presence of Sat was detected by ELISA in the supernatant of 93.7% of EAEC strains harboring sat and in none lacking the gene. The effect of Sat during infection was investigated in polarized Caco-2 cells infected with Sat-producing EAEC (CV323/77, O125ab:H21). This strain induced intense cell detachment, which was inhibited by PMSF or Sat antiserum. Also, sat transcription and Sat production were detected during infection. Here we demonstrate that Sat is internalized in polarized cells leading to F-actin disruption which preceded cell detachment. A comparative study of the toxin action in cell lines corresponding to the infection sites in which bacteria carrying the sat gene have been isolated was performed. Cells originating from the gastrointestinal tract (Caco-2), urinary (LLC-PK1) and endothelium (HUVEC) were incubated with purified Sat. The time required for observation of cell damage differed according to the cell line. HUVEC cells were more sensitive to Sat than cells derived from urinary and intestinal tracts. The intense activity of Sat on the endothelial cells suggests that Sat could also be a virulence factor for the bacteria in the bloodstream. In addition, this is the first work demonstrating that Sat induces cytotoxic effect during EAEC infection in vitro. The cell damage observed during infection indicates that Sat may be another toxin with cytotoxic role in the EAEC pathogenesis.
Highlights
Secreted autotransporter toxin (Sat) is a Class-1 serine protease autotransporters of Enterobacteriaceae (SPATE) whose cytopathic effect has been described in kidney and bladder cells [12], since the toxin was originally described in uropathogenic E. coli (UPEC) [10]
Among the diarrheagenic E. coli pathotypes the presence of sat has already been detected in enterotoxigenic E. coli (ETEC) [16], enteropathogenic E. coli (EPEC) [10,17,45], enteroaggregative Escherichia coli (EAEC) [10,16,19] and diffusely adherent E. coli (DAEC) [16,18,21]
Among these pathotypes cell damage has only been described in models of DAEC infection. [18,21]
Summary
SPATE comprises a large group of trypsin-like serine proteases secreted by enteropathogens such as Escherichia coli, Shigella, Salmonella and Citrobacter species [3,5,6,7]. These proteins are characterized by the presence of three domains: an N-terminal signal sequence; an extracellular passenger domain, surface exposed or secreted, which exhibits the serine protease GDSGS motif; and a C-terminal β-barrel domain, anchored to the outer membrane [4,8]. Phylogenetic analysis clustered SPATE members into two groups: class-1, including those with cytotoxic activities; and class-2, including proteases with mucinolytic and immunomodulatory activities [7]
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