Secreted autotransporter toxin (Sat) induces cell damage during enteroaggregative Escherichia coli infection

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.

Secreted Autotransporter Toxin (Sat) Mediates Innate Immune System Evasion

“…The subcutaneous route of inoculation used in our murine model highlights that the cytotoxic effects of Sat (38)(39)(40)(41)(42)(43)(44)(45) are also important for bacteria to access the bloodstream, since Sat causes endothelial damages (44). However, since our murine model of infection evaluates lethality only, further studies are necessary to fully understand the action of Sat in the bloodstream components.…”

“…Sat Was Purified as a Native Protein From EC071 Culture Supernatant EC071 culture supernatant was concentrated and submitted to an anionic exchange column for Sat purification. Elution fractions collected during this process were analyzed by SDS-PAGE and immunoblotting using anti-Sat serum (44). Silver nitrate staining revealed a single band of approximately 100 kDa in five of these fractions (Figure 4A), which were recognized by anti-Sat serum (Figure 4B).…”

Acylpolyamine Mygalin as a TLR4 Antagonist Based on Molecular Docking and In Vitro Analyses

“…Total RNA was extracted from the macrophages with 180 µL of QIAzol lysis reagent (Qiagen ® ) and purified using a Direct-zol kit (ZYMO RESEARCH) following the manufacturer's instructions. RNA was quantified by measuring the absorbance at 260 nm using a GE NanoVue Plus TM spectrophotometer (GE Lifesciences) and the integrity was visualized on 1.2% agarose gels stained with red GelRed TM (Biotium) [35]. Total RNA (100 ng) was converted into cDNA using a High Capacity RNA-to-cDNA kit (Applied Biosystems TM ).…”

Virulence Factors of Enteric Pathogenic Escherichia coli: A Review

“…Sat , a SPATE toxin with cytotoxic and enterotoxic activities, impairs the tight junction and mediates autophagy in the epithelial cells. The enteroaggregative heat-stable toxin has mechanistic and physical similarities to the enterotoxin STa secreted by ETEC ( Table 1 ) [ 105 ].…”