Laboratório de Bacteriologia, Instituto Butantan, São Paulo, SP, Brazil.
Laboratório de Bioquímica e Biofísica, Instituto Butantan, São Paulo, SP, Brazil.
PLoS One. 2020 Feb 21;15(2):e0228959. doi: 10.1371/journal.pone.0228959. eCollection 2020.
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.
分泌型自转运毒素（Sat）是肠杆菌科的一种 107kDa 丝氨酸蛋白酶自转运体（SPATE），在肾和膀胱细胞中具有细胞毒性。进一步的研究在肠聚集性大肠杆菌（EAEC）和从新生儿败血症和脑膜炎分离的大肠杆菌菌株中检测到 Sat 编码基因（sat）。在这里，我们研究了 Sat 作为 EAEC 细胞毒素的作用。Sat 从携带 sat 的大肠杆菌菌株（DEC/Sat+，O126:H2）中纯化，并在兔中用于产生抗体。ELISA 检测到 93.7%携带 sat 的 EAEC 菌株的上清液中存在 Sat，而缺乏该基因的菌株则不存在。在感染过程中，用产生 Sat 的 EAEC（CV323/77，O125ab:H21）感染极化的 Caco-2 细胞来研究 Sat 的作用。该菌株诱导强烈的细胞脱落，这种脱落被 PMSF 或 Sat 抗血清抑制。此外，在感染过程中检测到 sat 的转录和 Sat 的产生。在这里，我们证明 Sat 被内化到极化细胞中，导致 F-肌动蛋白断裂，这先于细胞脱落。对携带 sat 基因的细菌分离的感染部位相应的细胞系中毒素作用进行了比较研究。用纯化的 Sat 孵育来自胃肠道（Caco-2）、泌尿道（LLC-PK1）和内皮（HUVEC）的细胞系。观察到细胞损伤所需的时间根据细胞系而有所不同。HUVEC 细胞比来自泌尿道和肠道的细胞对 Sat 更敏感。Sat 对内皮细胞的强烈活性表明 Sat 也可能是细菌在血液中的一种毒力因子。此外，这是首次证明 Sat 在体外 EAEC 感染过程中诱导细胞毒性作用的研究。感染过程中观察到的细胞损伤表明 Sat 可能是 EAEC 发病机制中另一种具有细胞毒性作用的毒素。
