Carbonell G V, Amorim C R N, Furumura M T, Darini A L C, Fonseca B A L, Yano T
Departamento de Microbiologia e Imunologia, Instituto de Biologia, Universidade Estadual de Campinas, Campinas, SP, Brasil.
Braz J Med Biol Res. 2003 Mar;36(3):351-9. doi: 10.1590/s0100-879x2003000300010. Epub 2003 Mar 7.
Serratia marcescens cytotoxin was purified to homogeneity by ion-exchange chromatography on a DEAE Sepharose Fast Flow column, followed by gel filtration chromatography on a Sephadex G100 column. The molecular mass of the cytotoxin was estimated to be about 50 kDa. Some biological properties of the cytotoxin were analyzed and compared with well-characterized toxins, such as VT1, VT2 and CNF from Escherichia coli and hemolysin produced by S. marcescens. The sensitivity of the cell lines CHO, HeLa, HEp-2, Vero, BHK-21, MA 104 and J774 to the cytotoxin was determined by the cell viability assay using neutral red. CHO and HEp-2 were highly sensitive, with massive cellular death after 1 h of treatment, followed by BHK-21, HeLa, Vero and J774 cells, while MA 104 was insensitive to the toxin. Cytotoxin induced morphological changes such as cell rounding with cytoplasmic retraction and nuclear compactation which were evident 15 min after the addition of cytotoxin. The cytotoxic assays show that 15 min of treatment with the cytotoxin induced irreversible intoxication of the cells, determined by loss of cell viability. Concentrations of 2 CD50 (0.56 g/ml) of purified cytotoxin did not present any hemolytic activity, showing that the cytotoxin is distinct from S. marcescens hemolysin. Antisera prepared against S. marcescens cytotoxin did not neutralize the cytotoxic activity of VT1, VT2 or CNF toxin, indicating that these toxins do not share antigenic determinants with cytotoxin. Moreover, we did not detect gene sequences for any of these toxins in S. marcescens by PCR assay. These results suggest that S. marcescens cytotoxin is not related to any of these toxins from E. coli.
粘质沙雷氏菌细胞毒素通过在DEAE Sepharose Fast Flow柱上进行离子交换色谱，随后在Sephadex G100柱上进行凝胶过滤色谱，被纯化至同质。该细胞毒素的分子量估计约为50 kDa。分析了该细胞毒素的一些生物学特性，并与特征明确的毒素进行了比较，如大肠杆菌的VT1、VT2和CNF以及粘质沙雷氏菌产生的溶血素。使用中性红通过细胞活力测定法确定了CHO、HeLa、HEp-2、Vero、BHK-21、MA 104和J774细胞系对该细胞毒素的敏感性。CHO和HEp-2高度敏感，处理1小时后大量细胞死亡，其次是BHK-21、HeLa、Vero和J774细胞，而MA 104对该毒素不敏感。细胞毒素诱导形态学变化，如细胞变圆、细胞质收缩和核浓缩，在添加细胞毒素15分钟后很明显。细胞毒性测定表明，用细胞毒素处理15分钟会导致细胞不可逆中毒，这由细胞活力丧失来确定。纯化的细胞毒素浓度为2 CD50（0.56 μg/ml）时未表现出任何溶血活性，表明该细胞毒素与粘质沙雷氏菌溶血素不同。针对粘质沙雷氏菌细胞毒素制备的抗血清不能中和VT1、VT2或CNF毒素的细胞毒性活性，表明这些毒素与细胞毒素不共享抗原决定簇。此外，我们通过PCR测定在粘质沙雷氏菌中未检测到任何这些毒素的基因序列。这些结果表明，粘质沙雷氏菌细胞毒素与大肠杆菌的任何这些毒素均无关。
