Identification and characterization of Neurotoxic factor(s) present in Shigella

Abstract

Infection by Shigella is associated with a significant number of human deaths worldwide. Complications from Shigellosis include leukemoid reaction, hemolytic uremic syndrome (HUS), seizure, encephalopathy, abdominal cramps, rectal pain and septicemia, which significantly increase morbidity and mortality. Abnormalities in the central nervous system (CNS) and HUS are more common in children under 5 years of age. The majority of Shigella associated neurological findings were reported in patients with S. sonnei and S. flexneri. But both the species do not usually produce shiga toxin, as both are lacking the structural gene encoding shiga toxin production. However, neurotoxic principle(s) in S. flexneri has not yet been discovered. In this study an attempt was taken to identify neurotoxic factor(s) present in Shigella to understand the molecular basis of toxin induced mammalian cell death. A total of 385 clinical isolates of different serotypes of Shigella isolated between 1997 and 2013 from diarrhoeal patients attending the Dhaka treatment centre of icddr,b were included in this study. Of these, 238 were S. flexneri, 80 S. dysenteriae, 42 S. boydii and 25 S. sonnei. All these strains were characterized following standard Microbiological and Biochemical methods. These strains were screened for the presence of known toxin and virulence genes such as ipaH, ial, ipaBCD, set, sen and stx by PCR and plasmid profiling. Cytotoxic and neurotoxic activity of the toxin were determined using HeLa cells and cerebellar granule neurons from rat pups respectively. Fluid accumulation in Rabbit ileum and histopathological changes of the rabbit loop segments was studied for enterotoxic activity. DNA fragmentation and chromatin condensation assay were used as markers of apoptosis. The biological status of cells at different phases of cell cycle was analyzed by FACS at different time intervals. The toxin(s) preparations involved 40%, 60% and 80% saturation of the culture supernatant with ammonium sulphate. Toxin(s) containing fraction was prepared by column chromatography using sephadex G-75. Of 385 isolates, PCR and plasmid analysis showed that almost all serotype of Shigella contain the 140MDa plasmid and sen gene except 17 S. flexneri 1c strains. All S. dysenteriae 1 contained stx gene whereas the set gene is only present in S. flexneri 2a and rarely in S. flexneri 2b. Seventeen 140 MDa lacking S. flexneri 1c strains were also negative for the common toxin gene. These 17 strains were noninvasive by sereny and Congo red binding test. Therefore, these 17 S. flexneri 1c strains were extensively used for comparative analysis. S. flexneri 1c (K-314 and K-915) were used as a representative of these 17 strains for partially purified toxin fractions and source of novel toxic factor(s) in this study. The degree of cytotoxicity differs in different species of Shigella. Around 72% (172/238) strains of S. flexneri possessed the cytotoxic activity. Of S. flexneri, serotype 1c had severe effect (more than 85% cell death) on HeLa cell. Serotype 2a, 2b, X variant and type 4 had almost similar cytotoxic effect (>70% cell death) while serotype 3a, Y variant, 4X, 6a and 6b showed less cytotoxic effect (40-45% cell death). In case of S. dysenteriae (n=80), only serotype 1 and some strains of serotype 4 showed cytotoxic effect (>90% cell death). In contrast, S. boydii (n=42) supernatant caused elongation of the HeLa cells whereas S. sonnei (n=25) caused the rounding of the cell with less frequency. It is interesting to note that the 17 S. flexneri 1c strains lacking 140 MDa plasmid and toxin gene showed strong cytotoxic effect (>90% cell death) on HeLa cells in dose dependant manner. Further analysis showed that toxic factors in these S. flexneri 1c exhibited biochemical properties similar to those of protein. Heat treatment significantly reduced cytotoxic activity of S flexneri 1c (K-314). DNase and RNase treatment did not affect cytotoxic activity while protease treatment significantly reduced its cytotoxic activity. HeLa cells were also treated with 40%, 60% and 80% fractions of S. flexneri 1c K-314 strain. However, only 60% fraction showed strong cytotoxic activity. This fraction contains an active protein component molecular mass of 100–125kDa which is sensitive to heat and trypsin. In the rabbit ileal loops assay, S. flexneri 1c (K-314) was unable to accumulate fluid. But the histo-pathological report of the loop segments showed moderate inflammatory infiltrate in lamina propria, submucosa of the wall of the intestine and sheared off of tips villi in some places with Grade-3 inflammation. However, lysates S. dysenteriae 1 caused fluid accumulation and enterocyte necrosis and shearing of tip of villi with Grade-4 inflammation. In this report, the culture supernatant of S. flexneri exhibited not only the cytotoxic activity, but also impaired neurological function in mice. The LD50 dose was found to be 1 µg. The sign of toxic effect appeared after 30h and finally hind limb paralysis and rapid breathing were observed and all mice died within 36h. In primary rat brain (cerebellar) neurons culture model it was found that these strains showed strong neurotoxic activity. The neurite length (at least 100 neurons from random fields) was determined from both toxin treated and untreated neurons. Reduction of neurite length indicated the presence of neurotoxic factor. S. flexneri induced apoptosis in HeLa cells. The toxin fraction(s) has been shown to induce apoptosis as determined by morphological changes (rounding) inhibiting polymerization of actin filament, chromatin condensation, and chromosomal DNA fragmentation in HeLa cells. Phosphorylation of the H2AX and release of cytochrome C indicated that toxin from S. flexneri might activate additional pathways leading to apoptosis. The progression of HeLa cell was arrested at G 0 /G phase as the percentage of cells in that phase increased gradually from 17.25 to 51.52 with time. This study represents the first attempt for identification and characterization of neurotoxic factor present in S. flexneri. The toxin produced by S. flexneri 1c showed cytotoxic, enterotoxic and neurotoxic activity. The above evidences suggested that toxic factors in S. flexneri are different from that of stx, set, and sen. Identification and characterization of toxic factors in S. flexneri may provide critical information in understanding the molecular basis of Shigella-induced neurotoxic effect on different types of human cells.