Differential response of the human renal proximal tubular epithelial cell line HK-2 to Shiga toxin types 1 and 2.

Shiga toxins (Stxs) are expressed by the enteric pathogens Shigella dysenteriae serotype 1 and certain serotypes of Escherichia coli. Stx-producing bacteria cause bloody diarrhea with the potential to progress to acute renal failure. Stxs are potent protein synthesis inhibitors and are the primary virulence factors responsible for renal damage that may follow diarrheal disease. We explored the use of the immortalized human proximal tubule epithelial cell line HK-2 as an in vitro model of Stx-induced renal damage. We showed that these cells express abundant membrane Gb(3) and are differentially susceptible to the cytotoxic action of Stxs, being more sensitive to Shiga toxin type 1 (Stx1) than to Stx2. At early time points (24 h), HK-2 cells were significantly more sensitive to Stxs than Vero cells; however, by 72 h, Vero cell monolayers were completely destroyed while some HK-2 cells survived toxin challenge, suggesting that a subpopulation of HK-2 cells are relatively toxin resistant. Fluorescently labeled Stx1 B subunits localized to both lysosomal and endoplasmic reticulum (ER) compartments in HK-2 cells, suggesting that differences in intracellular trafficking may play a role in susceptibility to Stx-mediated cytotoxicity. Although proinflammatory cytokines were not upregulated by toxin challenge, Stx2 selectively induced the expression of two chemokines, macrophage inflammatory protein-1α (MIP-1α) and MIP-1ß. Stx1 and Stx2 differentially activated components of the ER stress response in HK-2 cells. Finally, we demonstrated significant poly(ADP-ribose) polymerase (PARP) cleavage after exposure to Stx1 or Stx2. However, procaspase 3 cleavage was undetectable, suggesting that HK-2 cells may undergo apoptosis in response to Stxs in a caspase 3-independent manner.

Isolation and characterization of minicell-producing mutants of Shigella spp.

Minicells are small, anucleate cells resulting from aberrant cell divisions at the polar ends of bacilli. We have isolated minicell-producing mutant strains of Shigella flexneri 2a (MC-I) and Shigella dysenteriae 1 (MC-V) after mutagenesis with N-methyl-N'-nitro-N-nitrosoguanidine. Microscopically, broth cultures of MC-I and MC-V were found to contain free minicells, normal cells, and filamentous cells with polar, attached minicells. Both strains retained their ability to provoke keratoconjunctivitis in guinea pigs and to invade HeLa cells. Purified suspensions of minicells containing less than one whole cell per 10(6) minicells were obtained by a combination of differential sedimentation and density gradient centrifugation (5 to 30% [wt/vol] linear sucrose gradients). Each MC-I minicell contained about 0.005 times the amount of deoxyribonucleic acid of one normal S. flexneri. The MC-V minicell had about 0.003 times the amount of deoxyribonucleic acid of one whole S. dysenteriae cell. Purified MC-V minicells were treated with polymyxin B to release Shiga toxin. Shiga toxin was readily detected in MC-V minicells by means of a microtiter HeLa cell cytotoxicity assay. Our findings indicate that such a minicell-producing alteration in the cell division cycle of shigellae has not significantly affected their virulence.