No abstract

The enteric pathogen Salmonella enterica serotype Typhimurium induces apoptosis in infected macrophages. This process is rapid, specific, and depends on the type III protein secretion system encoded within Salmonella pathogenicity island 1 (SPI1). Here, we demonstrate that serotype Typhimurium can activate programmed macrophage cell death independently of SPI1. SPI1 independent induction of apoptosis in infected macrophages is observed as early as 12 to 13 h postinfection, even in the absence of intracellular bacterial replication. Delayed activation of programmed macrophage cell death is not observed with serotype Typhimurium strains mutated in ompR or SPI2. Even though SPI2 mutants have a defect in intracellular proliferation, our results indicate that long-term intracellular survival and growth are not required for delayed macrophage killing per se, since Salmonella mutants that are severely defective in intracellular growth still induce delayed apoptosis. Inactivation of genes required for either rapid or delayed induction of apoptosis results in a conditional noncytotoxic phenotype, whereas simultaneous inactivation of genes required for both rapid and delayed induction of apoptosis renders serotype Typhimurium noncytotoxic under all conditions tested. Our hypothesis is that differential activation of programmed macrophage cell death by serotype Typhimurium occurs under discrete physiological conditions at distinct locations within an infected host.

Eschterichia coli K-12 strains producing high levels of Shiga-like toxin type II (SLT-II) but not SLT-I were previously shown to be virulent in an orally infected, streptomycin-treated mouse model. In this investigation, we tested the virulence of several SLT-II-producing enterohemorrhagic E. coli (EHEC) isolates from patients with hemorrhagic colitis or hemolytic uremic syndrome. All of the strains tested were able to colonize the mouse intestine. However, only two strains were consistently virulent for mice: 091:1121 strain B2F1(Str), which was previously shown to carry two copies of si-II-related toxins, and 091:H21 strain H414-36/89(Strr), which was found in this study to contain three genes from the si-Hi group. The oral 50%o lethal doses of strains B2F1(Strr) and H414-36/89(Strr) when fed to streptomycin-treated mice were less than 10 bacteria. Histological sections from moribund mice fed the 091:H21 strains demonstrated extensive renal tubular necrosis; however, hematological results were not consistent with a diagnosis of hemolytic uremic syndrome. The central role of SLT in the virulence of the 091:1121 EHEC strains was supported by the finding that streptomycin-treated mice preinoculated with monoclonal antibody specific for SLT-II survived oral challenge with either B2Fl(Strr) or H414-36/89(Strr). The basis for the variation in virulence among the SLT-II-producing EHEC strains tested was not determined. However, a correlation between the capacity of an EHEC strain to grow in small intestinal mucus and lethality in the streptomycin-treated mice was observed.

Characteristically, enterohemorrhagic Escherichia coli (EHEC) strains produce Shiga-like toxin type I (SLT-I), SLT-II, or both of these immunologically distinct cytotoxins. No antigenic or receptor-binding variants of SLT-I have been identified, but a number of SLT-II-related toxins have been described. Because EHEC 091:H21 strain B2F1, which produces two SLT-II-related toxins, is exquisitely virulent in an orally infected, streptomycin-treated mouse model (oral 50% lethal dose [LD501, <10 organisms), we asked whether the pathogenicity of strain B2F1 was a consequence of SLT-II-related toxin production. For this purpose, we compared the lethality of orally administered E. coli DH5ct(Strr) strains that produced different cytotoxic levels of SLT-II, SLT-Ilvha (cloned from B2F1), SLT-Ilvhb (also cloned from B2F1), or SLT-IIc (cloned from EHEC 0157:H7 strain E32511) on Vero cells. We also calculated the specific activities of purified SLT-Ilvhb and SLT-II in intraperitoneally injected mice and on Vero cells. The two purified toxins were equally toxic for mice, but SLT-Ilvhb was approximately 100-fold less active than SLT-II on Vero cells and bound to the glycolipid receptor Gb3 with lower affinity than did SLT-II. In addition, characterization of SLT-II-related toxin-binding (B) subunit mutants generated in this study revealed that the reduced in vitro cytotoxic levels of the SLT-II-related toxins were due to Asn-16 in the B subunit. Taken together, these findings do not support the idea that B2F1 is uniquely virulent because of the in vivo toxicity of SLT-II-related toxins but do demonstrate differences in in vitro cytotoxic activity among the SLT-II group produced by human EHEC isolates.