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The GRINgenes encoding N-methyl-D-aspartate receptor (NMDAR) subunits are remarkably intolerant to variation. Many pathogenic NMDAR variants result in their protein misfolding, inefficient assembly, reduced surface expression, and impaired function on neuronal membrane, causing neurological disorders including epilepsy and intellectual disability. Here, we investigated the proteostasis maintenance of NMDARs containing epilepsy-associated variations in the GluN2A subunit, including M705V and A727T. In the transfected HEK293T cells, we showed that the two variants were targeted to the proteasome for degradation and had reduced functional surface expression. We demonstrated that the application of BIX, a known small molecule activator of an HSP70 family chaperone BiP (binding immunoglobulin protein) in the endoplasmic reticulum (ER), dose-dependently enhanced the functional surface expression of the M705V and A727T variants in HEK293T cells. Moreover, BIX (10 μM) increased the surface protein levels of the M705V variant in human iPSC-derived neurons. We revealed that BIX promoted folding, inhibited degradation, and enhanced anterograde trafficking of the M705V variant by modest activation of the IRE1 pathway of the unfolded protein response. Our results suggest that adapting the ER proteostasis network restores the folding, trafficking, and function of pathogenic NMDAR variants, representing a potential treatment for neurological disorders resulting from NMDAR dysfunction.

Rapalogues are powerful therapeutic modalities for breast cancer; however, they suffer from low solubility and dose-limiting side effects. To overcome these challenges, we developed a long-circulating multiheaded drug carrier called 5FA, which contains rapamycin-binding domains linked with elastin-like polypeptides (ELPs). To target these “Hydra-ELPs” toward breast cancer, we here linked 5FA with four distinct peptides which are reported to engage the cell surface form of the 78 kDa glucose-regulated protein (csGRP78). To determine if these peptides affected the carrier solubility, this library was characterized by light scattering and mass spectrometry. To guide in vitro selection of the most potent functional carrier for rapamycin, its uptake and inhibition of mTORC1 were monitored in a ductal breast cancer model (BT474). Using flow cytometry to track cellular association, it was found that only the targeted carriers enhanced cellular uptake and were susceptible to proteolysis by SubA, which specifically targets csGRP78. The functional inhibition of mTOR was monitored by Western blot for pS6K, whereby the best carrier L-5FA reduced mTOR activity by 3-fold compared to 5FA or free rapamycin. L-5FA was further visualized using super-resolution confocal laser scanning microscopy, which revealed that targeting increased exposure to the carrier by ∼8-fold. This study demonstrates how peptide ligands for GRP78, such as the L peptide (RLLDTNRPLLPY), may be incorporated into protein-based drug carriers to enhance targeting.

Endoplasmic reticulum (ER) stress inhibits activation of AKT by insulin by depleting insulin receptors and interferes with the delivery of newly synthesized insulin receptors to the cell surface. Bypass of the secretory pathway in synthesis of the cytosolic protein tyrosine kinase domain of the insulin receptor negates the effects of ER stress on activation of AKT by insulin.

Hemophilia A, an X-linked bleeding disorder caused by deficiency of factor VIII (FVIII), is treated by protein replacement. Unfortunately, this regimen is costly due to the expense of producing recombinant FVIII as a consequence of its low-level secretion from mammalian host cells. FVIII expression activates the endoplasmic reticulum (ER) stress response, causes oxidative stress, and induces apoptosis. Importantly, little is known about the factors that cause protein misfolding and aggregation in metazoans. Here, we identified intrinsic and extrinsic factors that cause FVIII to form aggregates. We show that FVIII forms amyloid-like fibrils within the ER lumen upon increased FVIII synthesis or inhibition of glucose metabolism. Significantly, FVIII amyloids can be dissolved upon restoration of glucose metabolism to produce functional secreted FVIII. Two ER chaperone families and their cochaperones, immunoglobulin binding protein (BiP) and calnexin/calreticulin, promote FVIII solubility in the ER, where the former is also required for disaggregation. A short aggregation motif in the FVIII A1 domain (termed Aggron) is necessary and sufficient to seed β-sheet polymerization, and BiP binding to this Aggron prevents amyloidogenesis. Our findings provide novel insight into mechanisms that limit FVIII secretion and ER protein aggregation in general and have implication for ongoing hemophilia A gene-therapy clinical trials.

Hemophilia A, an X-linked bleeding disorder caused by deficiency of factor VIII (FVIII), is treated by protein replacement. Unfortunately, this regimen is costly due to the expense of producing recombinant FVIII as a consequence of its low-level secretion from mammalian host cells. FVIII expression activates the endoplasmic reticulum (ER) stress response, causes oxidative stress, and induces apoptosis. Importantly, little is known about the factors that cause protein misfolding and aggregation in metazoans. Here, we identified intrinsic and extrinsic factors that cause FVIII to form aggregates. We show that FVIII forms amyloid-like fibrils within the ER lumen upon increased FVIII synthesis or inhibition of glucose metabolism. Significantly, FVIII amyloids can be dissolved upon restoration of glucose metabolism to produce functional secreted FVIII. Two ER chaperone families and their cochaperones, immunoglobulin binding protein (BiP) and calnexin/calreticulin, promote FVIII solubility in the ER, where the former is also required for disaggregation. A short aggregation motif in the FVIII A1 domain (termed Aggron) is necessary and sufficient to seed β-sheet polymerization, and BiP binding to this Aggron prevents amyloidogenesis. Our findings provide novel insight into mechanisms that limit FVIII secretion and ER protein aggregation in general and have implication for ongoing hemophilia A gene-therapy clinical trials.

Neu5Ac, Neu5Gc, and KDN are three forms of sialic acids in vertebrates that possess distinct biological functions. Herein, we report the synthesis and metabolic incorporation of the 9-azido analogues of three sialic acid forms in mammalian cells. The incorporated sialic acid analogues enable fluorescent imaging of cell-surface sialoglycans and proteomic profiling of sialoglycoproteins. Furthermore, we apply them to metabolically engineer cell surfaces with sialoglycans terminated with distinct sialic acids or their 9-azido analogues. The remodeled cells expressing specific cell-surface sialoglycoforms show distinct binding affinity toward subtilase cytotoxin (SubAB), a toxin secreted by Shiga toxigenic Escherichia coli. The 9-azido analogues of sialic acid forms developed in this work provide a versatile tool for metabolic remodeling of cell-surface properties and modulating pathogen–host interactions.

Protein transport into the mammalian endoplasmic reticulum (ER) is mediated by the heterotrimeric Sec61 channel. The signal recognition particle (SRP) and TRC systems and Sec62 have all been characterized as membrane-targeting components for small presecretory proteins, whereas Sec63 and the lumenal chaperone BiP act as auxiliary translocation components. Here, we report the transport requirements of two natural, small presecretory proteins and engineered variants using semipermeabilized human cells after the depletion of specific ER components. Our results suggest that hSnd2, Sec62, and SRP and TRC receptor each provide alternative targeting pathways for short secretory proteins and define rules of engagement for the actions of Sec63 and BiP during their membrane translocation. We find that the Sec62/Sec63 complex plus BiP can facilitate Sec61 channel opening, thereby allowing precursors that have weak signal peptides or other inhibitory features to translocate. A Sec61 inhibitor can mimic the effect of BiP depletion on Sec61 gating, suggesting that they both act at the same essential membrane translocation step.

The endoplasmic reticulum (ER) stress response constitutes cellular reactions triggered by a wide variety of stimuli that disturb folding of proteins, often leading to apoptosis. ER stress-induced apoptotic cell death is thought to be an important contributor to many human pathological conditions. The molecular mechanism of this apoptosis process has been highly controversial with both the receptor and the mitochondrial pathways being implicated. Using knockout mouse models and RNAi-mediated gene silencing in cell lines, our group and others had demonstrated the importance of the mitochondrial apoptotic pathway in ER stress-induced cell death, particularly the role of the pro-apoptotic BH3-only BCL-2 family members, BIM and PUMA. However, a recent report suggested a central role for the death receptor, DR5, activated in a ligand-independent manner, and the initiator caspase, caspase-8, in ER stress-induced cell death. This prompted us to re-visit our previous observations and attempt to reproduce the newly published findings. Here we report that the mitochondrial apoptotic pathway, activated by BH3-only proteins, is essential for ER stress-induced cell death and that, in contrast to the previous report, DR5 as well as caspase-8 are not required for this process.

ABSTRACTEnterohemorrhagic Escherichia coli(EHEC) is an important cause of diarrhea and hemolytic-uremic syndrome (HUS) worldwide. Australia's worst outbreak of HUS occurred in Adelaide in 1995 and was one of the first major HUS outbreaks attributed to a non-O157 Shiga-toxigenic E. coli(STEC) strain. Molecular analyses conducted at the time suggested that the outbreak was caused by an O111:H−clone, with strains from later in the outbreak harboring an extra copy of the genes encoding the potent Shiga toxin 2 (Stx2). Two decades later, we have used next-generation sequencing to compare two isolates from early and late in this important outbreak. We analyzed genetic content, single-nucleotide polymorphisms (SNPs), and prophage insertion sites; for the latter, we demonstrate how paired-end sequence data can be leveraged to identify such insertion sites. The two strains are genetically identical except for six SNP differences and the presence of not one but two additional Stx2-converting prophages in the later isolate. Isolates from later in the outbreak were associated with higher levels of morbidity, suggesting that the presence of the additional Stx2-converting prophages is significant in terms of the virulence of this clone.

ABSTRACTStreptococcus pneumoniaeis the leading infectious cause of death in children in the world. However, the mechanisms that drive the progression from asymptomatic colonization to disease are poorly understood. Two virulence-associated genomic accessory regions (ARs) were deleted in a highly virulent serotype 1 clinical isolate (strain 4496) and examined for their contribution to pathogenesis. Deletion of a prophage encoding a platelet-binding protein (PblB) resulted in reduced adherence, biofilm formation, reduced initial infection within the lungs, and a reduction in the number of circulating platelets in infected mice. However, the region's overall contribution to the survival of mice was not significant. In contrast, deletion of the variable region of pneumococcal pathogenicity island 1 (vPPI1) was also responsible for a reduction in adherence and biofilm formation but also reduced survival and invasion of the pleural cavity, blood, and lungs. While the 4496ΔPPI1 strain induced higher expression of the genes encoding interleukin-10 (IL-10) and CD11b in the lungs of challenged mice than the wild-type strain, very few other genes exhibited altered expression. Moreover, while the level of IL-10 protein was increased in the lungs of 4496ΔPPI1 mutant-infected mice compared to strain 4496-infected mice, the levels of gamma interferon (IFN-γ), CXCL10, CCL2, and CCL4 were not different in the two groups. However, the 4496ΔPPI1 mutant was found to be more susceptible than the wild type to phagocytic killing by a macrophage-like cell line. Therefore, our data suggest that vPPI1 may be a major contributing factor to the heightened virulence of certain serotype 1 strains, possibly by influencing resistance to phagocytic killing.

Endoplasmic reticulum (ER)-associated degradation (ERAD) represents a principle quality control mechanism to clear misfolded proteins in the ER; however, its physiological significance and the nature of endogenous ERAD substrates remain largely unexplored. Here we discover that IRE1α, the sensor of the unfolded protein response (UPR), is a bona fide substrate of the Sel1L–Hrd1 ERAD complex. ERAD-mediated IRE1α degradation occurs under basal conditions in a BiP-dependent manner, requires both the intramembrane hydrophilic residues of IRE1α and the lectin protein OS9, and is attenuated by ER stress. ERAD deficiency causes IRE1α protein stabilization, accumulation and mild activation both in vitro and in vivo. Although enterocyte-specific Sel1L-knockout mice (Sel1LΔIEC) are viable and seem normal, they are highly susceptible to experimental colitis and inflammation-associated dysbiosis, in an IRE1α-dependent but CHOP-independent manner. Hence, Sel1L–Hrd1 ERAD serves a distinct, essential function in restraint of IRE1α signalling in vivo by managing its protein turnover.

Colon cancer stem cells (colon-CSCs) are more resistant to conventional chemotherapy than differentiated cancer cells. This subset of therapy refractory cells is therefore believed to play an important role in post-therapeutic tumor relapse. In order to improve the rate of sustained response to conventional chemotherapy, development of approaches is warranted that specifically sensitize colon-CSCs to treatment. Here, we report that ER-stress-induced activation of the unfolded protein response (UPR) forces colon-CSCs to differentiate, resulting in their enhanced sensitivity to chemotherapy in vitro and in vivo. Our data suggest that agents that induce activation of the UPR may be used to specifically increase sensitivity of colon-CSCs to the effects of conventional chemotherapy.

ABSTRACTStreptococcus pneumoniaeis a diverse species causing invasive as well as localized infections that result in massive global morbidity and mortality. Strains vary markedly in pathogenic potential, but the molecular basis is obscured by the diversity and plasticity of the pneumococcal genome. We have previously reported that S. pneumoniaeserotype 3 isolates belonging to the same multilocus sequence type (MLST) differed markedly in in vitroand in vivophenotypes, in accordance with the clinical site of isolation, suggesting stable niche adaptation within a clonal lineage. In the present study, we have extended our analysis to serotype 14 clinical isolates from cases of sepsis or otitis media that belong to the same MLST (ST15). In a murine intranasal challenge model, five ST15 isolates (three from blood and two from ears) colonized the nasopharynx to similar extents. However, blood and ear isolates exhibited significant differences in bacterial loads in other host niches (lungs, ear, and brain) at both 24 and 72 h postchallenge. In spite of these differences, blood and ear isolates were present in the lungs at similar levels at 6 h postchallenge, suggesting that early immune responses may underpin the distinct virulence phenotypes. Transcriptional analysis of lung tissue from mice infected for 6 h with blood isolates versus ear isolates revealed 8 differentially expressed genes. Two of these were exclusively expressed in response to infection with the ear isolate. These results suggest a link between the differential capacities to elicit early innate immune responses and the distinct virulence phenotypes of clonally related S. pneumoniaestrains.

ABSTRACTShiga-toxigenic Escherichia coli(STEC) causes severe gastrointestinal infections in humans that may lead to life-threatening systemic sequelae, such as the hemolytic uremic syndrome (HUS). Rapid diagnosis of STEC infection early in the course of disease opens a window of opportunity for therapeutic intervention, for example, by administration of agents that neutralize Shiga toxin (Stx) in the gut lumen. We previously developed a recombinant bacterium that expresses a mimic of the Stx receptor globotriaosyl ceramide (Gb3) on its surface through modification of the lipopolysaccharide (A. W. Paton, R. Morona, and J. C. Paton, Nat Med 6:265–270, 2000, http://dx.doi.org/10.1038/73111). This construct was highly efficacious in vivo, protecting mice from otherwise fatal STEC disease, but the fact that it is a genetically modified organism (GMO) has been a barrier to clinical development. In the present study, we have overcome this issue by development of Gb3 receptor mimic bacterial ghosts (BGs) that are not classified as GMOs. Gb3-BGs neutralized Stx1 and Stx2 in vitrowith high efficiency, whereas alternative Gb3-expressing non-GMO subbacterial particles (minicells and outer membrane blebs) were ineffective. Gb3-BGs were highly efficacious in a murine model of STEC disease. All mice (10/10) treated with Gb3-BGs survived challenge with a highly virulent O113:H21 STEC strain and showed no pathological signs of renal injury. In contrast, 6/10 mice treated with control BGs succumbed to STEC challenge, and survivors exhibited significant weight loss, neutrophilia, and histopathological evidence of renal damage. Thus, Gb3-BGs offer a non-GMO approach to treatment of STEC infection in humans, particularly in an outbreak setting.

ABSTRACTSubtilase cytotoxin (SubAB) is the prototype of a recently emerged family of AB5 cytotoxins produced by Shiga-toxigenic Escherichia coli(STEC). Its mechanism of action involves highly specific A-subunit-mediated proteolytic cleavage of the essential endoplasmic reticulum (ER) chaperone BiP. Our previous in vivostudies showed that intraperitoneal injection of purified SubAB causes a major redistribution of leukocytes and elevated leukocyte apoptosis in mice, as well as profound splenic atrophy. In the current study, we investigated selected chemokine and proinflammatory cytokine responses to treatment with SubAB, a nontoxic derivative (SubAA272B), or Shiga toxin 2 (Stx2) in human macrophage (U937), brain microvascular endothelial (HBMEC), and colonic epithelial (HCT-8) cell lines, at the levels of secreted protein, cell-associated protein, and gene expression. Stx2 treatment upregulated expression of chemokines and cytokines at both the protein and mRNA levels. In contrast, SubAB induced significant decreases in secreted interleukin-8 (IL-8) and monocyte chemoattractant protein 1 (MCP-1) in all three tested cell lines and a significant decrease in secreted IL-6 in HBMECs. The downregulation of secreted chemokines or cytokines was not observed in SubAA272B-treated cells, indicating a requirement for BiP cleavage. The downregulation of secreted chemokines and cytokines by SubAB was not reflected at the mRNA and cell-associated protein levels, suggesting a SubAB-induced export defect.

ABSTRACTShiga toxin-producing Escherichia coliO157:H7 (STEC) is by far the most prevalent serotype associated with hemolytic uremic syndrome (HUS) although many non-O157 STEC strains have been also isolated from patients with HUS. The main virulence factor of STEC is the Shiga toxin type 2 (Stx2) present in O157 and non-O157 strains. Recently, another toxin, named subtilase cytotoxin (SubAB), has been isolated from several non-O157 strains and may contribute to the pathogenesis of HUS. Here, we have demonstrated that an O113:H21 STEC strain expressing SubAB and Stx2 inhibits normal water absorption across human colon and causes damage to the surface epithelium, necrosis, mononuclear inflammatory infiltration, edema, and marked mucin depletion. This damage was less marked, but nevertheless significant, when purified SubAB or E. coliO113:H21 expressing only SubAB was assayed. This is the first study showing that SubAB may directly participate in the mechanisms of diarrhea in children infected with non-O157 STEC strains.

Stem cells generate rapidly dividing transit-amplifying cells that have lost the capacity for self-renewal but cycle for a number of times until they exit the cell cycle and undergo terminal differentiation. We know very little of the type of signals that trigger the earliest steps of stem cell differentiation and mediate a stem cell to transit-amplifying cell transition. We show that in normal intestinal epithelium, endoplasmic reticulum (ER) stress and activity of the unfolded protein response (UPR) are induced at the transition from stem cell to transit-amplifying cell. Induction of ER stress causes loss of stemness in a Perk-eIF2α-dependent manner. Inhibition of Perk-eIF2α signaling results in stem cell accumulation in organoid culture of primary intestinal epithelium. Our findings show that the UPR plays an important role in the regulation of intestinal epithelial stem cell differentiation.

ABSTRACTThe principal function of bacterial AB5 toxin B subunits is to interact with glycan receptors on the surfaces of target cells and mediate the internalization of holotoxin. However, B subunit-receptor interactions also have the potential to impact cell signaling pathways and, in so doing, contribute to pathogenesis independently of the catalytic (toxic) A subunits. Various Salmonella entericaserovars, including Salmonella entericaserovar Typhi, encode an AB5 toxin (ArtAB), the A subunit of which is an ADP-ribosyltransferase related to the S1 subunit of pertussis toxin. However, although the A subunit is able to catalyze ADP-ribosylation of host G proteins, a cytotoxic phenotype has yet to be identified for the holotoxin. We therefore examined the capacity of the purified B subunit (ArtB) from S. Typhi to elicit cytokine, chemokine, and adhesion molecule responses in human macrophage (U937), colonic epithelial (HCT-8) cell, and brain microvascular endothelial cell (HBMEC) lines. Secretion of the chemokines monocyte chemotactic protein 1 (MCP-1) and interleukin 8 (IL-8) was increased in all three tested cell lines, with macrophage inflammatory protein 1a (MIP-1a), MIP-1ß, and granulocyte colony-stimulating factor (G-CSF) also significantly increased in U937 cells. ArtB also upregulated the cytokines tumor necrosis factor alpha (TNF-a) and IL-6 in HBMECs and HCT-8 cells, but not in U937 cells, while intercellular adhesion molecule 1 (ICAM-1) was upregulated in HCT-8 and U937 cells and vascular cell adhesion molecule 1 (VCAM-1) was upregulated in HBMECs. Thus, ArtB may contribute to pathogenesis independently of the A subunit by promoting and maintaining a strong inflammatory response at the site of infection.

ABSTRACTStreptococcus pneumoniaeis a diverse species causing invasive as well as localized infections that result in massive global morbidity and mortality. Strains vary markedly in pathogenic potential, but the molecular basis is obscured by the diversity and plasticity of the pneumococcal genome. In the present study, S. pneumoniaeserotype 3 blood (n= 12) or ear (n= 13) isolates were multilocus sequence typed (MLST) and assessed for biofilm formation and virulence phenotype. Blood and ear isolates exhibited similar MLST distributions but differed markedly in phenotype. Blood isolates formed robust biofilms only at pH 7.4, which were enhanced in Fe(III)-supplemented medium. Conversely, ear isolates formed biofilms only at pH 6.8, and Fe(III) was inhibitory. Biofilm formation paralleled luxSexpression and genetic competence. In a mouse intranasal challenge model, blood isolates did not stably colonize the nasopharynx but spread to the blood; none spread to the ear. Ear isolates colonized the nasopharynx at higher levels and also spread to the ear compartment in a significant proportion of animals; none caused bacteremia. Thus, pneumococci of the same serotype and MLST exhibit distinct phenotypes in accordance with clinical site of isolation, indicative of stable niche adaptation within a clonal lineage.

During recovery from acute glomerulonephritis, cell proliferation, matrix expansion, and expression of the dedifferentiation marker α-smooth muscle actin (α-SMA) subside spontaneously. However, the molecular mechanisms underlying this recovery process remain elusive. In mesangioproliferative glomerulonephritis, the unfolded protein response (UPR) is induced in activated, dedifferentiated mesangial cells. We investigated the role of the UPR in mesangial cell deactivation and redifferentiation and found that, during experimental glomerulonephritis in rats, reinforcement of the UPR significantly attenuated mesangial cell proliferation, matrix expansion, and expression of α-SMA. Consistent with this in vivoresult, induction of the UPR suppressed cell proliferation and transcriptional expression of type IV collagen (ColIV) and α-SMA in activated mesangial cells. The UPR reduced phosphorylation of Akt in vitroand in vivo, and it was responsible for attenuation of cell proliferation. The UPR also preferentially depressed levels of total and phosphorylated Smads without affecting transcriptional levels, and it was responsible for suppression of ColIV and α-SMA. Translational suppression via the eIF2α pathway, but not proteasome-mediated protein degradation, was responsible for the down-regulation of Smads. These results suggest the novel potential of the UPR to facilitate a phenotypic shift of activated glomerular cells toward deactivation and redifferentiation. The UPR may serve as endogenous machinery that supports recovery of glomeruli from acute inflammation.

Misfolding mutations in rod opsin cause the blinding disease retinitis pigmentosa. The ER chaperone BiP is required to prevent rod opsin aggregation, and overexpression of BiP can improve mutant rod opsin mobility. This could be important for therapies based on manipulating rod opsin folding and aggregation.

ABSTRACTShiga-toxigenic Escherichia coli(STEC) O113:H21 strains that lack the locus of enterocyte effacement (LEE) efficiently invade eukaryotic cells in vitro, unlike LEE-positive O157:H7 strains. We used a fliCdeletion mutant of the O113:H21 STEC strain 98NK2 (98NK2?fliC) to show that invasion of colonic epithelial (HCT-8) cells is heavily dependent on production of flagellin, even though adherence to the cells was actually enhanced in the mutant. Flagellin binds and signals through Toll-like receptor 5 (TLR5), but there was no evidence that either TLR5, the adaptor protein myeloid differentiation primary response gene 88 (MyD88), or the serine kinase interleukin-1 receptor-associated kinase (IRAK) were required for invasion of HCT-8 cells by strain 98NK2, as judged by transfection, RNA knockdown, or inhibitor studies. However, pretreatment of cells with anti-asialo-GM1 significantly decreased 98NK2 invasion (by 40.8%), while neuraminidase treatment (which cleaves terminal sialic acid residues, thus converting GM1 into asialo-GM1) significantly increased invasion (by 70.7%). Pretreatment of HCT-8 cells with either the cholesterol-depleting agent methyl-ß-cyclodextrin (MßCD) or the tyrosine kinase inhibitor genistein significantly decreased invasion by 98NK2, indicating a potential role for lipid rafts in the invasion mechanism. Confocal microscopy also showed invading 98NK2 colocalized with lipid raft markers caveolin-1 and GM1. Interestingly, anti-asialo-GM1, neuraminidase, MßCD, and genistein have similar effects on the vestigial level of STEC invasion seen for STEC strain 98NK2?fliC, indicating that lipid rafts mediate a common step in flagellin-dependent and flagellin-independent cellular invasion.

ABSTRACTDuring infection, Streptococcus pneumoniaeexists mainly in sessile biofilms rather than in planktonic form, except during sepsis. The capacity to form biofilms is believed to be important for nasopharyngeal colonization as well as disease pathogenesis, but relatively little is known about the regulation of this process. Here, we investigated the effect of exogenous iron [Fe(III)] as well as the role of luxS(encoding S-ribosylhomocysteine lyase) on biofilm formation by S. pneumoniaeD39. Fe(III) strongly enhanced biofilm formation at concentrations of ≥50 μM, while Fe(III) chelation with deferoxamine was inhibitory. Importantly, Fe(III) also upregulated the expression of luxSin wild-type D39. A luxS-deficient mutant (D39luxS) failed to form a biofilm, even with Fe(III) supplementation, whereas a derivative overexpressing luxS(D39luxS+) exhibited enhanced biofilm formation capacity and could form a biofilm without added Fe(III). D39luxSexhibited reduced expression of the major Fe(III) transporter PiuA, and the cellular [Fe(III)] was significantly lower than that in D39; in contrast, D39luxS+ had a significantly higher cellular [Fe(III)] than the wild type. The release of extracellular DNA, which is an important component of the biofilm matrix, also was directly related to luxSexpression. Similarly, genetic competence, as measured by transformation frequency as well as the expression of competence genes comD, comX, comW, cglA, and dltAand the murein hydrolase cbpD, which is associated with fratricide-dependent DNA release, all were directly related to luxSexpression levels and were further upregulated by Fe(III). Moreover, mutagenesis of cbpDblocked biofilm formation. We propose that competence, fratricide, and biofilm formation are closely linked in pneumococci, and that luxSis a central regulator of these processes. We also propose that the stimulatory effects of Fe(III) on all of these parameters are due to the upregulation of luxSexpression, and that LuxS provides for a positive Fe(III)-dependent amplification loop by increasing iron uptake.

During infection, Streptococcus pneumoniae exists mainly in sessile biofilms rather than in planktonic form, except during sepsis. The capacity to form biofilms is believed to be important for nasopharyngeal colonization as well as disease pathogenesis, but relatively little is known about the regulation of this process. Here, we investigated the effect of exogenous iron [Fe(III)] as well as the role of luxS (encoding S-ribosylhomocysteine lyase) on biofilm formation by S. pneumoniae D39. Fe(III) strongly enhanced biofilm formation at concentrations of ≥50 µM, while Fe(III) chelation with deferoxamine was inhibitory. Importantly, Fe(III) also upregulated the expression of luxS in wild-type D39. A luxS-deficient mutant (D39luxS) failed to form a biofilm, even with Fe(III) supplementation, whereas a derivative overexpressing luxS (D39luxS+) exhibited enhanced biofilm formation capacity and could form a biofilm without added Fe(III). D39luxS exhibited reduced expression of the major Fe(III) transporter PiuA, and the cellular [Fe(III)] was significantly lower than that in D39; in contrast, D39luxS+ had a significantly higher cellular [Fe(III)] than the wild type. The release of extracellular DNA, which is an important component of the biofilm matrix, also was directly related to luxS expression. Similarly, genetic competence, as measured by transformation frequency as well as the expression of competence genes comD, comX, comW, cglA, and dltA and the murein hydrolase cbpD, which is associated with fratricide-dependent DNA release, all were directly related to luxS expression levels and were further upregulated by Fe(III). Moreover, mutagenesis of cbpD blocked biofilm formation. We propose that competence, fratricide, and biofilm formation are closely linked in pneumococci, and that luxS is a central regulator of these processes. We also propose that the stimulatory effects of Fe(III) on all of these parameters are due to the upregulation of luxS expression, and that LuxS provides for a positive Fe(III)-dependent amplification loop by increasing iron uptake.

Subtilase cytotoxin (SubAB) is the prototype of a new family of AB5cytotoxins produced by Shiga-toxigenic Escherichia coli. Its cytotoxicity is due to its capacity to enter cells and specifically cleave the essential endoplasmic reticulum chaperone BiP. Previous studies have shown that intraperitoneal injection of mice with purified SubAB causes a pathology that overlaps with that seen in human cases of hemolytic-uremic syndrome, as well as dramatic splenic atrophy, suggesting that leukocytes are targeted. Here we investigated SubAB-induced leukocyte changes in the peritoneal cavity, blood, and spleen. After intraperitoneal injection, SubAB bound peritoneal leukocytes (including T and B lymphocytes, neutrophils, and macrophages). SubAB elicited marked leukocytosis, which peaked at 24 h, and increased neutrophil activation in the blood and peritoneal cavity. It also induced a marked redistribution of leukocytes among the three compartments: increases in leukocyte subpopulations in the blood and peritoneal cavity coincided with a significant decline in splenic cells. SubAB treatment also elicited significant increases in the apoptosis rates of CD4+T cells, B lymphocytes, and macrophages. These findings indicate that apart from direct cytotoxic effects, SubAB interacts with cellular components of both the innate and the adaptive arm of the immune system, with potential consequences for disease pathogenesis.

ABSTRACTSubtilase cytotoxin (SubAB) is the prototype of a new family of AB5cytotoxins produced by Shiga-toxigenic Escherichia coli. Its cytotoxicity is due to its capacity to enter cells and specifically cleave the essential endoplasmic reticulum chaperone BiP. Previous studies have shown that intraperitoneal injection of mice with purified SubAB causes a pathology that overlaps with that seen in human cases of hemolytic-uremic syndrome, as well as dramatic splenic atrophy, suggesting that leukocytes are targeted. Here we investigated SubAB-induced leukocyte changes in the peritoneal cavity, blood, and spleen. After intraperitoneal injection, SubAB bound peritoneal leukocytes (including T and B lymphocytes, neutrophils, and macrophages). SubAB elicited marked leukocytosis, which peaked at 24 h, and increased neutrophil activation in the blood and peritoneal cavity. It also induced a marked redistribution of leukocytes among the three compartments: increases in leukocyte subpopulations in the blood and peritoneal cavity coincided with a significant decline in splenic cells. SubAB treatment also elicited significant increases in the apoptosis rates of CD4+T cells, B lymphocytes, and macrophages. These findings indicate that apart from direct cytotoxic effects, SubAB interacts with cellular components of both the innate and the adaptive arm of the immune system, with potential consequences for disease pathogenesis.

ABSTRACTSubtilase cytotoxin (SubAB) was first isolated from a Shiga toxigenic Escherichia coli(STEC) strain that was responsible for an outbreak of hemolytic-uremic syndrome and is the prototype of a new family of AB5cytotoxins. SubAB is a subtilase-like serine protease, and upon uptake by host cells, it is trafficked to the endoplasmic reticulum (ER), where it cleaves the essential ER chaperone BiP (GRP78) with high specificity. Previous work has shown that BiP cleavage by SubAB initiates ER stress-signaling pathways in host cells that eventuate in cell death associated with DNA fragmentation, a hallmark of apoptosis. The present study has investigated the role of the Bcl-2 protein family, which has been shown to regulate ER stress-induced apoptosis in other model systems. Examination of the cytotoxicity of SubAB for wild-type and bax−/−/bak−/−mouse embryonic fibroblasts and comparison of apoptotic markers in these cells revealed that SubAB cytotoxicity can be predominantly attributed to the activation of apoptotic pathways activated by Bax/Bak. The results of the present study further our understanding of the molecular mechanism whereby SubAB kills eukaryotic cells and contributes to STEC pathogenesis, in addition to consolidating the roles of Bcl-2 family members in the regulation of ER stress-induced apoptosis.

Subtilase cytotoxin (SubAB) was first isolated from a Shiga toxigenic Escherichia coli (STEC) strain that was responsible for an outbreak of hemolytic-uremic syndrome and is the prototype of a new family of AB5cytotoxins. SubAB is a subtilase-like serine protease, and upon uptake by host cells, it is trafficked to the endoplasmic reticulum (ER), where it cleaves the essential ER chaperone BiP (GRP78) with high specificity. Previous work has shown that BiP cleavage by SubAB initiates ER stress-signaling pathways in host cells that eventuate in cell death associated with DNA fragmentation, a hallmark of apoptosis. The present study has investigated the role of the Bcl-2 protein family, which has been shown to regulate ER stress-induced apoptosis in other model systems. Examination of the cytotoxicity of SubAB for wild-type and bax–/–/bak–/–mouse embryonic fibroblasts and comparison of apoptotic markers in these cells revealed that SubAB cytotoxicity can be predominantly attributed to the activation of apoptotic pathways activated by Bax/Bak. The results of the present study further our understanding of the molecular mechanism whereby SubAB kills eukaryotic cells and contributes to STEC pathogenesis, in addition to consolidating the roles of Bcl-2 family members in the regulation of ER stress-induced apoptosis.

Targeting endoplasmic reticulum stress-induced apoptosis may offer an alternative therapeutic strategy for metastatic melanoma. Fenretinide and bortezomib induce apoptosis of melanoma cells but their efficacy may be hindered by the unfolded protein response, which promotes survival by ameliorating endoplasmic reticulum stress. The aim of this study was to test the hypothesis that inhibition of GRP78, a vital unfolded protein response mediator, increases cell death in combination with endoplasmic reticulum stress-inducing agents. Down-regulation of GRP78 by small-interfering RNA increased fenretinide- or bortezomib-induced apoptosis. Treatment of cells with a GRP78-specific subtilase toxin produced a synergistic enhancement with fenretinide or bortezomib. These data suggest that combining endoplasmic reticulum stress-inducing agents with strategies to down-regulate GRP78, or other components of the unfolded protein response, may represent a novel therapeutic approach for metastatic melanoma.

ABSTRACTShiga toxins expressed in the intestinal lumen during infection with Shiga-toxigenic Escherichia colimust translocate across the epithelium and enter the systemic circulation to cause systemic (pathological) effects, including hemolytic uremic syndrome. The transepithelial migration of polymorphonuclear leukocytes in response to chemokine expression by intestinal epithelial cells is thought to promote uptake of Stx from the intestinal lumen by compromising the epithelial barrier. In the present study, we investigated the hypothesis that flagellin acts in conjunction with Shiga toxin to augment this chemokine expression. We investigated the relative contributions of nuclear factor κB (NF-κB) and mitogen-activated protein kinase (MAPK) signaling to transcription and translation of interleukin-8. Using reporter gene constructs, we showed that flagellin-mediated interleukin-8 gene transcription is heavily dependent on both NF-κB and extracellular signal-regulated kinase 1 and 2 (ERK-1/2) activation. In contrast, inhibition of p38 has no detectable effect on interleukin-8 gene transcription, even though flagellin-mediated activation of host p38 is critical for maximal interleukin-8 protein expression. Inhibition of MAPK-interacting kinase 1 suggests that p38 signaling affects the posttranscriptional regulation of interleukin-8 protein expression induced by flagellin. Cotreatment with Stx2 and flagellin results in a synergistic upregulation of c-Jun N-terminal protein kinases (JNKs), p38 activation, and a superinduction of interleukin-8 mRNA. This synergism was also evident at the protein level, with increased interleukin-8 protein detectable following cotreatment with flagellin and Stx2. We propose that flagellin, in conjunction with Shiga toxin, synergistically upregulates stress-activated protein kinases, resulting in superinduction of interleukin-8 and, ultimately, absorption of Stx into the systemic circulation.

Shiga toxins expressed in the intestinal lumen during infection with Shiga-toxigenic Escherichia coli must translocate across the epithelium and enter the systemic circulation to cause systemic (pathological) effects, including hemolytic uremic syndrome. The transepithelial migration of polymorphonuclear leukocytes in response to chemokine expression by intestinal epithelial cells is thought to promote uptake of Stx from the intestinal lumen by compromising the epithelial barrier. In the present study, we investigated the hypothesis that flagellin acts in conjunction with Shiga toxin to augment this chemokine expression. We investigated the relative contributions of nuclear factor B (NF-B) and mitogen-activated protein kinase (MAPK) signaling to transcription and translation of interleukin-8. Using reporter gene constructs, we showed that flagellin-mediated interleukin-8 gene transcription is heavily dependent on both NF-B and extracellular signal-regulated kinase 1 and 2 (ERK-1/2) activation. In contrast, inhibition of p38 has no detectable effect on interleukin-8 gene transcription, even though flagellin-mediated activation of host p38 is critical for maximal interleukin-8 protein expression. Inhibition of MAPK-interacting kinase 1 suggests that p38 signaling affects the posttranscriptional regulation of interleukin-8 protein expression induced by flagellin. Cotreatment with Stx2 and flagellin results in a synergistic upregulation of c-Jun N-terminal protein kinases (JNKs), p38 activation, and a superinduction of interleukin-8 mRNA. This synergism was also evident at the protein level, with increased interleukin-8 protein detectable following cotreatment with flagellin and Stx2. We propose that flagellin, in conjunction with Shiga toxin, synergistically upregulates stress-activated protein kinases, resulting in superinduction of interleukin-8 and, ultimately, absorption of Stx into the systemic circulation.

Many microbial pathogens recognise oligosaccharides displayed on the surface of host cells as receptors for toxins and adhesins. These ligand-receptor interactions are critical for disease pathogenesis, making them promising targets for novel anti-infectives. One strategy with particular utility against enteric infections involves expression of molecular mimics of host oligosaccharides on the surface of harmless bacteria capable of surviving in the gut. This can be achieved in Gram-negative bacteria by manipulating the outer core region of the lipopolysaccharide (LPS) through expression of cloned heterologous glycosyltransferases. The resultant chimeric LPS molecules are incorporated into the outer membrane by the normal assembly route and presented as a closely packed 2-D array of receptor mimics. Several such “designer probiotics” have been constructed, and these bind bacterial toxins in the gut lumen with very high avidity, blocking their uptake by host cells and thereby preventing disease.

Shiga-toxigenic Escherichia coli (STEC) strains cause serious gastrointestinal disease, which can lead to potentially life-threatening systemic complications such as hemolytic-uremic syndrome. Although the production of Shiga toxin has been considered to be the main virulence trait of STEC for many years, the capacity to colonize the host intestinal epithelium is a crucial step in pathogenesis. In this study, we have characterized a novel megaplasmid-encoded outer membrane protein in locus of enterocyte effacement (LEE)-negative O113:H21 STEC strain 98NK2, termed Sab (for STEC autotransporter [AT] contributing to biofilm formation). The 4,296-bp sab gene encodes a 1,431-amino-acid protein with the features of members of the AT protein family. When expressed in E. coli JM109, Sab contributed to the diffuse adherence to human epithelial (HEp-2) cells and promoted biofilm formation on polystyrene surfaces. A 98NK2 sab deletion mutant was also defective in biofilm formation relative to its otherwise isogenic wild-type parent, and this was complemented by transformation with a sab-carrying plasmid. Interestingly, an unrelated O113:H21 STEC isolate that had a naturally occurring deletion in sab was similarly defective in biofilm formation. PCR analysis indicated that sab is present in LEE-negative STEC strains belonging to serotypes/groups O113:H21, O23, and O82:H8. These findings raise the possibility that Sab may contribute to colonization in a subset of LEE-negative STEC strains.

ABSTRACTShiga-toxigenic Escherichia coli(STEC) strains cause serious gastrointestinal disease, which can lead to potentially life-threatening systemic complications such as hemolytic-uremic syndrome. Although the production of Shiga toxin has been considered to be the main virulence trait of STEC for many years, the capacity to colonize the host intestinal epithelium is a crucial step in pathogenesis. In this study, we have characterized a novel megaplasmid-encoded outer membrane protein in locus of enterocyte effacement (LEE)-negative O113:H21 STEC strain 98NK2, termed Sab (for STEC autotransporter [AT] contributing to biofilm formation). The 4,296-bp sabgene encodes a 1,431-amino-acid protein with the features of members of the AT protein family. When expressed in E. coliJM109, Sab contributed to the diffuse adherence to human epithelial (HEp-2) cells and promoted biofilm formation on polystyrene surfaces. A 98NK2 sabdeletion mutant was also defective in biofilm formation relative to its otherwise isogenic wild-type parent, and this was complemented by transformation with a sab-carrying plasmid. Interestingly, an unrelated O113:H21 STEC isolate that had a naturally occurring deletion in sabwas similarly defective in biofilm formation. PCR analysis indicated that sabis present in LEE-negative STEC strains belonging to serotypes/groups O113:H21, O23, and O82:H8. These findings raise the possibility that Sab may contribute to colonization in a subset of LEE-negative STEC strains.

We found that, in murine podocytes, expression of monocyte chemoattractant protein 1 (MCP-1) in response to TNF-α was suppressed by indomethacin but not by ibuprofen. This anti-inflammatory potential was correlated with induction of 78-kDa glucose-regulated protein (GRP78), a marker of unfolded protein response (UPR). Indomethacin, but not ibuprofen, also triggered expression of CHOP, another endogenous indicator of UPR, as well as repression of endoplasmic reticulum stress-responsive alkaline phosphatase, an exogenous indicator of UPR. Like ibuprofen, other nonsteroidal anti-inflammatory drugs including aspirin and sulindac also did not induce UPR, indicating that the induction of UPR by indomethacin was independent of cyclooxygenase inhibition. The induction of UPR by indomethacin was observed similarly in other cells including mesangial cells and tubular epithelial cells. In tumor necrosis factor (TNF)-α-treated cells, suppression of MCP-1by indomethacin was inversely correlated with induction of UPR, and other inducers of UPR including tunicamycin, thapsigargin, and A23187 reproduced the suppressive effect. Reporter assays showed that indomethacin as well as thapsigargin attenuated activation of NF-κB by TNF-α, and it was associated with enhanced degradation of TNF receptor-associated factor 2 (TRAF2) and blunted degradation of IκBβ. Subsequent experiments revealed that acute ablation of GRP78 protein by AB5subtilase cytotoxin caused reinforcement of MCP-1induction and NF-κB activation by TNF-α and that transfection with GRP78significantly suppressed the cytokine-induced activation of NF-κB. These results suggested that indomethacin suppressed the response of podocytes to TNF-α via UPR and that UPR-triggered induction of GRP78 and degradation of TRAF2 may be responsible, at least in part, for the suppressive effect of indomethacin.

The contribution of flagellin to the virulence of the O113:H21 Shiga-toxigenic Escherichia coli (STEC) strain 98NK2 was investigated in the streptomycin-treated mouse model. Groups of mice were challenged with either the wild-type STEC or a fliC deletion derivative thereof. There was no difference in the level of gut colonization by the two strains, but the fliC mutant was significantly less virulent than its parent; the overall survival rates were 43.7% and 81.2%, respectively (P < 0.025). This is the first report of a nontoxic accessory virulence factor contributing to a fatal outcome of STEC infection in this model. Although H21 FliC is known to be a potent inducer of CXC chemokines, including interleukin 8, there was no obvious difference in the recruitment of polymorphonuclear leukocytes to the intestinal epithelium of mice challenged with either strain. However, immunofluorescence microscopy suggested that the fliC mutant was less capable of forming a close association with the colonic epithelium. This may have reduced the uptake of Stx2 by mice infected with the mutant.

ABSTRACTThe contribution of flagellin to the virulence of the O113:H21 Shiga-toxigenic Escherichia coli(STEC) strain 98NK2 was investigated in the streptomycin-treated mouse model. Groups of mice were challenged with either the wild-type STEC or a fliCdeletion derivative thereof. There was no difference in the level of gut colonization by the two strains, but the fliCmutant was significantly less virulent than its parent; the overall survival rates were 43.7% and 81.2%, respectively (P< 0.025). This is the first report of a nontoxic accessory virulence factor contributing to a fatal outcome of STEC infection in this model. Although H21 FliC is known to be a potent inducer of CXC chemokines, including interleukin 8, there was no obvious difference in the recruitment of polymorphonuclear leukocytes to the intestinal epithelium of mice challenged with either strain. However, immunofluorescence microscopy suggested that the fliCmutant was less capable of forming a close association with the colonic epithelium. This may have reduced the uptake of Stx2 by mice infected with the mutant.

We have recently described a novel AB5 cytotoxin produced by certain Shiga toxin-producing Escherichia coli strains. The A subunit of this toxin is a subtilase-like serine protease, while the B pentamer mediates binding to host cell glycolipid receptors. The subtilase cytotoxin is lethal for mice, causing extensive microvascular thrombosis as well as necrosis in the brain, kidneys, and liver. In the present study, we have immunized mice with a purified derivative of the toxin with a Ser272 → Ala mutation in the A subunit which abolishes cytotoxicity. This elicited strong antibody responses, as judged by enzyme-linked immunosorbent assay, which conferred protection against intraperitoneal challenge with purified toxin. Immunized mice were also protected from weight loss resulting from oral challenge with an E. coli K-12 clone expressing the active toxin.

ABSTRACTWe have recently described a novel AB5cytotoxin produced by certain Shiga toxin-producing Escherichia colistrains. The A subunit of this toxin is a subtilase-like serine protease, while the B pentamer mediates binding to host cell glycolipid receptors. The subtilase cytotoxin is lethal for mice, causing extensive microvascular thrombosis as well as necrosis in the brain, kidneys, and liver. In the present study, we have immunized mice with a purified derivative of the toxin with a Ser272→ Ala mutation in the A subunit which abolishes cytotoxicity. This elicited strong antibody responses, as judged by enzyme-linked immunosorbent assay, which conferred protection against intraperitoneal challenge with purified toxin. Immunized mice were also protected from weight loss resulting from oral challenge with an E. coliK-12 clone expressing the active toxin.

ABSTRACTWe have recently described a novel AB5subtilase cytotoxin produced by certain Shiga toxigenic Escherichia coli(STEC) strains. This potentially lethal toxin may contribute to severe gastrointestinal and systemic disease in humans. In this study we have developed a trivalent PCR assay for the detection of the novel toxin A subunit gene subA, as well as stx1and stx2. The three primer pairs used in the assay do not interfere with each other and generate amplification products of 556, 180, and 255 bp, respectively. The assay can be used for determining the toxin genotype of STEC isolates, as well as for direct detection of toxin genes in primary fecal culture extracts.

The Shiga toxigenic Escherichia coli (STEC) O113:H21 strain 98NK2, which was responsible for an outbreak of hemolytic uremic syndrome, secretes a highly potent and lethal subtilase cytotoxin that is unrelated to any bacterial toxin described to date. It is the prototype of a new family of AB5 toxins, comprising a single 35-kilodalton (kD) A subunit and a pentamer of 13-kD B subunits. The A subunit is a subtilase-like serine protease distantly related to the BA_2875 gene product of Bacillus anthracis. The B subunit is related to a putative exported protein from Yersinia pestis, and binds to a mimic of the ganglioside GM2. Subtilase cytotoxin is encoded by two closely linked, cotranscribed genes (subA and subB), which, in strain 98NK2, are located on a large, conjugative virulence plasmid. Homologues of the genes are present in 32 out of 68 other STEC strains tested. Intraperitoneal injection of purified subtilase cytotoxin was fatal for mice and resulted in extensive microvascular thrombosis, as well as necrosis in the brain, kidneys, and liver. Oral challenge of mice with E. coli K-12–expressing cloned subA and subB resulted in dramatic weight loss. These findings suggest that the toxin may contribute to the pathogenesis of human disease.

There is increasing evidence that by facilitating translocation of Shiga toxin (Stx) across the intestinal epithelium and by transporting bound toxin to remote sites such as the renal endothelium, polymorphonuclear leukocytes (PMNs) play a key role in the pathogenesis of Shiga-toxigenic Escherichia coli (STEC) disease. Plasma levels of PMN-attracting CXC chemokines such as interleukin-8 (IL-8) also appear to correlate in humans with the severity of disease. Thus, the capacity of STEC strains to elicit CXC chemokine responses in intestinal epithelial cells may be a crucial step in pathogenesis. Accordingly, we attempted to determine which STEC factors are responsible for CXC chemokine induction in human colonic epithelial cells. Infection of Hct-8 cells with locus for enterocyte effacement (LEE)-negative STEC strains isolated from patients with severe STEC disease resulted in up-regulation of IL-8, macrophage inflammatory protein 2alpha (MIP-2alpha), MIP-2beta, and ENA-78 mRNA significantly higher and earlier than that elicited by several LEE-positive STEC strains, including the O157:H7 strain EDL933. Similarly, levels of IL-8 protein in LEE-negative STEC-infected Hct-8 culture supernatants were significantly higher than in LEE-positive STEC-infected culture supernatants. The difference in responses could not be attributed to the expression or nonexpression of LEE genes, the presence or absence of an STEC megaplasmid, or differences in O serogroups or in the type or amount of Stx produced. Interestingly, however, several of the LEE-negative STEC strains eliciting the strongest chemokine responses belonged to flagellar serotype H21. Incubation of Hct-8 cells with isolated H21 flagellin elicited IL-8 and MIP-2alpha responses similar to those seen in the presence of the most potent LEE-negative STEC strains. Deletion of the fliC gene, but not the stx(2) gene, largely abolished the capacity of O113:H21 LEE-negative STEC strain 98NK2 to elicit IL-8 and MIP-2alpha responses in Hct-8 cells. Taken together, these data suggest that although Stx is capable of inducing CXC chemokine responses, the elevated responses seen in cells infected with certain STEC strains are largely attributable to the production of flagellin.