Crystal structure of bacterial cytotoxic necrotizing factor CNFY reveals molecular building blocks for intoxication.

Cytotoxic necrotizing factors (CNFs) are bacterial single-chain exotoxins that modulate cytokinetic/oncogenic and inflammatory processes through activation of host cell Rho GTPases. To achieve this, they are secreted, bind surface receptors to induce endocytosis and translocate a catalytic unit into the cytosol to intoxicate host cells. A three-dimensional structure that provides insight into the underlying mechanisms is still lacking. Here, we determined the crystal structure of full-length Yersinia pseudotuberculosis CNFY . CNFY consists of five domains (D1-D5), and by integrating structural and functional data, we demonstrate that D1-3 act as export and translocation module for the catalytic unit (D4-5) and for a fused ß-lactamase reporter protein. We further found that D4, which possesses structural similarity to ADP-ribosyl transferases, but had no equivalent catalytic activity, changed its position to interact extensively with D5 in the crystal structure of the free D4-5 fragment. This liberates D5 from a semi-blocked conformation in full-length CNFY , leading to higher deamidation activity. Finally, we identify CNF translocation modules in several uncharacterized fusion proteins, which suggests their usability as a broad-specificity protein delivery tool.

Infection and antibiotic-associated changes in the fecal microbiota of C. rodentium ϕstx2dact-infected C57BL/6 mice.

Enterohemorrhagic Escherichia coli causes watery to bloody diarrhea, which may progress to hemorrhagic colitis and hemolytic-uremic syndrome. While early studies suggested that antibiotic treatment may worsen the pathology of an enterohemorrhagic Escherichia coli (EHEC) infection, recent work has shown that certain non-Shiga toxin-inducing antibiotics avert disease progression. Unfortunately, both intestinal bacterial infections and antibiotic treatment are associated with dysbiosis. This can alleviate colonization resistance, facilitate secondary infections, and potentially lead to more severe illness. To address the consequences in the context of an EHEC infection, we used the established mouse infection model organism Citrobacter rodentium ϕstx2dact and monitored changes in fecal microbiota composition during infection and antibiotic treatment. C. rodentium ϕstx2dact infection resulted in minor changes compared to antibiotic treatment. The infection caused clear alterations in the microbial community, leading mainly to a reduction of Muribaculaceae and a transient increase in Enterobacteriaceae distinct from Citrobacter. Antibiotic treatments of the infection resulted in marked and distinct variations in microbiota composition, diversity, and dispersion. Enrofloxacin and trimethoprim/sulfamethoxazole, which did not prevent Shiga toxin-mediated organ damage, had the least disruptive effects on the intestinal microbiota, while kanamycin and tetracycline, which rapidly cleared the infection without causing organ damage, caused a severe reduction in diversity. Kanamycin treatment resulted in the depletion of all but Bacteroidetes genera, whereas tetracycline effects on Clostridia were less severe. Together, these data highlight the need to address the impact of individual antibiotics in the clinical care of life-threatening infections and consider microbiota-regenerating therapies.IMPORTANCEUnderstanding the impact of antibiotic treatment on EHEC infections is crucial for appropriate clinical care. While discouraged by early studies, recent findings suggest certain antibiotics can impede disease progression. Here, we investigated the impact of individual antibiotics on the fecal microbiota in the context of an established EHEC mouse model using C. rodentium ϕstx2dact. The infection caused significant variations in the microbiota, leading to a transient increase in Enterobacteriaceae distinct from Citrobacter. However, these effects were minor compared to those observed for antibiotic treatments. Indeed, antibiotics that most efficiently cleared the infection also had the most detrimental effect on the fecal microbiota, causing a substantial reduction in microbial diversity. Conversely, antibiotics showing adverse effects or incomplete bacterial clearance had a reduced impact on microbiota composition and diversity. Taken together, our findings emphasize the delicate balance required to weigh the harmful effects of infection and antibiosis in treatment.

An RNA thermometer dictates production of a secreted bacterial toxin.

Frequent transitions of bacterial pathogens between their warm-blooded host and external reservoirs are accompanied by abrupt temperature shifts. A temperature of 37°C serves as reliable signal for ingestion by a mammalian host, which induces a major reprogramming of bacterial gene expression and metabolism. Enteric Yersiniae are Gram-negative pathogens accountable for self-limiting gastrointestinal infections. Among the temperature-regulated virulence genes of Yersinia pseudotuberculosis is cnfY coding for the cytotoxic necrotizing factor (CNFY), a multifunctional secreted toxin that modulates the host's innate immune system and contributes to the decision between acute infection and persistence. We report that the major determinant of temperature-regulated cnfY expression is a thermo-labile RNA structure in the 5'-untranslated region (5'-UTR). Various translational gene fusions demonstrated that this region faithfully regulates translation initiation regardless of the transcription start site, promoter or reporter strain. RNA structure probing revealed a labile stem-loop structure, in which the ribosome binding site is partially occluded at 25°C but liberated at 37°C. Consistent with translational control in bacteria, toeprinting (primer extension inhibition) experiments in vitro showed increased ribosome binding at elevated temperature. Point mutations locking the 5'-UTR in its 25°C structure impaired opening of the stem loop, ribosome access and translation initiation at 37°C. To assess the in vivo relevance of temperature control, we used a mouse infection model. Y. pseudotuberculosis strains carrying stabilized RNA thermometer variants upstream of cnfY were avirulent and attenuated in their ability to disseminate into mesenteric lymph nodes and spleen. We conclude with a model, in which the RNA thermometer acts as translational roadblock in a two-layered regulatory cascade that tightly controls provision of the CNFY toxin during acute infection. Similar RNA structures upstream of various cnfY homologs suggest that RNA thermosensors dictate the production of secreted toxins in a wide range of pathogens.

Variations in microbiota composition of laboratory mice influence Citrobacter rodentium infection via variable short-chain fatty acid production.

The composition of the intestinal microbiota influences the outcome of enteric infections in human and mice. However, the role of specific members and their metabolites contributing to disease severity is largely unknown. Using isogenic mouse lines harboring distinct microbiota communities, we observed highly variable disease kinetics of enteric Citrobacter rodentium colonization after infection. Transfer of communities from susceptible and resistant mice into germ-free mice verified that the varying susceptibilities are determined by microbiota composition. The strongest differences in colonization were observed in the cecum and could be maintained in vitro by coculturing cecal bacteria with C. rodentium. Cohousing of animals as well as the transfer of cultivable bacteria from resistant to susceptible mice led to variable outcomes in the recipient mice. Microbiome analysis revealed that a higher abundance of butyrate-producing bacteria was associated with the resistant phenotype. Quantification of short-chain fatty acid (SCFA) levels before and after infection revealed increased concentrations of acetate, butyrate and propionate in mice with delayed colonization. Addition of physiological concentrations of butyrate, but not of acetate and/or propionate strongly impaired growth of C. rodentium in vitro. In vivo supplementation of susceptible, antibiotic-treated and germ-free mice with butyrate led to the same level of protection, notably only when cecal butyrate concentration reached a concentration higher than 50 nmol/mg indicating a critical threshold for protection. In the recent years, commensal-derived primary and secondary bacterial metabolites emerged as potent modulators of hosts susceptibility to infection. Our results provide evidence that variations in SCFA production in mice fed fibre-rich chow-based diets modulate susceptibility to colonization with Enterobacteriaceae not only in antibiotic-disturbed ecosystems but even in undisturbed microbial communities. These findings emphasise the need for microbiota normalization across laboratory mouse lines for infection experiments with the model-pathogen C. rodentium independent of investigations of diet and antibiotic usage.

Identification of Translocation Inhibitors Targeting the Type III Secretion System of Enteropathogenic Escherichia coli.

Infections with enteropathogenic Escherichia coli (EPEC) cause severe diarrhea in children. The noninvasive bacteria adhere to enterocytes of the small intestine and use a type III secretion system (T3SS) to inject effector proteins into host cells to modify and exploit cellular processes in favor of bacterial survival and replication. Several studies have shown that the T3SSs of bacterial pathogens are essential for virulence. Furthermore, the loss of T3SS-mediated effector translocation results in increased immune recognition and clearance of the bacteria. The T3SS is, therefore, considered a promising target for antivirulence strategies and novel therapeutics development. Here, we report the results of a high-throughput screening assay based on the translocation of the EPEC effector protein Tir (translocated intimin receptor). Using this assay, we screened more than 13,000 small molecular compounds of six different compound libraries and identified three substances which showed a significant dose-dependent effect on translocation without adverse effects on bacterial or eukaryotic cell viability. In addition, these substances reduced bacterial binding to host cells, effector-dependent cell detachment, and abolished attaching and effacing lesion formation without affecting the expression of components of the T3SS or associated effector proteins. Moreover, no effects of the inhibitors on bacterial motility or Shiga-toxin expression were observed. In summary, we have identified three new compounds that strongly inhibit T3SS-mediated translocation of effectors into mammalian cells, which could be valuable as lead substances for treating EPEC and enterohemorrhagic E. coli infections.

Identification of Antibiotics That Diminish Disease in a Murine Model of Enterohemorrhagic Escherichia coli Infection.

Infections with enterohemorrhagic Escherichia coli (EHEC) cause disease ranging from mild diarrhea to hemolytic-uremic syndrome (HUS) and are the most common cause of renal failure in children in high-income countries. The severity of the disease derives from the release of Shiga toxins (Stx). The use of antibiotics to treat EHEC infections is generally avoided, as it can result in increased stx expression. Here, we systematically tested different classes of antibiotics and found that their influence on stx expression and release varies significantly. We assessed a selection of these antibiotics in vivo using the Citrobacter rodentium ϕstx2dact mouse model and show that stx2d-inducing antibiotics resulted in weight loss and kidney damage despite clearance of the infection. However, several non-Stx-inducing antibiotics cleared bacterial infection without causing Stx-mediated pathology. Our results suggest that these antibiotics might be useful in the treatment of EHEC-infected human patients and decrease the risk of HUS development.

A type III effector antagonizes death receptor signalling during bacterial gut infection.

Successful infection by enteric bacterial pathogens depends on the ability of the bacteria to colonize the gut, replicate in host tissues and disseminate to other hosts. Pathogens such as Salmonella, Shigella and enteropathogenic and enterohaemorrhagic (EPEC and EHEC, respectively) Escherichia coli use a type III secretion system (T3SS) to deliver virulence effector proteins into host cells during infection that promote colonization and interfere with antimicrobial host responses. Here we report that the T3SS effector NleB1 from EPEC binds to host cell death-domain-containing proteins and thereby inhibits death receptor signalling. Protein interaction studies identified FADD, TRADD and RIPK1 as binding partners of NleB1. NleB1 expressed ectopically or injected by the bacterial T3SS prevented Fas ligand or TNF-induced formation of the canonical death-inducing signalling complex (DISC) and proteolytic activation of caspase-8, an essential step in death-receptor-induced apoptosis. This inhibition depended on the N-acetylglucosamine transferase activity of NleB1, which specifically modified Arg 117 in the death domain of FADD. The importance of the death receptor apoptotic pathway to host defence was demonstrated using mice deficient in the FAS signalling pathway, which showed delayed clearance of the EPEC-like mouse pathogen Citrobacter rodentium and reversion to virulence of an nleB mutant. The activity of NleB suggests that EPEC and other attaching and effacing pathogens antagonize death-receptor-induced apoptosis of infected cells, thereby blocking a major antimicrobial host response.

Identification of a Distinct Substrate-binding Domain in the Bacterial Cysteine Methyltransferase Effectors NleE and OspZ.

The type III secretion system effector protein NleE from enteropathogenic Escherichia coli plays a key role in the inhibition of NF-κB activation during infection. NleE inactivates the ubiquitin chain binding activity of host proteins TAK1-binding proteins 2 and 3 (TAB2 and TAB3) by modifying the Npl4 zinc finger domain through S-adenosyl methionine-dependent cysteine methylation. Using yeast two-hybrid protein interaction studies, we found that a conserved region between amino acids 34 and 52 of NleE, in particular the motif (49)GITR(52), was critical for TAB2 and TAB3 binding. NleE mutants lacking (49)GITR(52) were unable to methylate TAB3, and wild type NleE but not NleE(49AAAA52) where each of GITR was replaced with alanine restored the ability of an nleE mutant to inhibit IL-8 production during infection. Another NleE target, ZRANB3, also associated with NleE through the (49)GITR(52) motif. Ectopic expression of an N-terminal fragment of NleE (NleE(34-52)) in HeLa cells showed competitive inhibition of wild type NleE in the suppression of IL-8 secretion during enteropathogenic E. coli infection. Similar results were observed for the NleE homologue OspZ from Shigella flexneri 6 that also bound TAB3 through the (49)GITR(52) motif and decreased IL-8 transcription through modification of TAB3. In summary, we have identified a unique substrate-binding motif in NleE and OspZ that is required for the ability to inhibit the host inflammatory response.

The Type III Effector NleD from Enteropathogenic Escherichia coli Differentiates between Host Substrates p38 and JNK.

Enteropathogenic Escherichia coli (EPEC) is a gastrointestinal pathogen that utilizes a type III secretion system (T3SS) to inject an array of virulence effector proteins into host enterocytes to subvert numerous cellular processes for successful colonization and dissemination. The T3SS effector NleD is a 26-kDa zinc metalloprotease that is translocated into host enterocytes, where it directly cleaves and inactivates the mitogen-activated protein kinase signaling proteins JNK and p38. Here a library of 91 random transposon-based, in-frame, linker insertion mutants of NleD were tested for their ability to cleave JNK and p38 during transient transfection of cultured epithelial cells. Immunoblot analysis of p38 and JNK cleavage showed that 7 mutant derivatives of NleD no longer cleaved p38 but maintained the ability to cleave JNK. Site-directed mutation of specific regions surrounding the insertion sites within NleD revealed that a single amino acid, R203, was essential for cleavage of p38 but not JNK in a direct in vitro cleavage assay, in transiently transfected cells, or in EPEC-infected cells. Mass spectrometry analysis narrowed the cleavage region to within residues 187 and 213 of p38. Mutation of residue R203 within NleD to a glutamate residue abolished the cleavage of p38 and impaired the ability of NleD to inhibit AP-1-dependent gene transcription of a luciferase reporter. Furthermore, the R203 mutation abrogated the ability of NleD to dampen interleukin-6 production in EPEC-infected cells. Overall, this work provides greater insight into substrate recognition and specificity by the type III effector NleD.

Anti-virulence Strategies to Target Bacterial Infections.

Resistance of important bacterial pathogens to common antimicrobial therapies and the emergence of multidrug-resistant bacteria are increasing at an alarming rate and constitute one of our greatest challenges in the combat of bacterial infection and accompanied diseases. The current shortage of effective drugs, lack of successful prevention measures and only a few new antibiotics in the clinical pipeline demand the development of novel treatment options and alternative antimicrobial therapies. Our increasing understanding of bacterial virulence strategies and the induced molecular pathways of the infectious disease provides novel opportunities to target and interfere with crucial pathogenicity factors or virulence-associated traits of the bacteria while bypassing the evolutionary pressure on the bacterium to develop resistance. In the past decade, numerous new bacterial targets for anti-virulence therapies have been identified, and structure-based tailoring of intervention strategies and screening assays for small-molecule inhibitors of such pathways were successfully established. In this chapter, we will take a closer look at the bacterial virulence-related factors and processes that present promising targets for anti-virulence therapies, recently discovered inhibitory substances and their promises and discuss the challenges, and problems that have to be faced.

Substrate recognition by the zinc metalloprotease effector NleC from enteropathogenic Escherichia coli.

Upon infection of epithelial cells, enteropathogenic Escherichia coli suppresses host cell inflammatory signalling in a type III secretion system (T3SS) dependent manner. Two key T3SS effector proteins involved in this response are NleE and NleC. NleC is a zinc metalloprotease effector that degrades the p65 subunit of NF-κB. Although the site of p65 cleavage by NleC is now well described, other areas of interaction have not been precisely defined. Here we constructed overlapping truncations of p65 to identify regions required for NleC cleavage. We determined that NleC cleaved both p65 and p50 within the Rel homology domain (RHD) and that two motifs, E22IIE25 and P177VLS180 , within the RHD of p65 were important for recognition and binding by NleC. Alanine substitution of one or both of these motifs protected p65 from binding and degradation by NleC. The E22IIE25 and P177VLS180 motifs were located within the structurally distinct N-terminal subdomain of the RHD involved in DNA binding by p65 on adjacent, parallel strands. Although these motifs have not been recognized previously, both were needed for the correct localization and function of p65. In summary, this work has identified two regions of p65 within the RHD needed for binding and cleavage by NleC and provides further insight into the molecular basis of substrate recognition by a T3SS effector.

The enteropathogenic E. coli (EPEC) Tir effector inhibits NF-κB activity by targeting TNFα receptor-associated factors.

Enteropathogenic Escherichia coli (EPEC) disease depends on the transfer of effector proteins into epithelia lining the human small intestine. EPEC E2348/69 has at least 20 effector genes of which six are located with the effector-delivery system genes on the Locus of Enterocyte Effacement (LEE) Pathogenicity Island. Our previous work implied that non-LEE-encoded (Nle) effectors possess functions that inhibit epithelial anti-microbial and inflammation-inducing responses by blocking NF-κB transcription factor activity. Indeed, screens by us and others have identified novel inhibitory mechanisms for NleC and NleH, with key co-operative functions for NleB1 and NleE1. Here, we demonstrate that the LEE-encoded Translocated-intimin receptor (Tir) effector has a potent and specific ability to inhibit NF-κB activation. Indeed, biochemical, imaging and immunoprecipitation studies reveal a novel inhibitory mechanism whereby Tir interaction with cytoplasm-located TNFα receptor-associated factor (TRAF) adaptor proteins induces their proteasomal-independent degradation. Infection studies support this Tir-TRAF relationship but reveal that Tir, like NleC and NleH, has a non-essential contribution in EPEC's NF-κB inhibitory capacity linked to Tir's activity being suppressed by undefined EPEC factors. Infections in a disease-relevant intestinal model confirm key NF-κB inhibitory roles for the NleB1/NleE1 effectors, with other studies providing insights on host targets. The work not only reveals a second Intimin-independent property for Tir and a novel EPEC effector-mediated NF-κB inhibitory mechanism but also lends itself to speculations on the evolution of EPEC's capacity to inhibit NF-κB function.

An innate immune sensor wandering around - NOD1 promotes cell migration via non-canonical signaling.

NOD1 is a cytosolic immune receptor well known for recognizing intracellular bacteria and inducing innate immune responses. Upon ligand binding, it usually forms a complex with the serine/threonine kinase RIPK2 to activate the transcription factor NF-κB. Next to its role in pathogen recognition, NOD1 has been associated with cancer progression. In this regard, Hezinger et al. investigated a non-canonical role of NOD1 in cell migration. They discovered that NOD1 is crucial for the migration and chemotaxis of HeLa cells and identified HAX-1 as a novel interaction partner.

Proteasome-independent degradation of canonical NFkappaB complex components by the NleC protein of pathogenic Escherichia coli.

The NFκB transcription factor is a key component of immune and inflammatory signaling as its activation induces the expression of antimicrobial reagents, chemokines, cytokines, and anti-apoptotic factors. Many pathogens encode effector proteins that target factors regulating NFκB activity and can provide novel insights on regulatory mechanisms. Given the link of NFκB dysfunction with inflammatory diseases and some cancers, these effectors have therapeutic potential. Here, screening enteropathogenic Escherichia coli proteins for those implicated in suppressing NFκB function revealed that eGFP-NleC, unlike eGFP, strongly inhibited basal and TNFα-induced NFκB reporter activity to prevent secretion of the chemokine, IL-8. Work involving NleC variants, chemical inhibitors, and immunoprecipitation studies support NleC being a zinc metalloprotease that degrades NFκB-IκBα complexes. The findings are consistent with features between residues 33-65 recruiting NFκB for proteasomal-independent degradation by a mechanism inhibited by metalloprotease inhibitors or disruption of a consensus zinc metalloprotease motif spanning NleC residues 183-187. This raises the prospect that mammalian cells, or other pathogens, employ a similar mechanism to modulate NFκB activity. Moreover, NleC represents a novel tool for validating NFκB as a therapeutic target and, indeed, as a possible therapeutic reagent.

Targeting bacterial pathogenesis by inhibiting virulence-associated Type III and Type IV secretion systems.

Infections caused by Gram-negative pathogens pose a major health burden. Both respiratory and gastrointestinal infections are commonly associated with these pathogens. With the increase in antimicrobial resistance (AMR) over the last decades, bacterial infections may soon become the threat they have been before the discovery of antibiotics. Many Gram-negative pathogens encode virulence-associated Type III and Type IV secretion systems, which they use to inject bacterial effector proteins across bacterial and host cell membranes into the host cell cytosol, where they subvert host cell functions in favor of bacterial replication and survival. These secretion systems are essential for the pathogens to cause disease, and secretion system mutants are commonly avirulent in infection models. Hence, these structures present attractive targets for anti-virulence therapies. Here, we review previously and recently identified inhibitors of virulence-associated bacterial secretions systems and discuss their potential as therapeutics.

The bacterial effectors EspG and EspG2 induce a destructive calpain activity that is kept in check by the co-delivered Tir effector.

Bacterial pathogens deliver multiple effector proteins into eukaryotic cells to subvert host cellular processes and an emerging theme is the cooperation between different effectors. Here, we reveal that a fine balance exists between effectors that are delivered by enteropathogenic E. coli (EPEC) which, if perturbed can have marked consequences on the outcome of the infection. We show that absence of the EPEC effector Tir confers onto the bacterium a potent ability to destroy polarized intestinal epithelia through extensive host cell detachment. This process was dependent on the EPEC effectors EspG and EspG2 through their activation of the host cysteine protease calpain. EspG and EspG2 are shown to activate calpain during EPEC infection, which increases significantly in the absence of Tir - leading to rapid host cell loss and necrosis. These findings reveal a new function for EspG and EspG2 and show that Tir, independent of its bacterial ligand Intimin, is essential for maintaining the integrity of the epithelium during EPEC infection by keeping the destructive activity of EspG and EspG2 in check.

Autophagy-A Story of Bacteria Interfering with the Host Cell Degradation Machinery.

Autophagy is a highly conserved and fundamental cellular process to maintain cellular homeostasis through recycling of defective organelles or proteins. In a response to intracellular pathogens, autophagy further acts as an innate immune response mechanism to eliminate pathogens. This review will discuss recent findings on autophagy as a reaction to intracellular pathogens, such as Salmonella typhimurium, Listeria monocytogenes, Mycobacterium tuberculosis, Staphylococcus aureus, and pathogenic Escherichia coli. Interestingly, while some of these bacteria have developed methods to use autophagy for their own benefit within the cell, others have developed fascinating mechanisms to evade recognition, to subvert the autophagic pathway, or to escape from autophagy.

The EspF effector, a bacterial pathogen's Swiss army knife.

Central to the pathogenesis of many bacterial pathogens is the ability to deliver effector proteins directly into the cells of their eukaryotic host. EspF is one of many effector proteins exclusive to the attaching and effacing pathogen family that includes enteropathogenic (EPEC) and enterohemorrhagic (EHEC) Escherichia coli. Work in recent years has revealed EspF to be one of the most multifunctional effector proteins known, with defined roles in several host cellular processes, including disruption of the epithelial barrier, antiphagocytosis, microvillus effacement, host membrane remodelling, modulation of the cytoskeleton, targeting and disruption of the nucleolus, intermediate filament disruption, cell invasion, mitochondrial dysfunction, apoptosis, and inhibition of several important epithelial transporters. Surprisingly, despite this high number of functions, EspF is a relatively small effector protein, and recent work has begun to decipher the molecular events that underlie its multifunctionality. This review focuses on the activities of EspF within the host cell and discusses recent findings and molecular insights relating to the virulence functions of this fascinating bacterial effector.

Attaching and effacing pathogens: the effector ABC of immune subversion.

The innate immune response resembles an essential barrier to bacterial infection. Many bacterial pathogens have, therefore, evolved mechanisms to evade from or subvert the host immune response in order to colonize, survive and multiply. The attaching and effacing pathogens enteropathogenic Escherichia coli, enterohaemorrhagic E. coli, Escherichia albertii and Citrobacter rodentium are Gram-negative extracellular gastrointestinal pathogens. They use a type III secretion system to inject effector proteins into the host cell to manipulate a variety of cellular processes. Over the last decade, considerable progress was made in identifying and characterizing the effector proteins of attaching and effacing pathogens that are involved in the inhibition of innate immune signaling pathways, in determining their host cell targets and elucidating the mechanisms they employ. Their functions will be reviewed here.

Treatment Strategies for Infections With Shiga Toxin-Producing Escherichia coli.

Infections with Shiga toxin-producing Escherichia coli (STEC) cause outbreaks of severe diarrheal disease in children and the elderly around the world. The severe complications associated with toxin production and release range from bloody diarrhea and hemorrhagic colitis to hemolytic-uremic syndrome, kidney failure, and neurological issues. As the use of antibiotics for treatment of the infection has long been controversial due to reports that antibiotics may increase the production of Shiga toxin, the recommended therapy today is mainly supportive. In recent years, a variety of alternative treatment approaches such as monoclonal antibodies or antisera directed against Shiga toxin, toxin receptor analogs, and several vaccination strategies have been developed and evaluated in vitro and in animal models. A few strategies have progressed to the clinical trial phase. Here, we review the current understanding of and the progress made in the development of treatment options against STEC infections and discuss their potential.