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ABSTRACT Enteric pathogens have complex interactions with the gut microbiota. Most of what is known about them has focused on microbiota-derived metabolites or small molecules that serve as nutrients and/or signals to aid in growth or transcriptionally regulate virulence gene expression. A common virulence strategy is to express a type III secretion system (T3SS), which is a molecular syringe deployed by many Gram-negative pathogens to hijack host cell function. Enterohemorrhagic Escherichia coli (EHEC) requires its T3SS to colonize the intestinal tract and cause disease. Here we report that a prominent member of the intestinal microbiota, Bacteroides thetaiotamicron (Bt), secretes proteases that cleave the translocon of the T3SS of EHEC to enhance effector translocation into host cells. This is in contrast from an endogenous protease from EHEC itself (namely, EspP) that cleaves the translocon protein EspB in a different site to limit effector translocation. The EspB protein forms the T3SS pore in mammalian cells, and pore proteins are conserved in the T3SSs from several pathogens. This is the first demonstration of a commensal species directly processing a pathogen’s T3SS, posing a new paradigm for how the microbiota can influence the severity of disease caused by bacterial pathogens. Because T3SSs are employed by many pathogens, this phenomenon has broad implications to commensal-pathogen relationships. IMPORTANCE The gut microbiota is usually regarded as providing colonization resistance against enteric pathogens. However, some pathogens evolved to thrive with the aid of certain members of the microbiota. Several Gram-negative bacteria employ type three secretion systems (T3SSs), which are molecular syringes that deliver effector proteins to host cells, hijacking host cell function. Here we show that the T3SS of enterohemorrhagic E. coli (EHEC) is cleaved by self and microbiota-derived proteases. Self-cleavage limits effector translocation, while cleavage by the microbiota member Bacteroides thetaiotamicron (Bt) exacerbates effector translocation and lesion formation on epithelial cells.

ABSTRACT Enteric pathogens such as enterohemorrhagic Escherichia coli (EHEC) and Citrobacter rodentium, which is largely used as a surrogate EHEC model for murine infections, are exposed to several host neurotransmitters in the gut. An important chemical exchange within the gut involves the neurotransmitters epinephrine and/or norepinephrine, extensively reported to increase virulence gene expression in EHEC, acting through two bacterial adrenergic sensors: QseC and QseE. However, EHEC is unable to establish itself and cause its hallmark lesions, attaching and effacing (AE) lesions, on murine enterocytes. To address the role of these neurotransmitters during enteric infection, we employed C. rodentium. Both EHEC and C. rodentium harbor the locus of enterocyte effacement (LEE) that is necessary for AE lesion formation. Here we show that expression of the LEE, as well as that of other virulence genes in C. rodentium, is also activated by epinephrine and/or norepinephrine. Both QseC and QseE are required for LEE gene activation in C. rodentium, and the qseC and qseE mutants are attenuated for murine infection. C. rodentium has a decreased ability to colonize dopamine β-hydroxylase knockout (Dbh−/−) mice, which do not produce epinephrine and norepinephrine. Both adrenergic sensors are required for C. rodentium to sense these neurotransmitters and activate the LEE genes during infection. These data indicate that epinephrine and norepinephrine are sensed by bacterial adrenergic receptors during enteric infection to promote activation of their virulence repertoire. This is the first report of the role of these neurotransmitters during mammalian gastrointestinal (GI) infection by a noninvasive pathogen. IMPORTANCE The epinephrine and norepinephrine neurotransmitters play important roles in gut physiology and motility. Of note, epinephrine and norepinephrine play a central role in stress responses in mammals, and stress has profound effects on GI function. Bacterial enteric pathogens exploit these neurotransmitters as signals to coordinate the regulation of their virulence genes. The bacterial QseC and QseE adrenergic sensors are at the center of this regulatory cascade. C. rodentium is a noninvasive murine pathogen with a colonization mechanism similar to that of EHEC, enabling the investigation of host signals in mice. The presence of these neurotransmitters in the gut is necessary for C. rodentium to fully activate its virulence program, in a QseC/QseE-dependent manner, to successfully colonize its murine host. Our study data provide the first example of epinephrine and norepinephrine signaling within the gut to stimulate infection by a bacterial pathogen in a natural animal infection.

ABSTRACT Invasive pathogens interface with the host and its resident microbiota through interkingdom signaling. The bacterial receptor QseC, which is a membrane-bound histidine sensor kinase, responds to the host stress hormones epinephrine and norepinephrine and the bacterial signal AI-3, integrating interkingdom signaling at the biochemical level. Importantly, the QseC signaling cascade is exploited by many bacterial pathogens to promote virulence. Here, we translated this basic science information into development of a potent small molecule inhibitor of QseC, LED209. Extensive structure activity relationship (SAR) studies revealed that LED209 is a potent prodrug that is highly selective for QseC. Its warhead allosterically modifies lysines in QseC, impairing its function and preventing the activation of the virulence program of several Gram-negative pathogens both in vitro and during murine infection. LED209 does not interfere with pathogen growth, possibly leading to a milder evolutionary pressure toward drug resistance. LED209 has desirable pharmacokinetics and does not present toxicity in vitro and in rodents. This is a unique antivirulence approach, with a proven broad-spectrum activity against multiple Gram-negative pathogens that cause mammalian infections. IMPORTANCE There is an imminent need for development of novel treatments for infectious diseases, given that one of the biggest challenges to medicine in the foreseeable future is the emergence of microbial antibiotic resistance. Here, we devised a broad-spectrum antivirulence approach targeting a conserved histidine kinase, QseC, in several Gram-negative pathogens that promotes their virulence expression. The LED209 QseC inhibitor has a unique mode of action by acting as a prodrug scaffold to deliver a warhead that allosterically modifies QseC, impeding virulence in several Gram-negative pathogens.

ABSTRACT Gastrointestinal (GI) bacteria sense diverse environmental signals as cues for differential gene regulation and niche adaptation. Pathogens such as enterohemorrhagic Escherichia coli (EHEC), which causes bloody diarrhea, use these signals for the temporal and energy-efficient regulation of their virulence factors. One of the main virulence strategies employed by EHEC is the formation of attaching and effacing (AE) lesions on enterocytes. Most of the genes necessary for the formation of these lesions are grouped within a pathogenicity island, the locus of enterocyte effacement (LEE), whose expression requires the LEE-encoded regulator Ler. Here we show that growth of EHEC in glycolytic environments inhibits the expression of ler and consequently all other LEE genes. Conversely, growth within a gluconeogenic environment activates expression of these genes. This sugar-dependent regulation is achieved through two transcription factors: KdpE and Cra. Both Cra and KdpE directly bind to the ler promoter, and Cra’s affinity to this promoter is catabolite dependent. Moreover, we show that the Cra and KdpE proteins interact in vitro and that KdpE’s ability to bind DNA is enhanced by the presence of Cra. Cra is important for AE lesion formation, and KdpE contributes to this Cra-dependent regulation. The deletion of cra and kdpE resulted in the ablation of AE lesions. One of the many challenges that bacteria face within the GI tract is to successfully compete for carbon sources. Linking carbon metabolism to the precise coordination of virulence expression is a key step in the adaptation of pathogens to the GI environment. IMPORTANCE An appropriate and prompt response to environmental cues is crucial for bacterial survival. Cra and KdpE are two proteins found in both nonpathogenic and pathogenic bacteria that regulate genes in response to differences in metabolite concentration. In this work, we show that, in the deadly pathogen enterohemorrhagic Escherichia coli (EHEC) O157:H7, which causes bloody diarrhea, these two proteins influence important virulence traits. We also propose that their control of one or more of these virulence traits is due to the direct interaction of the Cra and KdpE proteins with each other, as well as with their DNA targets. This work shows how EHEC coopts established mechanisms for sensing the metabolites and stress cues in the environment, to induce virulence factors in a temporal and energy-efficient manner, culminating in disease. Understanding how pathogens commandeer nonpathogenic systems can help us develop measures to control them.