Bacterial pathogens must be able to both recognize suitable niches within the host for colonization and successfully compete with commensal flora for nutrients in order to establish infection. Ethanolamine (EA) is a major component of mammalian and bacterial membranes and is used by pathogens as a carbon and/or nitrogen source in the gastrointestinal tract. The deadly human pathogen enterohemorrhagic Escherichia coli O157:H7 (EHEC) uses EA in the intestine as a nitrogen source as a competitive advantage for colonization over the microbial flora. Here we show that EA is not only important for nitrogen metabolism but that it is also used as a signaling molecule in cell-to-cell signaling to activate virulence gene expression in EHEC. EA in concentrations that cannot promote growth as a nitrogen source can activate expression of EHEC’s repertoire of virulence genes. The EutR transcription factor, known to be the receptor of EA, is only partially responsible for this regulation, suggesting that yet another EA receptor exists. This important link of EA with metabolism, cell-to-cell signaling, and pathogenesis, highlights the fact that a fundamental means of communication within microbial communities relies on energy production and processing of metabolites. Here we show for the first time that bacterial pathogens not only exploit EA as a metabolite but also coopt EA as a signaling molecule to recognize the gastrointestinal environment and promote virulence expression.

Remodeling of the host cytoskeleton is a common strategy employed by bacterial pathogens. Although there is vigorous investigation of the cell biology underlying these bacterially mediated cytoskeleton modifications, knowledge of the plasticity and dynamics of the bacterial signaling networks that regulate the expression of genes necessary for these phenotypes is lacking. Enterohemorrhagic Escherichia coli attaches to enterocytes, forming pedestal-like structures. Pedestal formation requires the expression of the locus-of-enterocyte-effacement (LEE) and espFu genes. The LEE encodes a molecular syringe, a type III secretion system (T3SS) used by pathogens to translocate effectors such as EspFu into the host cell. By using a combination of genetic, biochemical, and cell biology approaches, we show that pedestal formation relies on posttranscriptional regulation by two small RNAs (sRNAs), GlmY and GlmZ. The GlmY and GlmZ sRNAs are unique; they have extensive secondary structures and work in concert. Although these sRNAs may offer unique insights into RNA and posttranscriptional biology, thus far, only one target and one mechanism of action (exposure of the ribosome binding site from the glmS gene to promote its translation) has been described. Here we uncovered new targets and two different molecular mechanisms of action of these sRNAs. In the case of EspFu expression, they promote translation by cleavage of the transcript, while in regard to the LEE, they promote destabilization of the mRNA. Our findings reveal that two unique sRNAs act in concert through different molecular mechanisms to coordinate bacterial attachment to mammalian cells.

Enterohemorrhagic Escherichia coli O157:H7 (EHEC) causes bloody diarrhea and hemolytic-uremic syndrome. EHEC encodes the sRNA chaperone Hfq, which is important in posttranscriptional regulation. In EHEC strain EDL933, Hfq acts as a negative regulator of the locus of enterocyte effacement (LEE), which encodes most of the proteins involved in type III secretion and attaching and effacing (AE) lesions. Here, we deleted hfq in the EHEC strain 86-24 and compared global transcription profiles of the hfq mutant and wild-type (WT) strains in exponential growth phase. Deletion of hfq affected transcription of genes common to nonpathogenic and pathogenic strains of E. coli as well as pathogen-specific genes. Downregulated genes in the hfq mutant included ler, the transcriptional activator of all the LEE genes, as well as genes encoded in the LEE2 to -5 operons. Decreased expression of the LEE genes in the hfq mutant occurred at middle, late, and stationary growth phases. We also confirmed decreased regulation of the LEE genes by examining the proteins secreted and AE lesion formation by the hfq mutant and WT strains. Deletion of hfq also caused decreased expression of the two-component system qseBC, which is involved in interkingdom signaling and virulence gene regulation in EHEC, as well as an increase in expression of stx 2AB , which encodes the deadly Shiga toxin. Altogether, these data indicate that Hfq plays a regulatory role in EHEC 86-24 that is different from what has been reported for EHEC strain EDL933 and that the role of Hfq in EHEC virulence regulation extends beyond the LEE.Enterohemorrhagic Escherichia coli O157:H7 (EHEC) is a food-borne pathogen that causes severe bloody diarrhea and is often associated with complications, including hemolytic-uremic syndrome (HUS), seizures, cerebral edema, and/or coma (42). EHEC attaches intimately to intestinal epithelial cells and triggers extensive cytoskeletal rearrangements, resulting in attaching and effacing (AE) lesions and formation of a characteristic pedestal structure (40,41,46). Most of the genes involved in AE lesion formation are carried within a chromosomal pathogenicity island called the locus of enterocyte effacement (LEE) (53). The LEE contains five major operons (LEE1 to -5) that encode a type III secretion (TTS) system and effector proteins. EHEC's arsenal of virulence factors also includes non-LEE-encoded effector proteins that increase adherence or mediate colonization of host epithelial cells (8,16,26,27,32,84,87,88).The mortality associated with EHEC infections is due to the production and release of a Shiga toxin (Stx) by these bacteria. EHEC expresses Stx, and this potent inhibitor of protein synthesis can be absorbed systemically, where it binds to receptors found in the kidneys and the central nervous system, thus causing the complications associated with EHEC disease (42).Regulation of EHEC virulence genes is extremely complex and involves interkingdom signaling (12). EHEC senses the host-derived signals epinephrine and norepinephrine as well...

Enterohemorrhagic Escherichia coli (EHEC) is a significant human pathogen and is the cause of bloody diarrhea and hemolyticuremic syndrome. The virulence repertoire of EHEC includes the genes within the locus of enterocyte effacement (LEE) that are largely organized in five operons, LEE1 to LEE5, which encode a type III secretion system, several effectors, chaperones, and regulatory proteins. In addition, EHEC also encodes several non-LEE-encoded effectors and fimbrial operons. The virulence genes of this pathogen are under a large amount of posttranscriptional regulation. The small RNAs (sRNAs) GlmY and GlmZ activate the translation of glucosamine synthase (GlmS) in E. coli K-12, and in EHEC they destabilize the 3= fragments of the LEE4 and LEE5 operons and promote translation of the non-LEE-encoded effector EspFu. We investigated the global changes of EHEC gene expression governed by GlmY and GlmZ using RNA sequencing and gene arrays. This study extends the known effects of GlmY and GlmZ regulation to show that they promote expression of the curli adhesin, repress the expression of tryptophan metabolism genes, and promote the expression of acid resistance genes and the non-LEE-encoded effector NleA. In addition, seven novel EHEC-specific sRNAs were identified using RNA sequencing, and three of them-sRNA56, sRNA103, and sRNA350 -were shown to regulate urease, fimbria, and the LEE, respectively. These findings expand the knowledge of posttranscriptional regulation in EHEC. Enterohemorrhagic Escherichia coli (EHEC) is a major cause of hemorrhagic colitis and hemolytic-uremic syndrome. A defining characteristic of EHEC during infection is its ability to form attaching and effacing (AE) lesions on epithelial cells of the intestine, where the bacterium effaces the microvilli and forms an actin-rich pedestal structure that cups the bacterium (1). This process requires a type III secretion system encoded within the locus of enterocyte effacement (LEE) pathogenicity island (2, 3), as well as a non-LEE-encoded effector, EspFu/TccP (4, 5). The LEE contains 41 genes, the majority of which are organized in five major operons named LEE1 to LEE5. The first gene of the LEE1 operon encodes the master regulator Ler that activates transcription of all LEE genes (6). The LEE2 and LEE3 operons encode the major structural components of the type III secretion system, and LEE4 encodes the needle complex and the EspA filament that sheaths the needle and is part of the type III secretion system's translocon (7). The LEE5 operon encodes the adhesin intimin and its receptor Tir (8). In addition to the LEE, the EHEC genome contains many regions that are not present in the E. coli K-12 genome and are referred to as O-islands. Many of these regions contain genes encoding other virulence factors such as adhesins, toxins, and additional type III secreted effectors (9).To successfully infect the host, EHEC must tightly control expression of a complex array of virulence factors in response to several environmental signals (10). Bacteria use pos...

Numerous lethal stresses in bacteria including antibiotics, thymineless death, and MalE-LacZ expression trigger an increase in the production of reactive oxygen species. This results in the oxidation of the nucleotide pool by radicals produced by Fenton chemistry. Following the incorporation of these oxidized nucleotides into the genome, the cell's unsuccessful attempt to repair these lesions through base excision repair (BER) contributes causally to the lethality of these stresses. We review the evidence for this phenomenon of incomplete BER-mediated cell death and discuss how better understanding this pathway could contribute to the development of new antibiotics.