The yad and yeh fimbrial loci influence gene expression and virulence in enterohemorrhagic Escherichia coli O157:H7.

Fimbriae are essential virulence factors for many bacterial pathogens. Fimbriae are extracellular structures that attach bacteria to surfaces. Thus, fimbriae mediate a critical step required for any pathogen to establish infection by anchoring a bacterium to host tissue. The human pathogen enterohemorrhagic Escherichia coli (EHEC) O157:H7encodes 16 fimbriae that may be important for EHEC to initiate infection and allow for productive expression of virulence traits important in later stages of infection, including a type III secretion system (T3SS) and Shiga toxin; however, the roles of most EHEC fimbriae are largely uncharacterized. Here, we provide evidence that two EHEC fimbriae, Yad and Yeh, modulate expression of diverse genes including genes encoding T3SS and Shiga toxin and that these fimbriae are required for robust colonization of the gastrointestinal tract. These findings reveal a significant and previously unappreciated role for fimbriae in bacterial pathogenesis as important determinants of virulence gene expression.IMPORTANCEFimbriae are extracellular proteinaceous structures whose defining role is to anchor bacteria to surfaces. This is a fundamental step for bacterial pathogens to establish infection in a host. Here, we show that the contributions of fimbriae to pathogenesis are more complex. Specifically, we demonstrate that fimbriae influence expression of virulence traits essential for disease progression in the intestinal pathogen enterohemorrhagic Escherichia coli. Gram-positive and Gram-negative bacteria express multiple fimbriae; therefore, these findings may have broad implications for understanding how pathogens use fimbriae, beyond adhesion, to initiate infection and coordinate gene expression, which ultimately results in disease.

Flagellin outer domain dimerization modulates motility in pathogenic and soil bacteria from viscous environments.

Flagellar filaments function as the propellers of the bacterial flagellum and their supercoiling is key to motility. The outer domains on the surface of the filament are non-critical for motility in many bacteria and their structures and functions are not conserved. Here, we show the atomic cryo-electron microscopy structures for flagellar filaments from enterohemorrhagic Escherichia coli O157:H7, enteropathogenic E. coli O127:H6, Achromobacter, and Sinorhizobium meliloti, where the outer domains dimerize or tetramerize to form either a sheath or a screw-like surface. These dimers are formed by 180° rotations of half of the outer domains. The outer domain sheath (ODS) plays a role in bacterial motility by stabilizing an intermediate waveform and prolonging the tumbling of E. coli cells. Bacteria with these ODS and screw-like flagellar filaments are commonly found in soil and human intestinal environments of relatively high viscosity suggesting a role for the dimerization in these environments.

A pathogen-specific sRNA influences enterohemorrhagic Escherichia coli fitness and virulence in part by direct interaction with the transcript encoding the ethanolamine utilization regulatory factor EutR.

Enterohemorrhagic Escherichia coli (EHEC) O157:H7 relies on sRNAs to coordinate expression of metabolic and virulence factors to colonize the host. Here, we focus on the sRNA, named MavR (metabolism and virulence regulator), that is conserved among pathogenic Enterobacteriaceae. MavR is constitutively expressed under in vitro conditions that promote EHEC virulence gene expression. Using MS2-affinity purification coupled with RNA sequencing, the eutR transcript was identified as a putative target of MavR. EutR is a transcription factor that promotes expression of genes required for ethanolamine metabolism as well as virulence factors important for host colonization. MavR binds to the eutR coding sequence to protect the eutR transcript from RNase E-mediated degradation. Ultimately, MavR promotes EutR expression and in turn ethanolamine utilization and ethanolamine-dependent growth. RNAseq analyses revealed that MavR also affected expression of genes important for other metabolic pathways, motility, oxidative stress and attaching and effacing lesion formation, which contribute to EHEC colonization of the gastrointestinal tract. In support of the idea that MavR-dependent gene expression affects fitness during infection, deletion of mavR resulted in significant (∼10- to 100-fold) attenuation in colonization of the mammalian intestine. Altogether, these studies reveal an important, extensive, and robust phenotype for a bacterial sRNA in host-pathogen interactions.

The Ethanolamine-Sensing Transcription Factor EutR Promotes Virulence and Transmission during Citrobacter rodentium Intestinal Infection.

Enteric pathogens exploit chemical and nutrient signaling to gauge their location within a host and control expression of traits important for infection. Ethanolamine-containing molecules are essential in host physiology and play important roles in intestinal processes. The transcription factor EutR is conserved in the Enterobacteriaceae and is required for ethanolamine sensing and metabolism. In enterohemorrhagic Escherichia coli (EHEC) O157:H7, EutR responds to ethanolamine to activate expression of traits required for host colonization and disease; however, the importance of EutR to EHEC intestinal infection has not been examined. Because EHEC does not naturally colonize or cause disease in mice, we employed the natural murine pathogen Citrobacter rodentium as a model of EHEC virulence to investigate the importance of EutR in vivo EHEC and C. rodentium possess the locus of enterocyte effacement (LEE), which is the canonical virulence trait of attaching and effacing pathogens. Our findings demonstrate that ethanolamine sensing and EutR-dependent regulation of the LEE are conserved in C. rodentium Moreover, during infection, EutR is required for maximal LEE expression, colonization, and transmission efficiency. These findings reveal that EutR not only is important for persistence during the primary host infection cycle but also is required for maintenance in a host population.

Genetic, Inflammatory, and Epithelial Cell Differentiation Factors Control Expression of Human Calpain-14.

Eosinophilic esophagitis (EoE) is a chronic, food-driven allergic disease resulting in eosinophilic esophageal inflammation. We recently found that EoE susceptibility is associated with genetic variants in the promoter of CAPN14, a gene with reported esophagus-specific expression. CAPN14 is dynamically up-regulated as a function of EoE disease activity and after exposure of epithelial cells to interleukin-13 (IL-13). Herein, we aimed to explore molecular modulation of CAPN14 expression. We identified three putative binding sites for the IL-13-activated transcription factor STAT6 in the promoter and first intron of CAPN14 Luciferase reporter assays revealed that the two most distal STAT6 elements were required for the ∼10-fold increase in promoter activity subsequent to stimulation with IL-13 or IL-4, and also for the genotype-dependent reduction in IL-13-induced promoter activity. One of the STAT6 elements in the promoter was necessary for IL-13-mediated induction of CAPN14 promoter activity while the other STAT6 promoter element was necessary for full induction. Chromatin immunoprecipitation in IL-13 stimulated esophageal epithelial cells was used to further support STAT6 binding to the promoter of CAPN14 at these STAT6 binding sites. The highest CAPN14 and calpain-14 expression occurred with IL-13 or IL-4 stimulation of esophageal epithelial cells under culture conditions that allow the cells to differentiate into a stratified epithelium. This work corroborates a candidate molecular mechanism for EoE disease etiology in which the risk variant at 2p23 dampens CAPN14 expression in differentiated esophageal epithelial cells following IL-13/STAT6 induction of CAPN14 promoter activity.

After the Fact(or): Posttranscriptional Gene Regulation in Enterohemorrhagic Escherichia coli O157:H7.

To adapt to ever-changing environments, pathogens quickly alter gene expression. This can occur through transcriptional, posttranscriptional, or posttranslational regulation. Historically, transcriptional regulation has been thoroughly studied to understand pathogen niche adaptation, whereas posttranscriptional and posttranslational gene regulation has only relatively recently been appreciated to play a central role in bacterial pathogenesis. Posttranscriptional regulation may involve chaperones, nucleases, and/or noncoding small RNAs (sRNAs) and typically controls gene expression by altering the stability and/or translation of the target mRNA. In this review, we highlight the global importance of posttranscriptional regulation to enterohemorrhagic Escherichia coli (EHEC) gene expression and discuss specific mechanisms of how EHEC regulates expression of virulence factors critical to host colonization and disease progression. The low infectious dose of this intestinal pathogen suggests that EHEC is particularly well adapted to respond to the host environment.

Genomic characterization and comparison of seven Myoviridae bacteriophage infecting Bacillus thuringiensis.

Bacillus thuringiensis Kurstaki, a bacterium that is a source of biopesticides and a safe simulant for pathogenic Bacillus species, was used to isolate seven unique bacteriophages. The phage genomes were sequenced and ranged in size from 158,100 to 163,019 bp encoding 290-299 genes, and the GC content of ~38% was similar to that of the host bacterium. All phages had terminal repeats 2-3 kb long. Three of the phages encoded tRNAs and three contained a self-splicing intron in the DNA polymerase gene. They were categorized as a single cluster (>60% nucleotide conservation) containing three subclusters (>80% nucleotide conservation), supported by genomic synteny and phylogenetic analysis. Considering the published genomes of phages that infect the genus Bacillus and noting the ability of many of the Bacillus cereus group phages to infect multiple species, a clustering system based on gene content is proposed.