Gut microbes regroup to aid defence after infection.

Resident gut microbes can help to block infection, but the mechanisms involved are not fully understood. It has now been found that changes in the microbial community after infection boost the level of a molecule that combats harmful bacteria.

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 sRNA DicF integrates oxygen sensing to enhance enterohemorrhagic Escherichia coli virulence via distinctive RNA control mechanisms.

To establish infection, enteric pathogens integrate environmental cues to navigate the gastrointestinal tract (GIT) and precisely control expression of virulence determinants. During passage through the GIT, pathogens encounter relatively high levels of oxygen in the small intestine before transit to the oxygen-limited environment of the colon. However, how bacterial pathogens sense oxygen availability and coordinate expression of virulence traits is not resolved. Here, we demonstrate that enterohemorrhagic Escherichia coli O157:H7 (EHEC) regulates virulence via the oxygen-responsive small RNA DicF. Under oxygen-limited conditions, DicF enhances global expression of the EHEC type three secretion system, which is a key virulence factor required for host colonization, through the transcriptional activator PchA. Mechanistically, the pchA coding sequence (CDS) base pairs with the 5' untranslated region of the mRNA to sequester the ribosome binding site (RBS) and inhibit translation. DicF disrupts pchA cis-interactions by binding to the pchA CDS, thereby unmasking the pchA RBS and promoting PchA expression. These findings uncover a feed-forward regulatory pathway that involves distinctive mechanisms of RNA-based regulation and that provides spatiotemporal control of EHEC virulence.

Effect of Lipidation on the Localization and Activity of a Lysozyme Inhibitor in Neisseria gonorrhoeae.

The Gram-negative pathogen Neisseria gonorrhoeae (gonococcus [Gc]) colonizes lysozyme-rich mucosal surfaces. Lysozyme hydrolyzes peptidoglycan, leading to bacterial lysis. Gc expresses two proteins, SliC and NgACP, that bind and inhibit the enzymatic activity of lysozyme. SliC is a surface-exposed lipoprotein, while NgACP is found in the periplasm and also released extracellularly. Purified SliC and NgACP similarly inhibit lysozyme. However, whereas mutation of ngACP increases Gc susceptibility to lysozyme, the sliC mutant is only susceptible to lysozyme when ngACP is inactivated. In this work, we examined how lipidation contributes to SliC expression, cellular localization, and resistance of Gc to killing by lysozyme. To do so, we mutated the conserved cysteine residue (C18) in the N-terminal lipobox motif of SliC, the site for lipid anchor attachment, to alanine. SliC(C18A) localized to soluble rather than membrane fractions in Gc and was not displayed on the bacterial surface. Less SliC(C18A) was detected in Gc lysates compared to the wild-type protein. This was due in part to some release of the C18A mutant, but not wild-type, protein into the extracellular space. Surprisingly, Gc expressing SliC(C18A) survived better than SliC (wild type)-expressing Gc after exposure to lysozyme. We conclude that lipidation is not required for the ability of SliC to inhibit lysozyme, even though the lipidated cysteine is 100% conserved in Gc SliC alleles. These findings shed light on how members of the growing family of lysozyme inhibitors with distinct subcellular localizations contribute to bacterial defense against lysozyme.IMPORTANCENeisseria gonorrhoeae is one of many bacterial species that express multiple lysozyme inhibitors. It is unclear how inhibitors that differ in their subcellular localization contribute to defense from lysozyme. We investigated how lipidation of SliC, an MliC (membrane-bound lysozyme inhibitor of c-type lysozyme)-type inhibitor, contributes to its localization and lysozyme inhibitory activity. We found that lipidation was required for surface exposure of SliC and yet was dispensable for protecting the gonococcus from killing by lysozyme. To our knowledge, this is the first time the role of lipid anchoring of a lysozyme inhibitor has been investigated. These results help us understand how different lysozyme inhibitors are localized in bacteria and how this impacts resistance to lysozyme.

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.

The AraC Negative Regulator family modulates the activity of histone-like proteins in pathogenic bacteria.

The AraC Negative Regulators (ANR) comprise a large family of virulence regulators distributed among diverse clinically important Gram-negative pathogens, including Vibrio spp., Salmonella spp., Shigella spp., Yersinia spp., Citrobacter spp., and pathogenic E. coli strains. We have previously reported broad effects of the ANR members on regulators of the AraC/XylS family. Here, we interrogate possible broader effects of the ANR members on the bacterial transcriptome. Our studies focused on Aar (AggR-activated regulator), an ANR family archetype in enteroaggregative E. coli (EAEC) isolate 042. Transcriptome analysis of EAEC strain 042, 042aar and 042aar(pAar) identified more than 200 genes that were differentially expressed (+/- 1.5 fold, p<0.05). Most of those genes are located on the bacterial chromosome (195 genes, 92.85%), and are associated with regulation, transport, metabolism, and pathogenesis, based on the predicted annotation; a considerable number of Aar-regulated genes encoded for hypothetical proteins (46 genes, 21.9%) and regulatory proteins (25, 11.9%). Notably, the transcriptional expression of three histone-like regulators, H-NS (orf1292), H-NS homolog (orf2834) and StpA, was down-regulated in the absence of aar and may explain some of the effects of Aar on gene expression. By employing a bacterial two-hybrid system, LacZ reporter assays, pull-down and electrophoretic mobility shift assay (EMSA) analysis, we demonstrated that Aar binds directly to H-NS and modulates H-NS-induced gene silencing. Importantly, Aar was highly expressed in the mouse intestinal tract and was found to be necessary for maximal H-NS expression. In conclusion, this work further extends our knowledge of genes under the control of Aar and its biological relevance in vivo.

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.

The Ethanolamine Permease EutH Promotes Vacuole Adaptation of Salmonella enterica and Listeria monocytogenes during Macrophage Infection.

Ethanolamine is a ubiquitous and essential molecule within a host. Significantly, bacterial pathogens exploit ethanolamine during infection to promote growth and regulate virulence. The ethanolamine permease EutH is dispensable for growth in vitro under standard conditions, whereas EutH is required for ethanolamine utilization at low pH. These findings suggested a model in which EutH facilitates diffusion of ethanolamine into the bacterial cell in acidic environments. To date, the ecological significance of this model has not been thoroughly investigated, and the importance of EutH to bacterial growth under physiologically relevant conditions is not known. During infection, immune cells internalize invading bacteria within an acidic, nutrient-depleted vacuole called the phagosome. Here, we investigated the hypothesis that EutH promotes bacterial survival following phagocytosis. Our findings indicate that EutH is important for survival and replication of the facultative intracellular pathogens Salmonella enterica serovar Typhimurium and Listeria monocytogenes during prolonged or transient exposure to the phagosome, respectively. Furthermore, in agreement with EutH being important in the acidic environment, neutralization of the vacuole abolished the requirement for EutH. Significantly, consistent with a role for EutH in promoting intramacrophage survival, EutH was not required during S Typhimurium local intestinal infection but specifically conferred an advantage upon dissemination to peripheral organs. These findings reveal a physiologically relevant and conserved role for EutH in spatiotemporal niche adaptation during infection.

A large family of anti-activators accompanying XylS/AraC family regulatory proteins.

AraC Negative Regulators (ANR) suppress virulence genes by directly down-regulating AraC/XylS members in Gram-negative bacteria. In this study, we sought to investigate the distribution and molecular mechanisms of regulatory function for ANRs among different bacterial pathogens. We identified more than 200 ANRs distributed in diverse clinically important gram negative pathogens, including Vibrio spp., Salmonella spp., Shigella spp., Yersinia spp., Citrobacter spp., enterotoxigenic (ETEC) and enteroaggregative E. coli (EAEC), and members of the Pasteurellaceae. By employing a bacterial two hybrid system, pull down assays and surface plasmon resonance (SPR) analysis, we demonstrate that Aar (AggR-activated regulator), a prototype member of the ANR family in EAEC, binds with high affinity to the central linker domain of AraC-like member AggR. ANR-AggR binding disrupted AggR dimerization and prevented AggR-DNA binding. ANR homologs of Vibrio cholerae, Citrobacter rodentium, Salmonella enterica and ETEC were capable of complementing Aar activity by repressing aggR expression in EAEC strain 042. ANR homologs of ETEC and Vibrio cholerae bound to AggR as well as to other members of the AraC family, including Rns and ToxT. The predicted proteins of all ANR members exhibit three highly conserved predicted α-helices. Site-directed mutagenesis studies suggest that at least predicted α-helices 2 and 3 are required for Aar activity. In sum, our data strongly suggest that members of the novel ANR family act by directly binding to their cognate AraC partners.

Ethanolamine Signaling Promotes Salmonella Niche Recognition and Adaptation during Infection.

Chemical and nutrient signaling are fundamental for all cellular processes, including interactions between the mammalian host and the microbiota, which have a significant impact on health and disease. Ethanolamine is an essential component of cell membranes and has profound signaling activity within mammalian cells by modulating inflammatory responses and intestinal physiology. Here, we describe a virulence-regulating pathway in which the foodborne pathogen Salmonella enterica serovar Typhimurium (S. Typhimurium) exploits ethanolamine signaling to recognize and adapt to distinct niches within the host. The bacterial transcription factor EutR promotes ethanolamine metabolism in the intestine, which enables S. Typhimurium to establish infection. Subsequently, EutR directly activates expression of the Salmonella pathogenicity island 2 in the intramacrophage environment, and thus augments intramacrophage survival. Moreover, EutR is critical for robust dissemination during mammalian infection. Our findings reveal that S. Typhimurium co-opts ethanolamine as a signal to coordinate metabolism and then virulence. Because the ability to sense ethanolamine is a conserved trait among pathogenic and commensal bacteria, our work indicates that ethanolamine signaling may be a key step in the localized adaptation of bacteria within their mammalian hosts.

The Type Three Secretion System 2-Encoded Regulator EtrB Modulates Enterohemorrhagic Escherichia coli Virulence Gene Expression.

Enterohemorrhagic Escherichia coli O157:H7 (EHEC) is a foodborne pathogen that causes bloody diarrhea and hemolytic uremic syndrome throughout the world. A defining feature of EHEC pathogenesis is the formation of attaching and effacing (AE) lesions on colonic epithelial cells. Most of the genes that code for AE lesion formation, including a type three secretion system (T3SS) and effectors, are carried within a chromosomal pathogenicity island called the locus of enterocyte effacement (LEE). In this study, we report that a putative regulator, which is encoded in the cryptic E. coli type three secretion system 2 (ETT2) locus and herein renamed EtrB, plays an important role in EHEC pathogenesis. The etrB gene is expressed as a monocistronic transcript, and EtrB autoregulates expression. We provide evidence that EtrB directly interacts with the ler regulatory region to activate LEE expression and promote AE lesion formation. Additionally, we mapped the EtrB regulatory circuit in EHEC to determine a global role for EtrB. EtrB is regulated by the transcription factor QseA, suggesting that these proteins comprise a regulatory circuit important for EHEC colonization of the gastrointestinal tract.

Interkingdom Chemical Signaling in Enterohemorrhagic Escherichia coli O157:H7.

Escherichia coli is one of the most-studied species of bacteria due to its frequent incidence in diverse environments and hosts, as well as its use as a tool in molecular biology. Most E. coli strains are commensal, in that they colonize the host without causing disease; however, some strains of E. coli are pathogens and are able to cause diverse illnesses, including urinary tract infections, sepsis/meningitis, as well as intestinal disease that result in diarrhea (Kaper et al. 2004). Six categories of diarrheagenic E. coli are recognized, and these are classified in part based on how they interact with epithelial cells (Kaper et al. 2004). Of these, enterohemorrhagic E. coli O157:H7 (EHEC) is one of the most important pathogenic E. coli strains. EHEC causes major outbreaks of bloody diarrhea that can result in the development of fatal hemorrhagic colitis and hemolytic uremic syndrome (Karmali et al. 1983). EHEC colonizes the colon, where it forms attaching and effacing (AE) lesions on the intestinal epithelial cell. AE lesions are characterized by intimate attachment of EHEC to epithelial cells, effacement of the microvilli and rearrangement of the underlying cytoskeleton, which results in formation of a pedestal-like structure beneath the bacterium (Jerse et al. 1990; Jarvis et al. 1995; Kenny et al. 1997). Most of the genes involved in the formation of AE lesions are encoded within a chromosomal pathogenicity island termed the locus of enterocyte effacement (LEE) (McDaniel et al. 1995). The LEE contains 41 genes that are organized in five major operons (LEE1, LEE2, LEE3, LEE5, and LEE4) (Elliott et al. 1998, 1999; Mellies et al. 1999). The LEE encodes a type three secretion system (T3SS) (Jarvis et al. 1995), an adhesin (intimin) (Jerse et al. 1990) and its receptor (Tir) (Kenny et al. 1997), as well as effector proteins (Kenny et al. 1996; Abe et al. 1997; McNamara and Donnenberg 1998; Elliott et al. 2001; Tu et al. 2003; Kanack et al. 2005). EHEC also encodes an arsenal of effector proteins located outside of the LEE that are important in EHEC virulence (Campellone et al. 2004; Deng et al. 2004; Garmendia et al. 2004, 2005; Gruenheid et al. 2004; Tobe et al. 2006).

Ethanolamine and choline promote expression of putative and characterized fimbriae in enterohemorrhagic Escherichia coli O157:H7.

Enterohemorrhagic Escherichia coli (EHEC) O157:H7 is an important food-borne pathogen responsible for disease outbreaks worldwide. In order to colonize the human gastrointestinal (GI) tract and cause disease, EHEC must be able to sense the host environment and promote expression of virulence genes essential for adherence. Ethanolamine (EA) is an important metabolite for EHEC in the GI tract, and EA is also a signal that EHEC uses to activate virulence traits. Here, we report that EA influenced EHEC adherence to epithelial cells and fimbrial gene expression. Quantitative reverse transcriptase PCR indicated that EA promoted the transcription of the genes in characterized and putative fimbrial operons. Moreover, putative fimbrial structures were produced by EHEC cells grown with EA but not in medium lacking EA. Additionally, we defined two previously uncharacterized EA-regulated fimbrial operons, loc10 and loc11. We also tested whether choline or serine, both of which are also components of cell membranes, activated fimbrial gene expression. In addition to EA, choline activated fimbrial gene expression in EHEC. These findings describe for the first time the transcription of several putative fimbrial loci in EHEC. Importantly, the biologically relevant molecules EA and choline, which are abundant in the GI tract, promoted expression of these fimbriae.

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.

EutR is a direct regulator of genes that contribute to metabolism and virulence in enterohemorrhagic Escherichia coli O157:H7.

Ethanolamine (EA) metabolism is a trait associated with enteric pathogens, including enterohemorrhagic Escherichia coli O157:H7 (EHEC). EHEC causes severe bloody diarrhea and hemolytic uremic syndrome. EHEC encodes the ethanolamine utilization (eut) operon that allows EHEC to metabolize EA and gain a competitive advantage when colonizing the gastrointestinal tract. The eut operon encodes the transcriptional regulator EutR. Genetic studies indicated that EutR expression is induced by EA and vitamin B12 and that EutR promotes expression of the eut operon; however, biochemical evidence for these interactions has been lacking. We performed EA-binding assays and electrophoretic mobility shift assays (EMSAs) to elucidate a mechanism for EutR gene regulation. These studies confirmed EutR interaction with EA, as well as direct binding to the eutS promoter. EutR also contributes to expression of the locus of enterocyte effacement (LEE) in an EA-dependent manner. We performed EMSAs to examine EutR activation of the LEE. The results demonstrated that EutR directly binds the regulatory region of the ler promoter. These results present the first mechanistic description of EutR gene regulation and reveal a novel role for EutR in EHEC pathogenesis.

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.

Hfq virulence regulation in enterohemorrhagic Escherichia coli O157:H7 strain 86-24.

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.