Metabolism of L-arabinose converges with virulence regulation to promote enteric pathogen fitness.

Virulence and metabolism are often interlinked to control the expression of essential colonisation factors in response to host-associated signals. Here, we identified an uncharacterised transporter of the dietary monosaccharide ʟ-arabinose that is widely encoded by the zoonotic pathogen enterohaemorrhagic Escherichia coli (EHEC), required for full competitive fitness in the mouse gut and highly expressed during human infection. Discovery of this transporter suggested that EHEC strains have an enhanced ability to scavenge ʟ-arabinose and therefore prompted us to investigate the impact of this nutrient on pathogenesis. Accordingly, we discovered that ʟ-arabinose enhances expression of the EHEC type 3 secretion system, increasing its ability to colonise host cells, and that the underlying mechanism is dependent on products of its catabolism rather than the sensing of ʟ-arabinose as a signal. Furthermore, using the murine pathogen Citrobacter rodentium, we show that ʟ-arabinose metabolism provides a fitness benefit during infection via virulence factor regulation, as opposed to supporting pathogen growth. Finally, we show that this mechanism is not restricted to ʟ-arabinose and extends to other pentose sugars with a similar metabolic fate. This work highlights the importance integrating central metabolism with virulence regulation in order to maximise competitive fitness of enteric pathogens within the host-niche.

A new family of bacterial ribosome hibernation factors.

To conserve energy during starvation and stress, many organisms use hibernation factor proteins to inhibit protein synthesis and protect their ribosomes from damage1,2. In bacteria, two families of hibernation factors have been described, but the low conservation of these proteins and the huge diversity of species, habitats and environmental stressors have confounded their discovery3-6. Here, by combining cryogenic electron microscopy, genetics and biochemistry, we identify Balon, a new hibernation factor in the cold-adapted bacterium Psychrobacter urativorans. We show that Balon is a distant homologue of the archaeo-eukaryotic translation factor aeRF1 and is found in 20% of representative bacteria. During cold shock or stationary phase, Balon occupies the ribosomal A site in both vacant and actively translating ribosomes in complex with EF-Tu, highlighting an unexpected role for EF-Tu in the cellular stress response. Unlike typical A-site substrates, Balon binds to ribosomes in an mRNA-independent manner, initiating a new mode of ribosome hibernation that can commence while ribosomes are still engaged in protein synthesis. Our work suggests that Balon-EF-Tu-regulated ribosome hibernation is a ubiquitous bacterial stress-response mechanism, and we demonstrate that putative Balon homologues in Mycobacteria bind to ribosomes in a similar fashion. This finding calls for a revision of the current model of ribosome hibernation inferred from common model organisms and holds numerous implications for how we understand and study ribosome hibernation.

The novel anti-phage system Shield co-opts an RmuC domain to mediate phage defense across Pseudomonas species.

Competitive bacteria-bacteriophage interactions have resulted in the evolution of a plethora of bacterial defense systems preventing phage propagation. In recent years, computational and bioinformatic approaches have underpinned the discovery of numerous novel bacterial defense systems. Anti-phage systems are frequently encoded together in genomic loci termed defense islands. Here we report the identification and characterisation of a novel anti-phage system, that we have termed Shield, which forms part of the Pseudomonas defensive arsenal. The Shield system comprises the core component ShdA, a membrane-bound protein harboring an RmuC domain. Heterologous production of ShdA alone is sufficient to mediate bacterial immunity against several phages. We demonstrate that Shield and ShdA confer population-level immunity and that they can also decrease transformation efficiency. We further show that ShdA homologues can degrade DNA in vitro and, when expressed in a heterologous host, can alter the organisation of the host chromosomal DNA. Use of comparative genomic approaches identified how Shield can be divided into four subtypes, three of which contain additional components that in some cases can negatively affect the activity of ShdA and/or provide additional lines of phage defense. Collectively, our results identify a new player within the Pseudomonas bacterial immunity arsenal that displays a novel mechanism of protection, and reveals a role for RmuC domains in phage defense.

Distinct ecological fitness factors coordinated by a conserved Escherichia coli regulator during systemic bloodstream infection.

The ability of bacterial pathogens to adapt to host niches is driven by the carriage and regulation of genes that benefit pathogenic lifestyles. Genes that encode virulence or fitness-enhancing factors must be regulated in response to changing host environments to allow rapid response to challenges presented by the host. Furthermore, this process can be controlled by preexisting transcription factors (TFs) that acquire new roles in tailoring regulatory networks, specifically in pathogens. However, the mechanisms underlying this process are poorly understood. The highly conserved Escherichia coli TF YhaJ exhibits distinct genome-binding dynamics and transcriptome control in pathotypes that occupy different host niches, such as uropathogenic E. coli (UPEC). Here, we report that this important regulator is required for UPEC systemic survival during murine bloodstream infection (BSI). This advantage is gained through the coordinated regulation of a small regulon comprised of both virulence and metabolic genes. YhaJ coordinates activation of both Type 1 and F1C fimbriae, as well as biosynthesis of the amino acid tryptophan, by both direct and indirect mechanisms. Deletion of yhaJ or the individual genes under its control leads to attenuated survival during BSI. Furthermore, all three systems are up-regulated in response to signals derived from serum or systemic host tissue, but not urine, suggesting a niche-specific regulatory trigger that enhances UPEC fitness via pleiotropic mechanisms. Collectively, our results identify YhaJ as a pathotype-specific regulatory aide, enhancing the expression of key genes that are collectively required for UPEC bloodstream pathogenesis.

Control of resistance against bacteriophage killing by a metabolic regulator in meningitis-associated Escherichia coli.

Ecologically beneficial traits in bacteria are encoded by intrinsic and horizontally acquired genes. However, such traits are not universal, and the highly mosaic nature of bacterial genomes requires control at the transcriptional level to drive these processes. It has emerged that regulatory flexibility is widespread in the Escherichia coli species, whereby preexisting transcription factors can acquire new and unrelated roles in regulating beneficial traits. DsdC is the regulator of D-serine tolerance in E. coli, is essential for D-serine catabolism, and is often encoded by two copies in neonatal meningitis-associated E. coli (NMEC). Here, we reveal that DsdC is a global regulator of transcription in NMEC and does not require D-serine for the control of novel beneficial traits. We show that DsdC binds the chromosome in an unusual manner, with many binding sites arranged in clusters spanning entire operons and within gene coding sequences, such as neuO. Importantly, we identify neuO as the most significantly down-regulated gene in a strain deleted for both dsdC copies, in both the presence and absence of D-serine. NeuO is prophage encoded in several NMEC K1 isolates and mediates capsule O-acetylation but has no effect on attachment to or invasion of human brain endothelial cells. Instead, we demonstrate that NeuO provides resistance against K1 bacteriophage attack and that this critical function is regulated by DsdC. This work highlights how a horizontally acquired enzyme that functions in cell-surface modulation can be controlled by an intrinsic regulator to provide a key ecological benefit to an E. coli pathotype.

d-Serine induces distinct transcriptomes in diverse Escherichia coli pathotypes.

Appropriate interpretation of environmental signals facilitates niche specificity in pathogenic bacteria. However, the responses of niche-specific pathogens to common host signals are poorly understood. d-Serine (d-ser) is a toxic metabolite present in highly variable concentrations at different colonization sites within the human host that we previously found is capable of inducing changes in gene expression. In this study, we made the striking observation that the global transcriptional response of three Escherichia coli pathotypes - enterohaemorrhagic E. coli (EHEC), uropathogenic E. coli (UPEC) and neonatal meningitis-associated E. coli (NMEC) - to d-ser was highly distinct. In fact, we identified no single differentially expressed gene common to all three strains. We observed the induction of ribosome-associated genes in extraintestinal pathogens UPEC and NMEC only, and the induction of purine metabolism genes in gut-restricted EHEC, and UPEC indicating distinct transcriptional responses to a common signal. UPEC and NMEC encode dsdCXA - a genetic locus required for detoxification and hence normal growth in the presence of d-ser. Specific transcriptional responses were induced in strains accumulating d-ser (WT EHEC and UPEC/NMEC mutants lacking the d-ser-responsive transcriptional activator DsdC), corroborating the notion that d-ser is an unfavourable metabolite if not metabolized. Importantly, many of the UPEC-associated transcriptome alterations correlate with published data on the urinary transcriptome, supporting the hypothesis that d-ser sensing forms a key part of urinary niche adaptation in this pathotype. Collectively, our results demonstrate distinct pleiotropic responses to a common metabolite in diverse E. coli pathotypes, with important implications for niche selectivity.

Going virtual: a report from the sixth Young Microbiologists Symposium on 'Microbe Signalling, Organisation and Pathogenesis'.

The sixth Young Microbiologists Symposium on 'Microbe Signalling, Organisation and Pathogenesis' was scheduled to be held at the University of Southampton, UK, in late August 2020. However, due to the health and safety guidelines and travel restrictions as a response to the COVID-19 pandemic, the symposium was transitioned to a virtual format, a change embraced enthusiastically as the meeting attracted over 200 microbiologists from 40 countries. The event allowed junior scientists to present their work to a broad audience and was supported by the European Molecular Biology Organization, the Federation of European Microbiological Societies, the Society of Applied Microbiology, the Biochemical Society, the Microbiology Society and the National Biofilms Innovation Centre. Sessions covered recent advances in all areas of microbiology including: Secretion and transport across membranes, Gene regulation and signalling, Host-microbe interactions, and Microbial communities and biofilm formation. This report focuses on several of the highlights and exciting developments communicated during the talks and poster presentations.

Prokaryotic life finds a way: insights from evolutionary experimentation in bacteria.

While evolution proceeds through the generation of random variant alleles, the application of selective pressures can select for subsets of mutations that confer fitness-improving physiological benefits. This, in essence, defines the process of adaptive evolution. The rapid replication rate of bacteria has allowed for the design of experiments to study these processes over a reasonable timeframe within a laboratory setting. This has been greatly assisted by advances in tractability of diverse microorganisms, next generation sequencing technologies and bioinformatic analysis pipelines. Examining the processes by which organisms adapt their genetic code to cope with sub-optimal growth conditions has yielded a wealth of molecular insight into diverse biological processes. Here we discuss how the study of adaptive evolutionary trajectories in bacteria has allowed for improved understanding of stress responses, revealed important insight into microbial physiology, allowed for the production of highly optimised strains for use in biotechnology and increased our knowledge of the role of genomic plasticity in chronic infections.

Genomic plasticity of pathogenic Escherichia coli mediates d-serine tolerance via multiple adaptive mechanisms.

The molecular environment of the host can have profound effects on the behavior of resident bacterial species. We recently established how the sensing and response of enterohemorrhagic Escherichia coli (EHEC) to d-serine (d-Ser) resulted in down-regulation of type 3 secretion system-dependent colonization, thereby avoiding unfavorable environments abundant in this toxic metabolite. However, this model ignores a key determinant of the success of bacterial pathogens, adaptive evolution. In this study, we have explored the adaptation of EHEC to d-Ser and its consequences for pathogenesis. We rapidly isolated multiple, independent, EHEC mutants whose growth was no longer compromised in the presence of d-Ser. Through a combination of whole-genome sequencing and transcriptomics, we showed that tolerance could be attributed to disruption of one of two d-Ser transporters and/or activation of a previously nonfunctional d-Ser deaminase. While the implication of cytoplasmic transport in d-Ser toxicity was unsurprising, disruption of a single transporter, CycA, was sufficient to completely overcome the repression of type 3 secretion system activity normally associated with exposure to d-Ser. Despite the fact that this reveals a mechanism by which evolution could drive a pathogen to colonize new niches, interrogation of sequenced E. coli O157:H7 genomes showed a high level of CycA conservation, highlighting a strong selective pressure for functionality. Collectively, these data show that CycA is a critically important conduit for d-Ser uptake that is central to the niche restriction of EHEC.

Widespread Strain-Specific Distinctions in Chromosomal Binding Dynamics of a Highly Conserved Escherichia coli Transcription Factor.

Bacterial gene regulation is governed by often hundreds of transcription factors (TFs) that bind directly to targets on the chromosome. Global studies of TFs usually make assumptions that regulatory targets within model strains will be conserved between members of the same species harboring common genetic targets. We recently discovered that YhaJ of Escherichia coli is crucial for virulence in two different pathotypes but binds to distinct regions of their genomes and regulates no common genes. This surprising result leads to strain-specific mechanisms of virulence regulation, but the implications for other E. coli pathotypes or commensals were unclear. Here, we report that heterogenous binding of YhaJ is widespread within the E. coli species. We analyzed the global YhaJ binding dynamics of four evolutionarily distinct E. coli isolates under two conditions, revealing 78 significant sites on the core genome as well as horizontally acquired loci. Condition-dependent dosage of YhaJ correlated with the number of occupied sites in vivo but did not significantly alter its enrichment at regions bound in both conditions, explaining the availability of this TF to occupy accessory sites in response to the environment. Strikingly, only ∼15% of YhaJ binding sites were common to all strains. Furthermore, differences in enrichment of uncommon sites were observed largely in chromosomal regions found in all strains and not explained exclusively by binding to strain-specific horizontally acquired elements or mutations in the DNA binding sequence. This observation suggests that intraspecies distinctions in TF binding dynamics are a widespread phenomenon and represent strain-specific gene regulatory potential.IMPORTANCE In bacterial cells, hundreds of transcription factors coordinate gene regulation and thus are a major driver of cellular processes. However, the immense diversity in bacterial genome structure and content makes deciphering regulatory networks challenging. This is particularly apparent for the model organism Escherichia coli as evolution has driven the emergence of species members with highly distinct genomes, which occupy extremely different niches in nature. While it is well-known that transcription factors must integrate horizontally acquired DNA into the regulatory network of the cell, the extent of regulatory diversity beyond single model strains is unclear. We have explored this concept in four evolutionarily distinct E. coli strains and show that a highly conserved transcription factor displays unprecedented diversity in chromosomal binding sites. Importantly, this diversity is not restricted to strain-specific DNA or mutation in binding sites. This observation suggests that strain-specific regulatory networks are potentially widespread within individual bacterial species.

Plastic Circuits: Regulatory Flexibility in Fine Tuning Pathogen Success.

Bacterial pathogens employ diverse fitness and virulence mechanisms to gain an advantage in competitive niches. These lifestyle-specific traits require integration into the regulatory network of the cell and are often controlled by pre-existing transcription factors. In this review, we highlight recent advances that have been made in characterizing this regulatory flexibility in prominent members of the Enterobacteriaceae. We focus on the direct global interactions between transcription factors and their target genes in pathogenic Escherichia coli and Salmonella revealed using chromatin immunoprecipitation coupled with next-generation sequencing. Furthermore, the implications and advantages of such regulatory adaptations in benefiting distinct pathogenic lifestyles are discussed.

A highly conserved complete accessory Escherichia coli type III secretion system 2 is widespread in bloodstream isolates of the ST69 lineage.

Bacterial type III secretion systems (T3SSs) play an important role in pathogenesis of Gram-negative infections. Enteropathogenic and enterohemorrhagic Escherichia coli contain a well-defined T3SS but in addition a second T3SS termed E. coli T3SS 2 (ETT2) has been described in a number of strains of E. coli. The majority of pathogenic E. coli contain elements of a genetic locus encoding ETT2, but which has undergone significant mutational attrition rendering it without predicted function. Only a very few strains have been reported to contain an intact ETT2 locus. To investigate the occurrence of the ETT2 locus in strains of human pathogenic E. coli, we carried out genomic sequencing of 162 isolates obtained from patient blood cultures in Scotland. We found that 22 of 26 sequence type (ST) 69 isolates from this collection contained an intact ETT2 together with an associated eip locus which encodes putative secreted ETT2 effectors as well as eilA, a gene encoding a putative transcriptional regulator of ETT2 associated genes. Using a reporter gene for eilA activation, we defined conditions under which this gene was differentially activated. Analysis of published E. coli genomes with worldwide representation showed that ST69 contained an intact ETT2 in these strains as well. The conservation of the genes encoding ETT2 in human pathogenic ST69 strains strongly suggests it has importance in infection, although its exact functional role remains obscure.

Propionic Acid Promotes the Virulent Phenotype of Crohn's Disease-Associated Adherent-Invasive Escherichia coli.

Propionic acid (PA) is a bacterium-derived intestinal antimicrobial and immune modulator used widely in food production and agriculture. Passage of Crohn's disease-associated adherent-invasive Escherichia coli (AIEC) through a murine model, in which intestinal PA levels are increased to mimic the human intestine, leads to the recovery of AIEC with significantly increased virulence. Similar phenotypic changes are observed outside the murine model when AIEC is grown in culture with PA as the sole carbon source; such PA exposure also results in AIEC that persists at 20-fold higher levels in vivo. RNA sequencing identifies an upregulation of genes involved in biofilm formation, stress response, metabolism, membrane integrity, and alternative carbon source utilization. PA exposure also increases virulence in a number of E. coli isolates from Crohn's disease patients. Removal of PA is sufficient to reverse these phenotypic changes. Our data indicate that exposure to PA results in AIEC resistance and increased virulence in its presence.

Distinct intraspecies virulence mechanisms regulated by a conserved transcription factor.

Tailoring transcriptional regulation to coordinate the expression of virulence factors in tandem with the core genome is a hallmark of bacterial pathogen evolution. Bacteria encode hundreds of transcription factors forming the base-level control of gene regulation. Moreover, highly homologous regulators are assumed to control conserved genes between members within a species that harbor the same genetic targets. We have explored this concept in 2 Escherichia coli pathotypes that employ distinct virulence mechanisms that facilitate specification of a different niche within the host. Strikingly, we found that the transcription factor YhaJ actively regulated unique gene sets between intestinal enterohemorrhagic E. coli (EHEC) and extraintestinal uropathogenic E. coli (UPEC), despite being very highly conserved. In EHEC, YhaJ directly activates expression of type 3 secretion system components and effectors. Alternatively, YhaJ enhances UPEC virulence regulation by binding directly to the phase-variable type 1 fimbria promoter, driving its expression. Additionally, YhaJ was found to override the universal GAD acid tolerance system but exclusively in EHEC, thereby indirectly enhancing type 3 secretion pleiotropically. These results have revealed that within a species, conserved regulators are actively repurposed in a "personalized" manner to benefit particular lifestyles and drive virulence via multiple distinct mechanisms.

Genomic and transcriptomic characterization of Pseudomonas aeruginosa small colony variants derived from a chronic infection model.

Phenotypic change is a hallmark of bacterial adaptation during chronic infection. In the case of chronic Pseudomonas aeruginosa lung infection in patients with cystic fibrosis, well-characterized phenotypic variants include mucoid and small colony variants (SCVs). It has previously been shown that SCVs can be reproducibly isolated from the murine lung following the establishment of chronic infection with mucoid P. aeruginosa strain NH57388A. Using a combination of single-molecule real-time (PacBio) and Illumina sequencing we identify a large genomic inversion in the SCV through recombination between homologous regions of two rRNA operons and an associated truncation of one of the 16S rRNA genes and suggest this may be the genetic switch for conversion to the SCV phenotype. This phenotypic conversion is associated with large-scale transcriptional changes distributed throughout the genome. This global rewiring of the cellular transcriptomic output results in changes to normally differentially regulated genes that modulate resistance to oxidative stress, central metabolism and virulence. These changes are of clinical relevance because the appearance of SCVs during chronic infection is associated with declining lung function.

Characterization of the Mode of Action of Aurodox, a Type III Secretion System Inhibitor from Streptomyces goldiniensis.

Recent work has demonstrated that the polyketide natural product Aurodox from Streptomyces goldiniensis is able to block the pathogenesis of the murine pathogen Citrobacter rodentium In this work, we aimed to gain a better understanding of the mechanism of action of the compound. We show that Aurodox downregulates the expression of the type III secretion systems of enteropathogenic and enterohemorrhagic Escherichia coli Furthermore, we have used transcriptomic analysis to show that Aurodox inhibits the expression at the transcriptional level by repressing the master regulator, ler Our data support a model in which Aurodox acts upstream of ler and not directly on the secretion system itself. Finally, we have shown that Aurodox, unlike some traditional antibiotics, does not induce expression of RecA, which is essential for the production of Shiga toxin. We propose that these properties nominate Aurodox as a promising antivirulence therapy for the treatment of these infections.

Control freaks-signals and cues governing the regulation of virulence in attaching and effacing pathogens.

Enterohaemorrhagic Escherichia coli (EHEC) mediates disease using a type 3 secretion system (T3SS), which is encoded on the locus of enterocyte effacement (LEE) and is tightly controlled by master regulators. This system is further modulated by a number of signals that help to fine-tune virulence, including metabolic, environmental and chemical signals. Since the LEE and its master regulator, Ler, were established, there have been numerous scientific advancements in understanding the regulation and expression of virulence factors in EHEC. This review will discuss the recent advancements in this field since our previous review, with a focus on the transcriptional regulation of the LEE.

Author Correction: Host-associated niche metabolism controls enteric infection through fine-tuning the regulation of type 3 secretion.

The original version of this Article contained an error in the spelling of the author David Ruano-Gallego, which was incorrectly given as David R. Gallego. This has now been corrected in both the PDF and HTML versions of the Article.

Host-associated niche metabolism controls enteric infection through fine-tuning the regulation of type 3 secretion.

Niche-adaptation of a bacterial pathogen hinges on the ability to recognize the complexity of signals from the environment and integrate that information with the regulation of genes critical for infection. Here we report the transcriptome of the attaching and effacing pathogen Citrobacter rodentium during infection of its natural murine host. Pathogen gene expression in vivo was heavily biased towards the virulence factor repertoire and was found to be co-ordinated uniquely in response to the host. Concordantly, we identified the host-specific induction of a metabolic pathway that overlapped with the regulation of virulence. The essential type 3 secretion system and an associated suite of distinct effectors were found to be modulated co-ordinately through a unique mechanism involving metabolism of microbiota-derived 1,2-propanediol, which dictated the ability to colonize the host effectively. This study provides novel insights into how host-specific metabolic adaptation acts as a cue to fine-tune virulence.

Antibiotics induce sustained dysregulation of intestinal T cell immunity by perturbing macrophage homeostasis.

Macrophages in the healthy intestine are highly specialized and usually respond to the gut microbiota without provoking an inflammatory response. A breakdown in this tolerance leads to inflammatory bowel disease (IBD), but the mechanisms by which intestinal macrophages normally become conditioned to promote microbial tolerance are unclear. Strong epidemiological evidence linking disruption of the gut microbiota by antibiotic use early in life to IBD indicates an important role for the gut microbiota in modulating intestinal immunity. Here, we show that antibiotic use causes intestinal macrophages to become hyperresponsive to bacterial stimulation, producing excess inflammatory cytokines. Re-exposure of antibiotic-treated mice to conventional microbiota induced a long-term, macrophage-dependent increase in inflammatory T helper 1 (TH1) responses in the colon and sustained dysbiosis. The consequences of this dysregulated macrophage activity for T cell function were demonstrated by increased susceptibility to infections requiring TH17 and TH2 responses for clearance (bacterial Citrobacter rodentium and helminth Trichuris muris infections), corresponding with increased inflammation. Short-chain fatty acids (SCFAs) were depleted during antibiotic administration; supplementation of antibiotics with the SCFA butyrate restored the characteristic hyporesponsiveness of intestinal macrophages and prevented T cell dysfunction. Butyrate altered the metabolic behavior of macrophages to increase oxidative phosphorylation and also promoted alternative macrophage activation. In summary, the gut microbiota is essential to maintain macrophage-dependent intestinal immune homeostasis, mediated by SCFA-dependent pathways. Oral antibiotics disrupt this process to promote sustained T cell-mediated dysfunction and increased susceptibility to infections, highlighting important implications of repeated broad-spectrum antibiotic use.