James P. R. Connolly

From ingestion to colonization: the influence of the host environment on regulation of the LEE encoded type III secretion system in enterohaemorrhagic Escherichia coli.

Enterohaemorrhagic Escherichia coli (EHEC) binds to host tissue and intimately attaches to intestinal cells using a dedicated type III secretion system (T3SS). This complex multi-protein organelle is encoded within a large pathogenicity island called the locus of enterocyte effacement (LEE), which is subject to extensive regulatory control. Over the past 15 years we have gained a wealth of knowledge concerning how the LEE is regulated transcriptionally by specific, global and phage encoded regulators. More recently, significant advances have been made in our understanding of how specific signals, including host or microbiota derived metabolic products and various nutrient sources, can affect how the LEE-encoded T3SS is regulated. In this review we discuss regulation of the LEE, focusing on how these physiologically relevant signals are sensed and how they affect the expression of this major virulence factor. The implications for understanding the disease process by specific regulatory mechanisms are also discussed.

The host metabolite D-serine contributes to bacterial niche specificity through gene selection

Escherichia coli comprise a diverse array of both commensals and niche-specific pathotypes. The ability to cause disease results from both carriage of specific virulence factors and regulatory control of these via environmental stimuli. Moreover, host metabolites further refine the response of bacteria to their environment and can dramatically affect the outcome of the host–pathogen interaction. Here, we demonstrate that the host metabolite, D-serine, selectively affects gene expression in E. coli O157:H7. Transcriptomic profiling showed exposure to D-serine results in activation of the SOS response and suppresses expression of the Type 3 Secretion System (T3SS) used to attach to host cells. We also show that concurrent carriage of both the D-serine tolerance locus (dsdCXA) and the locus of enterocyte effacement pathogenicity island encoding a T3SS is extremely rare, a genotype that we attribute to an ‘evolutionary incompatibility’ between the two loci. This study demonstrates the importance of co-operation between both core and pathogenic genetic elements in defining niche specificity.

The metabolic enzyme AdhE controls the virulence of Escherichia coli O157:H7

Classical studies have focused on the role that individual regulators play in controlling virulence gene expression. An emerging theme, however, is that bacterial metabolism also plays a key role in this process. Our previous work identified a series of proteins that were implicated in the regulation of virulence. One of these proteins was AdhE, a bi‐functional acetaldehyde‐CoA dehydrogenase and alcohol dehydrogenase. Deletion of its gene (adhE) resulted in elevated levels of extracellular acetate and a stark pleiotropic phenotype: strong suppression of the Type Three Secretion System (T3SS) and overexpression of non‐functional flagella. Correspondingly, the adhE mutant bound poorly to host cells and was unable to swim. Furthermore, the mutant was significantly less virulent than its parent when tested in vivo, which supports the hypothesis that attachment and motility are central to the colonization process. The molecular basis by which AdhE affects virulence gene regulation was found to be multifactorial, involving acetate‐stimulated transcription of flagella expression and post‐transcriptional regulation of the T3SS through Hfq. Our study reveals fascinating insights into the links between bacterial physiology, the expression of virulence genes, and the underlying molecular mechanism mechanisms by which these processes are regulated.

A Highly Conserved Bacterial D-Serine Uptake System Links Host Metabolism and Virulence.

The ability of any organism to sense and respond to challenges presented in the environment is critically important for promoting or restricting colonization of specific sites. Recent work has demonstrated that the host metabolite D-serine has the ability to markedly influence the outcome of infection by repressing the type III secretion system of enterohaemorrhagic Escherichia coli (EHEC) in a concentration-dependent manner. However, exactly how EHEC monitors environmental D-serine is not understood. In this work, we have identified two highly conserved members of the E. coli core genome, encoding an inner membrane transporter and a transcriptional regulator, which collectively help to “sense” levels of D-serine by regulating its uptake from the environment and in turn influencing global gene expression. Both proteins are required for full expression of the type III secretion system and diversely regulated prophage-encoded effector proteins demonstrating an important infection-relevant adaptation of the core genome. We propose that this system acts as a key safety net, sampling the environment for this metabolite, thereby promoting colonization of EHEC to favorable sites within the host.

Novel Compounds Targeting the Enterohaemorrhagic Escherichia coli Type Three Secretion System Reveal Insights into Mechanisms of Secretion Inhibition

Anti‐virulence (AV) compounds are a promising alternative to traditional antibiotics for fighting bacterial infections. The Type Three Secretion System (T3SS) is a well‐studied and attractive AV target, given that it is widespread in more than 25 species of Gram‐negative bacteria, including enterohemorrhagic E. coli (EHEC), and as it is essential for host colonization by many pathogens. In this work, we designed, synthesized and tested a new series of compounds that block the functionality of the T3SS of EHEC. Affinity chromatography experiments identified the primary target of the compounds as the T3SS needle pore protein EspD, which is essential for effector protein translocation into host cells. These data were supported by mechanistic studies that determined the coiled‐coil domain 1 of EspD as a key compound‐binding site, thereby preventing correct assembly of the T3SS complex on the cell surface. However, binding of inhibitors to EspD or deletion of EspD itself did not result in transcriptional down‐regulation of effector proteins. Instead, we found the compounds to exhibit dual‐functionality by also down‐regulating transcription of the entire chromosomal locus encoding the T3SS, further demonstrating their desirability and effectiveness.

The Highly Conserved Escherichia coli Transcription Factor YhaJ Regulates Aromatic Compound Degradation.

The aromatic compound 2,4-dinitrotoluene (DNT), a common impurity in 2,4,6-trinitrotoluene (TNT) production, has been suggested as a tracer for the presence of TNT-based landmines due to its stability and high volatility. We have previously described an Escherichia coli bioreporter capable of detecting the presence of DNT vapors, harboring a fusion of the yqjF gene promoter to a reporter element. However, the DNT metabolite which is the direct inducer of yqjF, has not yet been identified, nor has the regulatory mechanism of the induction been clarified. We demonstrate here that the YhaJ protein, a member of the LysR type family, acts as a transcriptional regulator of yqjF activation, as well as of a panel of additional E. coli genes. This group of genes share a common sequence motif in their promoters, which is suggested here as a putative YhaJ-box. In addition, we have linked YhaJ to the regulation of quinol-like compound degradation in the cell, and identified yhaK as playing a role in the degradation of DNT.

When and where? Pathogenic Escherichia coli differentially sense host D-serine using a universal transporter system to monitor their environment

Sensing environmental stimuli is critically important for bacteria when faced with the multitude of adversities presented within the host. Responding appropriately to these signals and in turn integrating these responses into the regulatory network of the cell allows bacteria to control precisely when and where they should establish colonization. D-serine is an abundant metabolite of the human urinary tract but is a toxic metabolite for Escherichia coli that lack a D-serine tolerance locus. Enterohaemorrhagic E. coli (EHEC) cannot catabolize D-serine for this reason and colonize the large intestine specifically, an environment low in D-serine. EHEC can however use D-serine sensing to repress colonization thus signaling the presence of an unfavorable environment. In our recent work (Connolly, et al. PLoS Pathogens (2016) 12(1): e1005359), we describe the discovery of a functional and previously uncharacterized D-serine uptake system in E. coli. The genes identified are highly conserved in all E. coli lineages but are regulated differentially in unique pathogenic backgrounds. The study identified that EHEC, counter-intuitively, increase D-serine uptake in its presence but that this is a tolerated process and is used to increase the transcriptional response to this signal. It was also found that the system has been integrated into the transcriptional network of EHEC-specific virulence genes, demonstrating an important pathotype-specific adaptation of core genome components.

Identification and Characterization of Novel Compounds Blocking Shiga Toxin Expression in Escherichia coli O157:H7

Infections caused by Shiga toxin (Stx)-producing E. coli strains constitute a health problem, as they are problematic to treat. Stx production is a key virulence factor associated with the pathogenicity of enterohaemorrhagic E. coli (EHEC) and can result in the development of haemolytic uremic syndrome in infected patients. The genes encoding Stx are located on temperate lysogenic phages integrated into the bacterial chromosome and expression of the toxin is generally coupled to phage induction through the SOS response. We aimed to find new compounds capable of blocking expression of Stx type 2 (Stx2) as this subtype of Stx is more strongly associated with human disease. High-throughput screening of a small-molecule library identified a lead compound that reduced Stx2 expression in a dose-dependent manner. We show that the optimized compound interferes with the SOS response by directly affecting the activity and oligomerization of RecA, thus limiting phage activation and Stx2 expression. Our work suggests that RecA is highly susceptible to inhibition and that targeting this protein is a viable approach to limiting production of Stx2 by EHEC. This type of approach has the potential to limit production and transfer of other phage induced and transduced determinants.

Propionic acid enhances the virulence of Crohn's disease-associated adherent-invasive Escherichia coli

Short chain fatty acids (SCFA), such as propionic acid (PA), are natural human intestinal antimicrobials and immune modulators that are also used in Western food production and agriculture. Here we examine the effect of PA on the pathogenicity of the Crohn’s disease-associated microbe, adherent-invasive Escherichia coli (AIEC). We show that AIEC is insensitive to the antimicrobial effects of PA and adapts to utilize it for efficient growth. Repeated exposure of AIEC to PA significantly increases AIEC adherence to human intestinal epithelial cells, acid tolerance and biofilm formation. RNA-sequencing identified transcriptional changes in response to PA with upregulation of genes involved in biofilm formation, stress responses, metabolism, membrane integrity and use of alternative carbon sources. Finally, after pre-exposure to PA, AIEC demonstrated an increased ability to colonize (>5-fold) and persist (>50-fold) in an in vivo model where the low murine intestinal PA concentrations were increased to mimic those found in humans. Our data indicates that exposure of AIEC to PA evolves bacteria that are both resistant to this natural human intestinal antimicrobial, and increasingly virulent in its presence. Importance Propionic acid (PA) is a common agricultural and industrial antimicrobial in the Western world. Its use is increasing as efforts intensify to reduce reliance on antibiotics, particularly in food production. Here we show that exposure of adherent-invasive Escherichia coli (AIEC) to PA induced significant virulence associated phenotypic changes. AIEC exhibited greater adherence and biofilm formation in vitro, translating into greater colonization and persistence in an in vivo mouse model. RNAseq analysis identified transcriptional changes directly related to these phenotypic changes. While PA is generally regarded as safe for both human and animal use, the significance of SCFAs such as PA to maintaining human intestinal immune health means their widespread use requires further scrutiny. This will allow us to understand if, in a similar manner to antibiotics, SCFAs are facilitating horizontal transmission of microorganisms.

Genomic inversion drives small colony variant formation and increased virulence in P. aeruginosa

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-characterised 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. Here we show, using a combination of singlemolecule real-time (PacBio) and Illumina sequencing that the genetic switch for conversion to the SCV phenotype is a large genomic inversion through recombination between homologous regions of two rRNA operons. 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 since the appearance of SCVs during chronic infection is associated with declining lung function.