Characterizing the Pathogenic Potential of Crohn's Disease-Associated Adherent-Invasive Escherichia coli.

The microbiome of Crohn's disease (CD) patients is composed of a microbial community that is considered dysbiotic and proinflammatory in nature. The overrepresentation of Enterobacteriaceae species is a common feature of the CD microbiome, and much attention has been given to understanding the pathogenic role this feature plays in disease activity. Over 2 decades ago, a new Escherichia coli subtype called adherent-invasive E. coli (AIEC) was isolated and linked to ileal Crohn's disease. Since the isolation of the first AIEC strain, additional AIEC strains have been isolated from both inflammatory bowel disease (IBD) patients and non-IBD individuals using the original in vitro phenotypic characterization methods. Identification of a definitive molecular marker of the AIEC pathotype has been elusive; however, significant advancements have been made in understanding the genetic, metabolic, and virulence determinants of AIEC infection biology. Here, we review the current knowledge of AIEC pathogenesis to provide additional, objective measures that could be considered in defining AIEC and their pathogenic potential.

Psychological stress impairs IL22-driven protective gut mucosal immunity against colonising pathobionts.

Crohn's disease is an inflammatory disease of the gastrointestinal tract characterized by an aberrant response to microbial and environmental triggers. This includes an altered microbiome dominated by Enterobacteriaceae and in particular adherent-invasive E. coli (AIEC). Clinical evidence implicates periods of psychological stress in Crohn's disease exacerbation, and disturbances in the gut microbiome might contribute to the pathogenic mechanism. Here we show that stress-exposed mice develop ileal dysbiosis, dominated by the expansion of Enterobacteriaceae. In an AIEC colonisation model, stress-induced glucocorticoids promote apoptosis of CD45+CD90+ cells that normally produce IL-22, a cytokine that is essential for the maintenance of ileal mucosal barrier integrity. Blockade of glucocorticoid signaling or administration of recombinant IL-22 restores mucosal immunity, prevents ileal dysbiosis, and blocks AIEC expansion. We conclude that psychological stress impairs IL-22-driven protective immunity in the gut, which creates a favorable niche for the expansion of pathobionts that have been implicated in Crohn's disease. Importantly, this work also shows that immunomodulation can counteract the negative effects of psychological stress on gut immunity and hence disease-associated dysbiosis.

Low dietary fiber promotes enteric expansion of a Crohn's disease-associated pathobiont independent of obesity.

Obesity is associated with metabolic, immunological, and infectious disease comorbidities, including an increased risk of enteric infection and inflammatory bowel disease such as Crohn's disease (CD). Expansion of intestinal pathobionts such as adherent-invasive Escherichia coli (AIEC) is a common dysbiotic feature of CD, which is amplified by prior use of oral antibiotics. Although high-fat, high-sugar diets are associated with dysbiotic expansion of E. coli, it is unknown if the content of fat or another dietary component in obesogenic diets is sufficient to promote AIEC expansion. Here, we found that administration of an antibiotic combined with feeding mice an obesogenic low-fiber, high-sucrose, high-fat diet (HFD) that is typically used in rodent-obesity studies promoted AIEC intestinal expansion. Even a short-term (i.e., 1 day) pulse of HFD feeding before infection was sufficient to promote AIEC expansion, indicating that the magnitude of obesity was not the main driver of AIEC expansion. Controlled-diet experiments demonstrated that neither dietary fat nor sugar were the key determinants of AIEC colonization, but that lowering dietary fiber from approximately 13% to 5%-6% was sufficient to promote the intestinal expansion of AIEC when combined with antibiotics in mice. When combined with antibiotics, lowering fiber promoted AIEC intestinal expansion to a similar extent as widely used HFDs in mice. However, lowering dietary fiber was sufficient to promote AIEC intestinal expansion without affecting body mass. Our results show that low dietary fiber combined with oral antibiotics are environmental factors that promote the expansion of Crohn's disease-associated pathobionts in the gut.NEW & NOTEWORTHY It is commonly thought that obesity or a high-fat diet alters pathogenic bacteria and promotes inflammatory gut diseases. We found that lower dietary fiber is a key factor that expands a gut pathobiont linked to Crohn's disease, independent of obesity status in mice.

Emerging and divergent roles of pyrophosphorylated nucleotides in bacterial physiology and pathogenesis.

Bacteria inhabit diverse environmental niches and consequently must modulate their metabolism to adapt to stress. The nucleotide second messengers guanosine tetraphosphate (ppGpp) and guanosine pentaphosphate (pppGpp) (collectively referred to as (p)ppGpp) are essential for survival during nutrient starvation. (p)ppGpp is synthesized by the RelA-SpoT homologue (RSH) protein family and coordinates the control of cellular metabolism through its combined effect on over 50 proteins. While the role of (p)ppGpp has largely been associated with nutrient limitation, recent studies have shown that (p)ppGpp and related nucleotides have a previously underappreciated effect on different aspects of bacterial physiology, such as maintaining cellular homeostasis and regulating bacterial interactions with a host, other bacteria, or phages. (p)ppGpp produced by pathogenic bacteria facilitates the evasion of host defenses such as reactive nitrogen intermediates, acidic pH, and the complement system. Additionally, (p)ppGpp and pyrophosphorylated derivatives of canonical adenosine nucleotides called (p)ppApp are emerging as effectors of bacterial toxin proteins. Here, we review the RSH protein family with a focus on its unconventional roles during host infection and bacterial competition.

High-throughput fitness screening and transcriptomics identify a role for a type IV secretion system in the pathogenesis of Crohn's disease-associated Escherichia coli.

Adherent-invasive Escherichia coli (AIEC) are pathogenic bacteria frequently isolated from patients who have Crohn's disease (CD). Despite the phenotypic differences between AIEC and commensal E. coli, comparative genomic approaches have been unable to differentiate these two groups, making the identification of key virulence factors a challenge. Here, we conduct a high-resolution, in vivo genetic screen to map AIEC genes required for intestinal colonization of mice. In addition, we use in vivo RNA-sequencing to define the host-associated AIEC transcriptome. We identify diverse metabolic pathways required for efficient gut colonization by AIEC and show that a type IV secretion system (T4SS) is required to form biofilms on the surface of epithelial cells, thereby promoting AIEC persistence in the gut. E. coli isolated from CD patients are enriched for a T4SS, suggesting a possible connection to disease activity. Our findings establish the T4SS as a principal AIEC colonization factor and highlight the use of genome-wide screens in decoding the infection biology of CD-associated bacteria that otherwise lack a defined genetic signature.

The Unique Lifestyle of Crohn's Disease-Associated Adherent-Invasive Escherichia coli.

Escherichia coli is one of the most genetically and phenotypically diverse species of bacteria. This remarkable diversity produces a plethora of clinical outcomes following infection and has informed much of what we currently know about host-pathogen interactions for a wide range of bacteria-host relationships. In studying the role of microbes in disease, adherent-invasive E. coli (AIEC) has emerged as having a strong association with Crohn's disease (CD). Thus, there has been an equally strong effort to uncover the root origins of AIEC, to appreciate how AIEC differs from other well-known pathogenic E. coli variants, and to understand its connection to disease. Emerging from a growing body of research on AIEC is the understanding that AIEC itself is remarkably diverse, both in phylogenetic origins, genetic makeup, and behavior in the host setting. Here, we describe the unique lifestyle of CD-associated AIEC and review recent research that is uncovering the inextricable link between AIEC and its host in the context of CD.

Duodenal bacterial proteolytic activity determines sensitivity to dietary antigen through protease-activated receptor-2.

Microbe-host interactions are generally homeostatic, but when dysfunctional, they can incite food sensitivities and chronic diseases. Celiac disease (CeD) is a food sensitivity characterized by a breakdown of oral tolerance to gluten proteins in genetically predisposed individuals, although the underlying mechanisms are incompletely understood. Here we show that duodenal biopsies from patients with active CeD have increased proteolytic activity against gluten substrates that correlates with increased Proteobacteria abundance, including Pseudomonas. Using Pseudomonas aeruginosa producing elastase as a model, we show gluten-independent, PAR-2 mediated upregulation of inflammatory pathways in C57BL/6 mice without villus blunting. In mice expressing CeD risk genes, P. aeruginosa elastase synergizes with gluten to induce more severe inflammation that is associated with moderate villus blunting. These results demonstrate that proteases expressed by opportunistic pathogens impact host immune responses that are relevant to the development of food sensitivities, independently of the trigger antigen.

Host-Specific Adaptive Diversification of Crohn's Disease-Associated Adherent-Invasive Escherichia coli.

Crohn's disease (CD) is an inflammatory bowel disease influenced by bacteria. Adherent-invasive E. coli (AIEC) is associated with CD, yet the adaptations facilitating AIEC gut colonization are unknown. AIEC isolates exhibit high genetic diversity, suggesting strains evolve independently across different gut environments. We tracked the adaptive evolution of AIEC in a murine model of chronic colonization across multiple hosts and transmission events. We detected evolved lineages that outcompeted the ancestral strain in the host through independent mechanisms. One lineage was hypermotile because of a mobile insertion sequence upstream of the master flagellar regulator, flhDC, which enhanced AIEC invasion and establishment of a mucosal niche. Another lineage outcompeted the ancestral strain through improved use of acetate, a short-chain fatty acid in the gut. The presence of hypermotile and acetate-consuming lineages discriminated E. coli isolated from CD patients from healthy controls, suggesting an evolutionary trajectory that distinguishes AIEC from commensal E. coli.

A macrophage-based screen identifies antibacterial compounds selective for intracellular Salmonella Typhimurium.

Salmonella Typhimurium (S. Tm) establishes systemic infection in susceptible hosts by evading the innate immune response and replicating within host phagocytes. Here, we sought to identify inhibitors of intracellular S. Tm replication by conducting parallel chemical screens against S. Tm growing in macrophage-mimicking media and within macrophages. We identify several compounds that inhibit Salmonella growth in the intracellular environment and in acidic, ion-limited media. We report on the antimicrobial activity of the psychoactive drug metergoline, which is specific against intracellular S. Tm. Screening an S. Tm deletion library in the presence of metergoline reveals hypersensitization of outer membrane mutants to metergoline activity. Metergoline disrupts the proton motive force at the bacterial cytoplasmic membrane and extends animal survival during a systemic S. Tm infection. This work highlights the predictive nature of intracellular screens for in vivo efficacy, and identifies metergoline as a novel antimicrobial active against Salmonella.

Antibiotics Potentiate Adherent-Invasive E. coli Infection and Expansion.

BACKGROUND: Crohn's disease (CD) is an inflammatory bowel disease with a complex etiology. Paradoxically, CD is associated with the use of antibiotics and with an increased abundance of an unusual phenotypic group of Escherichia coli known as adherent-invasive E. coli (AIEC). However, the impact of antibiotics on AIEC infection has not been well studied in controlled models of infection. METHODS: We infected mice with AIEC before or after treatment with a variety of different classes of antibiotics. We assessed levels of AIEC in the feces and tissues, AIEC localization by immunofluorescence microscopy, and tissue pathology. RESULTS: We found that a wide range of antibiotic classes strongly potentiated initial AIEC infection and expanded AIEC in chronically infected mice. We found that the ability of antibiotics to potentiate AIEC infection did not correlate with a stereotyped shift in the gut bacterial community but was correlated with a decrease in overall diversity and a divergence from the pre-antibiotic state. We found that antibiotic-induced inflammation provided a fitness advantage for AIEC expansion through their use of oxidized metabolites in the postantibiotic period. CONCLUSIONS: Our results show that antibiotics can render hosts more susceptible to initial AIEC infection and can worsen infection in previously colonized hosts. AIEC appears to exploit host inflammatory responses that arise in the postantibiotic period, highlighting a previously unknown interaction between CD risk factors.

A polymicrobial view of disease potential in Crohn's-associated adherent-invasive E. coli.

The human gut is home to trillions of bacteria and provides the scaffold for one of the most complex microbial ecosystems in nature. Inflammatory bowel diseases, such as Crohn's disease, involve a compositional shift in the microbial constituents of this ecosystem with a marked expansion of Enterobacteriaceae, particularly Escherichia coli. Adherent-invasive E. coli (AIEC) strains are frequently isolated from the biopsies of Crohn's patients, where their ability to elicit inflammation suggests a possible role in Crohn's pathology. Here, we consider the origins of the AIEC pathovar and discuss how risk factors associated with Crohn's disease might influence AIEC colonization dynamics within the host to alter the overall disease potential of the microbial community.

Evolution of Salmonella-Host Cell Interactions through a Dynamic Bacterial Genome.

Salmonella Typhimurium has a broad arsenal of genes that are tightly regulated and coordinated to facilitate adaptation to the various host environments it colonizes. The genome of Salmonella Typhimurium has undergone multiple gene acquisition events and has accrued changes in non-coding DNA that have undergone selection by regulatory evolution. Together, at least 17 horizontally acquired pathogenicity islands (SPIs), prophage-associated genes, and changes in core genome regulation contribute to the virulence program of Salmonella. Here, we review the latest understanding of these elements and their contributions to pathogenesis, emphasizing the regulatory circuitry that controls niche-specific gene expression. In addition to an overview of the importance of SPI-1 and SPI-2 to host invasion and colonization, we describe the recently characterized contributions of other SPIs, including the antibacterial activity of SPI-6 and adhesion and invasion mediated by SPI-4. We further discuss how these fitness traits have been integrated into the regulatory circuitry of the bacterial cell through cis-regulatory evolution and by a careful balance of silencing and counter-silencing by regulatory proteins. Detailed understanding of regulatory evolution within Salmonella is uncovering novel aspects of infection biology that relate to host-pathogen interactions and evasion of host immunity.

Acute Infectious Gastroenteritis Potentiates a Crohn's Disease Pathobiont to Fuel Ongoing Inflammation in the Post-Infectious Period.

Crohn's disease (CD) is a chronic inflammatory condition of diverse etiology. Exposure to foodborne pathogens causing acute gastroenteritis produces a long-term risk of CD well into the post-infectious period but the mechanistic basis for this ongoing relationship to disease onset is unknown. We developed two novel models to study the comorbidity of acute gastroenteritis caused by Salmonella Typhimurium or Citrobacter rodentium in mice colonized with adherent-invasive Escherichia coli (AIEC), a bacterial pathobiont linked to CD. Here, we show that disease activity in the post-infectious period after gastroenteritis is driven by the tissue-associated expansion of the resident AIEC pathobiont, with an attendant increase in immunopathology, barrier defects, and delays in mucosal restitution following pathogen clearance. These features required AIEC resistance to host defense peptides and a fulminant inflammatory response to the enteric pathogen. Our results suggest that individuals colonized by AIEC at the time of acute infectious gastroenteritis may be at greater risk for CD onset. Importantly, our data identify AIEC as a tractable disease modifier, a finding that could be exploited in the development of therapeutic interventions following infectious gastroenteritis in at-risk individuals.

Multiple histidines in the periplasmic domain of the Salmonella enterica sensor kinase SsrA enhance signaling in response to extracellular acidification.

The two-component regulatory system SsrA-SsrB in Salmonella enterica controls expression of a virulence gene program required for intracellular survival in host cells. SsrA signaling is induced within the acidic host vacuole in which the bacteria reside; however, the mechanism by which SsrA senses this intracellular environment is unknown. Here, we show that the periplasmic sensor domain of SsrA is enriched in histidine residues that increase SsrA signaling below external pH of 6. While no single histidine accounted for the full acid-responsiveness of SsrA, we localized the acid-responsiveness principally to five histidines in the C-terminal end of the periplasmic sensor domain, with input from additional histidines in the N-terminal end of the senor. A sensor mutant lacking critical pH-responsive histidines was defective for acid-promoted activity, yet retained basal activity similar to wild type at neutral pH, indicating that the role of these histidines is to enhance signaling in response to acidification. In support of this, a pH-blind mutant was insensitive to the vacuole acidification blocking activity of bafilomycin, and was attenuated for competitive fitness during infection of mice. Our data demonstrate that SsrA contains a histidine-rich periplasmic sensor that enhances signaling in response to the innate host defense of vacuolar acidification.

Identification of the docking site between a type III secretion system ATPase and a chaperone for effector cargo.

A number of Gram-negative pathogens utilize type III secretion systems (T3SSs) to inject bacterial effector proteins into the host. An important component of T3SSs is a conserved ATPase that captures chaperone-effector complexes and energizes their dissociation to facilitate effector translocation. To date, there has been limited work characterizing the chaperone-T3SS ATPase interaction despite it being a fundamental aspect of T3SS function. In this study, we present the 2.1 Å resolution crystal structure of the Salmonella enterica SPI-2-encoded ATPase, SsaN. Our structure revealed a local and functionally important novel feature in helix 10 that we used to define the interaction domain relevant to chaperone binding. We modeled the interaction between the multicargo chaperone, SrcA, and SsaN and validated this model using mutagenesis to identify the residues on both the chaperone and ATPase that mediate the interaction. Finally, we quantified the benefit of this molecular interaction on bacterial fitness in vivo using chromosomal exchange of wild-type ssaN with mutants that retain ATPase activity but no longer capture the chaperone. Our findings provide insight into chaperone recognition by T3SS ATPases and demonstrate the importance of the chaperone-T3SS ATPase interaction for the pathogenesis of Salmonella.

Host defense peptide resistance contributes to colonization and maximal intestinal pathology by Crohn's disease-associated adherent-invasive Escherichia coli.

Host defense peptides secreted by colonocytes and Paneth cells play a key role in innate host defenses in the gut. In Crohn's disease, the burden of tissue-associated Escherichia coli commonly increases at epithelial surfaces where host defense peptides concentrate, suggesting that this bacterial population might actively resist this mechanism of bacterial killing. Adherent-invasive E. coli (AIEC) is associated with Crohn's disease; however, the colonization determinants of AIEC in the inflamed gut are undefined. Here, we establish that host defense peptide resistance contributes to host colonization by Crohn's-associated AIEC. We identified a plasmid-encoded genomic island (called PI-6) in AIEC strain NRG857c that confers high-level resistance to α-helical cationic peptides and α- and ß-defensins. Deletion of PI-6 sensitized strain NRG857c to these host defense molecules, reduced its competitive fitness in a mouse model of infection, and attenuated its ability to induce cecal pathology. This phenotype is due to two genes in PI-6, arlA, which encodes a Mig-14 family protein implicated in defensin resistance, and arlC, an OmpT family outer membrane protease. Implicit in these findings are new bacterial targets whose inhibition might limit AIEC burden and disease in the gut.

Persistent infection with Crohn's disease-associated adherent-invasive Escherichia coli leads to chronic inflammation and intestinal fibrosis.

Crohn's disease is a chronic inflammatory condition of the gastrointestinal tract in which alterations to the bacterial community contribute to disease. Adherent-invasive Escherichia coli are associated with human Crohn's disease; however, their role in intestinal immunopathology is unclear because of the lack of an animal model compatible with chronic timescales. Here we establish chronic adherent-invasive Escherichia coli infection in streptomycin-treated conventional mice (CD1, DBA/2, C3H, 129e and C57BL/6), enabling the study of host response and immunopathology. Adherent-invasive Escherichia coli induces an active T-helper 17 response, heightened levels of proinflammatory cytokines and fibrotic growth factors, with transmural inflammation and fibrosis. Depletion of CD8+ T cells increases caecal bacterial load, pathology and intestinal fibrosis in C57BL/6 mice, suggesting a protective role. Our findings provide evidence that chronic adherent-invasive Escherichia coli infections result in immunopathology similar to that seen in Crohn's disease. With this model, research into the host and bacterial genetics associated with adherent-invasive Escherichia coli-induced disease becomes more widely accessible.

Mapping and regulation of genes within Salmonella pathogenicity island 12 that contribute to in vivo fitness of Salmonella enterica Serovar Typhimurium.

Salmonella pathogenicity island 12 (SPI-12) of Salmonella enterica serovar Typhimurium is a 15-kb region that encompasses genes STM2230 to STM2245 and encodes a remnant phage known to contribute to bacterial virulence. In mouse infection experiments and replication assays in macrophages, we demonstrated a role for four genes in SPI-12 for bacterial survival in the host. STM2239, a potential Q antiterminator, showed a prominent contribution to bacterial fitness. Transcriptional reporter experiments, quantitative reverse transcription-PCR (RT-PCR), and immunoblotting demonstrated that the virulence regulator SsrB and STM2239 contribute to transcriptional activation of genes in SPI-12. SsrB was found to indirectly regulate this locus by transcriptional read-through from the sspH2 (STM2241) promoter. Chromatin immunoprecipitation showed that STM2239 copurified with the promoter regulating STM2237, suggesting that STM2239 may function as an antiterminator to activate adjacent genes. These results demonstrate that bacteriophage genes may be adapted by pathogenic bacteria to improve fitness in the host.

Type VI secretion system-associated gene clusters contribute to pathogenesis of Salmonella enterica serovar Typhimurium.

The enteropathogen Salmonella enterica serovar Typhimurium employs a suite of tightly regulated virulence factors within the intracellular compartment of phagocytic host cells resulting in systemic dissemination in mice. A type VI secretion system (T6SS) within Salmonella pathogenicity island 6 (SPI-6) has been implicated in this process; however, the regulatory inputs and the roles of noncore genes in this system are not well understood. Here we describe four clusters of noncore T6SS genes in SPI-6 based on a comparative relationship with the T6SS-3 of Burkholderia mallei and report that the disruption of these genes results in defects in intracellular replication and systemic dissemination in mice. In addition, we show that the expression of the SPI-6-encoded Hcp and VgrG orthologs is enhanced during late stages of macrophage infection. We identify six regions that are transcriptionally active during cell infections and that have regulatory contributions from the regulators of virulence SsrB, PhoP, and SlyA. We show that levels of protein expression are very weak under in vitro conditions and that expression is not enhanced upon the deletion of ssrB, phoP, slyA, qseC, ompR, or hfq, suggesting an unknown activating factor. These data suggest that the SPI-6 T6SS has been integrated into the Salmonella Typhimurium virulence network and customized for host-pathogen interactions through the action of noncore genes.

Characterization of DalS, an ATP-binding cassette transporter for D-alanine, and its role in pathogenesis in Salmonella enterica.

Expansion into new host niches requires bacterial pathogens to adapt to changes in nutrient availability and to evade an arsenal of host defenses. Horizontal acquisition of Salmonella Pathogenicity Island (SPI)-2 permitted the expansion of Salmonella enterica serovar Typhimurium into the intracellular environment of host cells by allowing it to deliver bacterial effector proteins across the phagosome membrane. This is facilitated by the SsrA-SsrB two-component regulatory system and a type III secretion system encoded within SPI-2. SPI-2 acquisition was followed by evolution of existing regulatory DNA, creating an expanded SsrB regulon involved in intracellular fitness and host infection. Here, we identified an SsrB-regulated operon comprising an ABC transporter in Salmonella. Biochemical and structural studies determined that the periplasmic solute-binding component, STM1633/DalS, transports D-alanine and that DalS is required for intracellular survival of the bacteria and for fitness in an animal host. This work exemplifies the role of nutrient exchange at the host-pathogen interface as a critical determinant of disease outcome.