Endocannabinoids Inhibit the Induction of Virulence in Enteric Pathogens.

Endocannabinoids are host-derived lipid hormones that fundamentally impact gastrointestinal (GI) biology. The use of cannabis and other exocannabinoids as anecdotal treatments for various GI disorders inspired the search for mechanisms by which these compounds mediate their effects, which led to the discovery of the mammalian endocannabinoid system. Dysregulated endocannabinoid signaling was linked to inflammation and the gut microbiota. However, the effects of endocannabinoids on host susceptibility to infection has not been explored. Here, we show that mice with elevated levels of the endocannabinoid 2-arachidonoyl glycerol (2-AG) are protected from enteric infection by Enterobacteriaceae pathogens. 2-AG directly modulates pathogen function by inhibiting virulence programs essential for successful infection. Furthermore, 2-AG antagonizes the bacterial receptor QseC, a histidine kinase encoded within the core Enterobacteriaceae genome that promotes the activation of pathogen-associated type three secretion systems. Taken together, our findings establish that endocannabinoids are directly sensed by bacteria and can modulate bacterial function.

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.

Glucuronic acid confers colonization advantage to enteric pathogens.

Glucuronidation is a detoxification process to eliminate endo- and xeno-biotics and neurotransmitters from the host circulation. Glucuronosyltransferase binds these compounds to glucuronic acid (GlcA), deactivating them and allowing their elimination through the gastrointestinal (GI) tract. However, the microbiota produces ß-glucuronidases that release GlcA and reactivate these compounds. Enteric pathogens such as enterohemorrhagic Escherichia coli (EHEC) and Citrobacter rodentium sense and utilize galacturonic acid (GalA), an isomer of GlcA, to outcompete the microbiota promoting gut colonization. However, the role of GlcA in pathogen colonization has not been explored. Here, we show that treatment of mice with a microbial ß-glucuronidase inhibitor (GUSi) decreased C. rodentium's colonization of the GI tract, without modulating bacterial virulence or host inflammation. Metagenomic studies indicated that GUSi did not change the composition of the intestinal microbiota in these animals. GlcA confers an advantage for pathogen expansion through its utilization as a carbon source. Congruently mutants unable to catabolize GlcA depict lower GI colonization compared to wild type and are not sensitive to GUSi. Germfree mice colonized with a commensal E. coli deficient for ß-glucuronidase production led to a decrease of C. rodentium tissue colonization, compared to animals monocolonized with an E. coli proficient for production of this enzyme. GlcA is not sensed as a signal and doesn't activate virulence expression but is used as a metabolite. Because pathogens can use GlcA to promote their colonization, inhibitors of microbial ß-glucuronidases could be a unique therapeutic against enteric infections without disturbing the host or microbiota physiology.

l-Arginine sensing regulates virulence gene expression and disease progression in enteric pathogens.

Microbiota, host and dietary metabolites/signals compose the rich gut chemical environment, which profoundly impacts virulence of enteric pathogens. Enterohemorrhagic Escherichia coli (EHEC) engages a syringe-like machinery named type-III secretion system (T3SS) to inject effectors within host cells that lead to intestinal colonization and disease. We previously conducted a high-throughput screen to identify metabolic pathways that affect T3SS expression. Here we show that in the presence of arginine, the arginine sensor ArgR, identified through this screen, directly activates expression of the genes encoding the T3SS. Exogenously added arginine induces EHEC virulence gene expression in vitro. Congruently, a mutant deficient in arginine transport (ΔartP) had decreased virulence gene expression. ArgR also augments murine disease caused by Citrobacter rodentium, which is a murine pathogen extensively employed as a surrogate animal model for EHEC. The source of arginine sensed by C. rodentium is not dietary. At the peak of C. rodentium infection, increased arginine concentration in the colon correlated with down-regulation of the host SLC7A2 transporter. This increase in the concentration of colonic arginine promotes virulence gene expression in C. rodentium Arginine is an important modulator of the host immune response to pathogens. Here we add that arginine also directly impacts bacterial virulence. These findings suggest that a delicate balance between host and pathogen responses to arginine occur during disease progression.

Interactions between the microbiota and pathogenic bacteria in the gut.

The microbiome has an important role in human health. Changes in the microbiota can confer resistance to or promote infection by pathogenic bacteria. Antibiotics have a profound impact on the microbiota that alters the nutritional landscape of the gut and can lead to the expansion of pathogenic populations. Pathogenic bacteria exploit microbiota-derived sources of carbon and nitrogen as nutrients and regulatory signals to promote their own growth and virulence. By eliciting inflammation, these bacteria alter the intestinal environment and use unique systems for respiration and metal acquisition to drive their expansion. Unravelling the interactions between the microbiota, the host and pathogenic bacteria will produce strategies for manipulating the microbiota against infectious diseases.

Redox, amino acid, and fatty acid metabolism intersect with bacterial virulence in the gut.

The gut metabolic landscape is complex and is influenced by the microbiota, host physiology, and enteric pathogens. Pathogens have to exquisitely monitor the biogeography of the gastrointestinal tract to find a suitable niche for colonization. To dissect the important metabolic pathways that influence virulence of enterohemorrhagic Escherichia coli (EHEC), we conducted a high-throughput screen. We generated a dataset of regulatory pathways that control EHEC virulence expression under anaerobic conditions. This unraveled that the cysteine-responsive regulator, CutR, converges with the YhaO serine import pump and the fatty acid metabolism regulator FadR to optimally control virulence expression in EHEC. CutR activates expression of YhaO to increase activity of the YhaJ transcription factor that has been previously shown to directly activate the EHEC virulence genes. CutR enhances FadL, which is a pump for fatty acids that represses inhibition of virulence expression by FadR, unmasking a feedback mechanism responsive to metabolite fluctuations. Moreover, CutR and FadR also augment murine infection by Citrobacter rodentium, which is a murine pathogen extensively employed as a surrogate animal model for EHEC. This high-throughput approach proved to be a powerful tool to map the web of cellular circuits that allows an enteric pathogen to monitor the gut environment and adjust the levels of expression of its virulence repertoire toward successful infection of the host.

Taming the Beast: Interplay between Gut Small Molecules and Enteric Pathogens.

The overuse of antibiotics has led to the evolution of drug-resistant bacteria that are becoming increasingly dangerous to human health. According to the Centers for Disease Control and Prevention, antibiotic-resistant bacteria cause at least 2 million illnesses and 23,000 deaths in the United States annually. Traditionally, antibiotics are bactericidal or bacteriostatic agents that place selective pressure on bacteria, leading to the expansion of antibiotic-resistant strains. In addition, antibiotics that are effective against some pathogens can also exacerbate their pathogenesis and may lead to severe progression of the disease. Therefore, alternative strategies are needed to treat antibiotic-resistant bacterial infections. One novel approach is to target bacterial virulence to prevent or limit pathogen colonization, while also minimizing tissue damage and disease comorbidities in the host. This review focuses on the interactions between enteric pathogens and naturally occurring small molecules in the human gut as potential therapeutic targets for antivirulence strategies. Individual small molecules in the intestines modulate enteric pathogen virulence and subsequent intestinal fitness and colonization. Targeted interruption of pathogen sensing of these small molecules could therefore attenuate their virulence. This review highlights the paths of discovery for new classes of antimicrobials that could potentially mitigate the urgent problem of antibiotic resistance.

The QseG Lipoprotein Impacts the Virulence of Enterohemorrhagic Escherichia coli and Citrobacter rodentium and Regulates Flagellar Phase Variation in Salmonella enterica Serovar Typhimurium.

The QseEF histidine kinase/response regulator system modulates expression of enterohemorrhagic Escherichia coli (EHEC) and Salmonella enterica serovar Typhimurium virulence genes in response to the host neurotransmitters epinephrine and norepinephrine. qseG, which encodes an outer membrane lipoprotein, is cotranscribed with qseEF in these enteric pathogens, but there is little knowledge of its role in virulence. Here, we found that in EHEC QseG interacts with the type III secretion system (T3SS) gate protein SepL and modulates the kinetics of attaching and effacing (AE) lesion formation on tissue-cultured cells. Moreover, an EHEC ΔqseG mutant had reduced intestinal colonization in an infant rabbit model. Additionally, in Citrobacter rodentium, an AE lesion-forming pathogen like EHEC, QseG is required for full virulence in a mouse model. In S Typhimurium, we found that QseG regulates the phase switch between the two flagellin types, FliC and FljB. In an S Typhimurium ΔqseG mutant, the phase-variable promoter for fljB is preferentially switched into the "on" position, leading to overproduction of this phase two flagellin. In infection of tissue-cultured cells, the S Typhimurium ΔqseG mutant provokes increased inflammatory cytokine production versus the wild type; in vivo, in a murine infection model, the ΔqseG strain caused a more severe inflammatory response and was attenuated versus the wild-type strain. Collectively, our findings demonstrate that QseG is important for full virulence in several enteric pathogens and controls flagellar phase variation in S Typhimurium, and they highlight both the complexity and conservation of the regulatory networks that control the virulence of enteric pathogens.

Fucose sensing regulates bacterial intestinal colonization.

The mammalian gastrointestinal tract provides a complex and competitive environment for the microbiota. Successful colonization by pathogens requires scavenging nutrients, sensing chemical signals, competing with the resident bacteria and precisely regulating the expression of virulence genes. The gastrointestinal pathogen enterohaemorrhagic Escherichia coli (EHEC) relies on inter-kingdom chemical sensing systems to regulate virulence gene expression. Here we show that these systems control the expression of a novel two-component signal transduction system, named FusKR, where FusK is the histidine sensor kinase and FusR the response regulator. FusK senses fucose and controls expression of virulence and metabolic genes. This fucose-sensing system is required for robust EHEC colonization of the mammalian intestine. Fucose is highly abundant in the intestine. Bacteroides thetaiotaomicron produces multiple fucosidases that cleave fucose from host glycans, resulting in high fucose availability in the gut lumen. During growth in mucin, B. thetaiotaomicron contributes to EHEC virulence by cleaving fucose from mucin, thereby activating the FusKR signalling cascade, modulating the virulence gene expression of EHEC. Our findings suggest that EHEC uses fucose, a host-derived signal made available by the microbiota, to modulate EHEC pathogenicity and metabolism.

Genetic and Mechanistic Analyses of the Periplasmic Domain of the Enterohemorrhagic Escherichia coli QseC Histidine Sensor Kinase.

The histidine sensor kinase (HK) QseC senses autoinducer 3 (AI-3) and the adrenergic hormones epinephrine and norepinephrine. Upon sensing these signals, QseC acts through three response regulators (RRs) to regulate the expression of virulence genes in enterohemorrhagic Escherichia coli (EHEC). The QseB, QseF, and KdpE RRs that are phosphorylated by QseC constitute a tripartite signaling cascade having different and overlapping targets, including flagella and motility, the type three secretion system encoded by the locus of enterocyte effacement (LEE), and Shiga toxin. We modeled the tertiary structure of QseC's periplasmic sensing domain and aligned the sequences from 12 different species to identify the most conserved amino acids. We selected eight amino acids conserved in all of these QseC homologues. The corresponding QseC site-directed mutants were expressed and still able to autophosphorylate; however, four mutants demonstrated an increased basal level of phosphorylation. These mutants have differential flagellar, motility, LEE, and Shiga toxin expression phenotypes. We selected four mutants for more in-depth analyses and found that they differed in their ability to phosphorylate QseB, KdpE, and QseF. This suggests that these mutations in the periplasmic sensing domain affected the region downstream of the QseC signaling cascade and therefore can influence which pathway QseC regulates.IMPORTANCE In the foodborne pathogen EHEC, QseC senses AI-3, epinephrine, and norepinephrine, increases its autophosphorylation, and then transfers its phosphate to three RRs: QseB, QseF, and KdpE. QseB controls expression of flagella and motility, KdpE controls expression of the LEE region, and QseF controls the expression of Shiga toxin. This tripartite signaling pathway must be tightly controlled, given that flagella and the type three secretion system (T3SS) are energetically expensive appendages and Shiga toxin expression leads to bacterial cell lysis. Our data suggest that mutations in the periplasmic sensing loop of QseC differentially affect the expression of the three arms of this signaling cascade. This suggests that these point mutations may change QseC's phosphotransfer preferences for its RRs.

Bacterial Chat: Intestinal Metabolites and Signals in Host-Microbiota-Pathogen Interactions.

Intestinal bacteria employ microbial metabolites from the microbiota and chemical signaling during cell-to-cell communication to regulate several cellular functions. Pathogenic bacteria are extremely efficient in orchestrating their response to these signals through complex signaling transduction systems. Precise coordination and interpretation of these multiple chemical cues is important within the gastrointestinal (GI) tract. Enteric foodborne pathogens, such as enterohemorrhagic Escherichia coli (EHEC) and Salmonella enterica serovar Typhimurium, or the surrogate murine infection model for EHEC, Citrobacter rodentium, are all examples of microorganisms that modulate the expression of their virulence repertoire in response to signals from the microbiota or the host, such as autoinducer-3 (AI-3), epinephrine (Epi), and norepinephrine (NE). The QseBC and QseEF two-component systems, shared by these pathogens, are involved in sensing these signals. We review how these signaling systems sense and relay these signals to drive bacterial gene expression; specifically, to modulate virulence. We also review how bacteria chat via chemical signals integrated with metabolite recognition and utilization to promote successful associations among enteric pathogens, the microbiota, and the host.

In vivo influence of in vitro up-regulated genes in the virulence of an APEC strain associated with swollen head syndrome.

Avian Pathogenic Escherichia coli is responsible for significant economic losses in the poultry industry by causing a range of systemic or localized diseases collectively termed colibacillosis. The virulence mechanisms of these strains that are pathogenic in poultry and possibly pathogenic in humans have not yet been fully elucidated. This work was developed to study if over-expressed genes in a microarray assay could be potentially involved in the pathogenicity of an Avian Pathogenic Escherichia coli strain isolated from a swollen head syndrome case. For this study, five over-expressed genes were selected for the construction of null mutants [flgE (flagellar hook), tyrR (transcriptional regulator), potF (putrescine transporter), yehD (putative adhesin) and bfr (bacterioferritin)]. The constructed mutants were evaluated for their capacity for the adhesion and invasion of in vitro cultured cells, their motility capacity, and their pathogenic potential in one-day-old chickens compared with the wild-type strain (WT). The Δbfr strain showed a decreased adhesion capacity on avian fibroblasts compared with WT, in the presence and absence of alpha-D-mannopyranoside, and the ΔpotF strain showed decreased adhesion only in the absence of alpha-D-mannopyranoside. The ΔtyrR mutant had a reduced ability to invade Hep-2 cells. No mutant showed changes in invading CEC-32 cells. The mutants ΔflgE and ΔtyrR showed a decreased ability to survive in HD-11 cells. The motility of the mutant strains Δbfr, ΔyehD and ΔpotF was increased, while the ΔtyrR mutant showed reduction, and the ΔflgE became non-motile. No mutant strain caused the same mortality of the WT in one-day-old chickens, showing attenuation to different degrees.

The Epinephrine/Norepinephrine/Autoinducer-3 Interkingdom Signaling System in Escherichia coli O157:H7.

Epinephrine/norepinephrine/AI-3 signaling is used as an interkingdom chemical signaling system between microbes and their hosts. This system is also exploited by pathogens to regulate virulence traits. In enterohemorrhagic E. coli (EHEC) O157:H7, it is essential for pathogenesis and flagella motility. These three signals activate expression of a pathogenicity island named locus of enterocyte effacement (LEE), Shiga toxin, and the flagella regulon. These signals are sensed by the two-component system QseBC, whereas the bacterial membrane receptor QseC autophosphorylates and phosphorylates the QseB response regulator initiating a complex phosphorelay signaling cascade that activates the expression of a second two-component system, QseEF. The QseEF two-component system is also involved in the expression of the virulence genes, and it senses epinephrine, phosphate, and sulfate. This complex signaling cascade still needs to be completely elucidated.

Virulence and stress-related periplasmic protein (VisP) in bacterial/host associations.

Gram-negative bacteria have an outer membrane containing LPS. LPS is constituted of an oligosaccharide portion and a lipid-A moiety that embeds this molecule within the outer membrane. LPS is a pathogen-associated molecular pattern, and several pathogens modify their lipid-A as a stealth strategy to avoid recognition by the innate immune system and gain resistance to host factors that disrupt the bacterial cell envelope. An essential feature of Salmonella enterica Typhimurium pathogenesis is its ability to replicate within vacuoles in professional macrophages. S. Typhimurium modifies its lipid-A by hydroxylation by the Fe2+/α-ketoglutarate-dependent dioxygenase enzyme (LpxO). Here, we show that a periplasmic protein of the bacterial oligonucleotide/oligosaccharide-binding fold family, herein named virulence and stress-related periplasmic protein (VisP), on binding to the sugar moiety of peptidoglycan interacts with LpxO. This interaction inhibits LpxO function, leading to decreased LpxO-dependent lipid-A modifications and increasing resistance to stressors within the vacuole environment during intramacrophage replication promoting systemic disease. Consequently, ΔvisP is avirulent in systemic murine infections, where VisP acts through LpxO. Several Gram-negative pathogens harbor both VisP and LpxO, suggesting that this VisP-LpxO mechanism of lipid-A modifications has broader implications in bacterial pathogenesis. Bacterial species devoid of LpxO (e.g., Escherichia coli) have no lipid-A phenotypes associated with the lack of VisP; however, VisP also controls LpxO-independent phenotypes. VisP and LpxO act independently in the S. Typhimurium murine colitis model, with both mutants being attenuated for diverging reasons; ΔvisP is less resistant to cationic antimicrobial peptides, whereas ΔlpxO is deficient for epithelial cell invasion. VisP converges bacterial cell wall homeostasis, stress responses, and pathogenicity.

Global analysis of posttranscriptional regulation by GlmY and GlmZ in enterohemorrhagic Escherichia coli O157:H7.

Enterohemorrhagic Escherichia coli (EHEC) is a significant human pathogen and is the cause of bloody diarrhea and hemolytic-uremic syndrome. The virulence repertoire of EHEC includes the genes within the locus of enterocyte effacement (LEE) that are largely organized in five operons, LEE1 to LEE5, which encode a type III secretion system, several effectors, chaperones, and regulatory proteins. In addition, EHEC also encodes several non-LEE-encoded effectors and fimbrial operons. The virulence genes of this pathogen are under a large amount of posttranscriptional regulation. The small RNAs (sRNAs) GlmY and GlmZ activate the translation of glucosamine synthase (GlmS) in E. coli K-12, and in EHEC they destabilize the 3' fragments of the LEE4 and LEE5 operons and promote translation of the non-LEE-encoded effector EspFu. We investigated the global changes of EHEC gene expression governed by GlmY and GlmZ using RNA sequencing and gene arrays. This study extends the known effects of GlmY and GlmZ regulation to show that they promote expression of the curli adhesin, repress the expression of tryptophan metabolism genes, and promote the expression of acid resistance genes and the non-LEE-encoded effector NleA. In addition, seven novel EHEC-specific sRNAs were identified using RNA sequencing, and three of them--sRNA56, sRNA103, and sRNA350--were shown to regulate urease, fimbria, and the LEE, respectively. These findings expand the knowledge of posttranscriptional regulation in EHEC.

The efficacy of immediate versus delayed antibiotic administration on bacterial growth and biofilm production of selected strains of uropathogenic Escherichia coli and Pseudomonas aeruginosa.

PURPOSE: The treatment of urinary tract infections (UTI) with antibiotics is commonly used, but recurrence and antibiotic resistance have been growing and concerning clinicians. We studied whether the rapid onset of a protective biofilm may be responsible for the lack of effectiveness of antibiotics against selected bacteria. MATERIALS AND METHODS: Two established uropathogenic Escherichia coli strains, UTI89 and CFT073, and two Pseudomonas aeruginosa strains, PA01 and Boston-41501, were studied to establish a reliable biofilm formation process. Bacterial growth (BG) was determined by optical density at 600 nm (OD 600) using a spectrophotometer, while biofilm formation (BF) using crystal violet staining was measured at OD 550. Next, these bacterial strains were treated with clinically relevant antibiotics, ciprofloxacin HCl (200 ng/mL and 2 µg/mL), nitrofurantoin (20 µg/mL and 40 µg/mL) and ampicillin (50 µg/mL) at time points of 0 (T0) or after 6 hours of culture (T6). All measurements, including controls (bacteria -1% DMSO), were done in triplicates and repeated three times for consistency. RESULTS: The tested antibiotics effectively inhibited both BG and BF when administered at T0 for UPEC strains, but not when the antibiotic administration started 6 hours later. For Pseudomonas strains, only Ciprofloxacin was able to significantly inhibit bacterial growth at T0 but only at the higher concentration of 2 µg/mL for T6. CONCLUSION: When established UPEC and Pseudomonas bacteria were allowed to culture for 6 hours before initialization of treatment, the therapeutic effect of selected antibiotics was greatly suppressed when compared to immediate treatment, probably as a result of the protective nature of the biofilm.

The American Academy of Microbiology discusses gain-of-function research of concern (GOFROC) and enhanced potential pandemic pathogens (ePPP).

The American Academy of Microbiology convened a workshop bringing together scientists with varied opinions on the conduct of gain-of-function research of concern (GOFROC) and enhanced pathogen with pandemic potential (ePPP) research. Five findings were: (1) research on infectious agents is necessary for understanding, monitoring, and developing treatments and prevention measures against these agents; (2) gain-of-function research of concern or ePPP research makes up a very small fraction of all biological research; (3) clearly defined terminologies for research of concern should be developed by the scientific community to avoid public confusion and highlight its practical benefits; (4) harmonized biorisk management standardization, training, mentoring, and reporting can help improve safety and security for laboratory workers and the public; and (5) expanded engagement and collaboration of scientists with policymakers and the public, including increased transparency on the risks and rewards of research with infectious agents, is needed.

Expression of the locus of enterocyte effacement genes during the invasion process of the atypical enteropathogenic Escherichia coli 1711-4 strain of serotype O51:H40.

Atypical enteropathogenic Escherichia coli (aEPEC) is a significant cause of diarrhea in developing countries. Some aEPEC strains, including the Brazilian representative strain of serotype O51:H40 called aEPEC 1711-4, can use flagella to attach to, invade, and persist in T84 and Caco-2 intestinal cells. They can even translocate from the gut to extraintestinal sites in a rat model. Although various aspects of the virulence of this strain were studied and the requirement of the T3SS for the efficiency of the invasion process was demonstrated, the expression of the LEE genes during the invasion and intracellular persistence remains unclear. To address this, the expression of flagella and the different LEE operons was evaluated during kinetic experiments of the interaction of aEPEC 1711-4 with enterocytes in vitro. The genome of the strain was also sequenced. The results showed that flagella expression remained unchanged, but the expression of eae and escJ increased during the early interaction and invasion of aEPEC 1711-4 into Caco-2 cells, and there was no change 24 hours post-infection during the persistence period. The number of pedestal-like structures formed on HeLa cells also increased during the 24-hour analysis. No known gene related to the invasion process was identified in the genome of aEPEC 1711-4, which was shown to belong to the global EPEC lineage 10. These findings suggest that LEE components and the intimate adherence promoted by intimin are necessary for the invasion and persistence of aEPEC 1711-4, but the detailed mechanism needs further study.

Enterococcus faecalis-derived adenine enhances enterohaemorrhagic Escherichia coli Type 3 Secretion System-dependent virulence.

Interactions between microbiota and enteric pathogens can promote colonization resistance or enhance pathogenesis. The pathobiont Enterococcus faecalis increases enterohaemorrhagic E. coli (EHEC) virulence by upregulating Type 3 Secretion System (T3SS) expression, effector translocation, and attaching and effacing (AE) lesion formation on enterocytes, but the mechanisms underlying this remain unknown. Using co-infection of organoids, metabolomics, supplementation experiments and bacterial genetics, here we show that co-culture of EHEC with E. faecalis increases the xanthine-hypoxanthine pathway activity and adenine biosynthesis. Adenine or E. faecalis promoted T3SS gene expression, while transcriptomics showed upregulation of adeP expression, which encodes an adenine importer. Mechanistically, adenine relieved High hemolysin activity (Hha)-dependent repression of T3SS gene expression in EHEC and promoted AE lesion formation in an AdeP-dependent manner. Microbiota-derived purines, such as adenine, support multiple beneficial host responses; however, our data show that this metabolite also increases EHEC virulence, highlighting the complexity of pathogen-microbiota-host interactions in the gut.