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.

Lactobacillus acidophilus LA-5 Ameliorates Inflammation and Alveolar Bone Loss Promoted by A. actinomycetemcomitans and S. gordonii in Mice and Impacts Oral and Gut Microbiomes.

The benefits of probiotics on dysbiotic microbiomes and inflammation are dependent on the tested strain, host factors, and the resident microbiome. There is limited knowledge on the effects of probiotics in A. actinomycetemcomitans-associated periodontitis. Thus, Lactobacillus acidophilus LA5 (LA5) was orally inoculated for 30 days in C57Bl/6 mice infected with A. actinomycetemcomitans JP2 (Aa) and S. gordonii (Sg). Alveolar bone loss, gingival gene expression, and oral and gut microbiomes were determined. LA5 controlled bone loss in Aa+Sg-infected mice, downregulated the expression of Il-1ß and upregulated Il-10 in gingival tissues, and altered the oral and gut microbiomes. LA5 increased the diversity of the oral microbiome of Aa+Sg infected mice, and Aa+Sg and Aa+Sg+LA5 oral or gut microbiomes clustered apart. LA5 induced shifts in Aa+Sg infected mice by increasing the abundance of Muribaculaceae and decreasing Bifidobacteriaceae in the oral cavity and increasing the abundance of Verrucomicrobiae and Eggerthellales in the gut. In conclusion, LA5 oral administration controls experimental Aa-associated periodontitis by altering inflammatory gene expression and the oral and gut microbiomes.

Interactions between Enterohemorrhagic Escherichia coli (EHEC) and Gut Commensals at the Interface of Human Colonoids.

The interactions between the gut microbiota and pathogens are complex and can determine the outcome of an infection. Enterohemorrhagic Escherichia coli (EHEC) is a major human enteric pathogen that colonizes the colon through attaching and effacing (AE) lesions and uses microbiota-derived molecules as cues to control its virulence. Different gut commensals can modulate EHEC virulence. However, the lack of an animal model that recapitulates the human pathophysiology of EHEC infection makes it challenging to investigate how variations in microbiota composition could affect host susceptibility to this pathogen. Here, we addressed these interactions building from simple to more complex in vitro systems, culminating with the use of the physiological relevant human colonoids as a model to study the interactions between EHEC and different gut commensals. We demonstrated that Bacteroides thetaiotaomicron and Enterococcus faecalis enhance virulence expression and AE lesion formation in cultured epithelial cells, as well as on the colonic epithelium, while commensal E. coli did not affect these phenotypes. Importantly, in the presence of these three commensals together, virulence and AE lesion are enhanced. Moreover, we identified specific changes in the metabolic landscape promoted by different members of the gut microbiota and showed that soluble factors released by E. faecalis can increase EHEC virulence gene expression. Our study highlights the importance of interspecies bacterial interactions and chemical exchange in the modulation of EHEC virulence. IMPORTANCE Enterohemorrhagic E. coli (EHEC) is a natural human pathogen that poorly colonizes mice. Hence, the use of murine models to understand features of EHEC infection is a challenge. In this study, we use human colonoids as a physiologically relevant model to study interactions between EHEC and gut commensals. We demonstrate that the ability of EHEC to form AE lesions on the intestinal epithelium is enhanced by the presence of certain gut commensals, such as B. thetaiotaomicron and E. faecalis, while it is not affected by commensal E. coli. Furthermore, we show that commensal bacteria differently impact the metabolic landscape. These data suggest that microbiota compositions can differentially modulate EHEC-mediated disease.

Citrobacter rodentium infection at the gut-brain axis interface.

The gut-brain axis plays a critical role in the maintenance of the gastrointestinal tract homeostasis. Several enteric pathogens have developed strategies to sense neurochemical molecules to regulate their virulence in the gut. Additionally, there is growing evidence that gut dysbiosis can strongly affect host brain responses. Here we review different mechanisms that have been proposed to mediate gut-brain axis communication using Citrobacter rodentium, a natural murine enteric pathogen and one of the most widely used small animal models for studying host-microbe interactions. We highlight studies that have identified-specific pathways used by C. rodentium to sense host neurochemicals during colonization as well as behavioral responses and brain pathologies affected by pathogen colonization of the gut.

EspFu-Mediated Actin Assembly Enhances Enteropathogenic Escherichia coli Adherence and Activates Host Cell Inflammatory Signaling Pathways.

The translocation of effectors into the host cell through type 3 secretion systems (T3SS) is a sophisticated strategy employed by pathogenic bacteria to subvert host responses and facilitate colonization. Enteropathogenic Escherichia coli (EPEC) and enterohemorrhagic E. coli (EHEC) utilize the Tir and EspFu (also known as TccP) effectors to remodel the host cytoskeleton, culminating in the formation of attaching and effacing (AE) lesions on enterocytes. While some EPEC strains require tyrosine phosphorylation of Tir and recruitment of the host Nck to trigger actin polymerization, EHEC and certain EPEC strains, whose Tir is not phosphorylated, rely on the effector EspFu for efficient actin remodeling. Here, we investigated the role played by Tir-Nck and Tir-EspFu actin polymerization pathways during the infection of epithelial cells, as well as the host transcriptional response to the AE lesion formation induced by EPEC. We found that EspFu-mediated actin assembly promotes bacterial attachment and epithelial colonization more efficiently than Tir-Nck. Moreover, we showed that both actin polymerization mechanisms can activate inflammatory pathways and reverse the anti-inflammatory response induced by EPEC in epithelial cells. However, this activity is remarkably more evident in infections with EspFu-expressing EPEC strains. This study demonstrates the complex interactions between effector-mediated actin remodeling and inflammation. Different strains carry different combinations of these two effectors, highlighting the plasticity of pathogenic E. coli enteric infections.IMPORTANCE EPEC is among the leading causes of diarrheal disease worldwide. The colonization of the gut mucosa by EPEC results in actin pedestal formation at the site of bacterial attachment. These pedestals are referred to as attaching and effacing (AE) lesions. Here, we exploit the different molecular mechanisms used by EPEC to induce AE lesions on epithelial cells, showing that the effector EspFu is associated with increased bacterial attachment and enhanced epithelial colonization compared to the Tir-Nck pathway. Moreover, we also showed that actin pedestal formation can counterbalance the anti-inflammatory activity induced by EPEC, especially when driven by EspFu. Collectively, our findings provide new insights into virulence mechanisms employed by EPEC to colonize epithelial cells, as well as the host response to this enteric pathogen.

Genomic Properties and Temporal Analysis of the Interaction of an Invasive Escherichia albertii With Epithelial Cells.

Diarrhea is one of the main causes of infant mortality worldwide, mainly in the developing world. Among the various etiologic agents, Escherichia albertii is emerging as an important human enteropathogen. E. albertii promote attaching and effacing (AE) lesions due to the presence of the locus of enterocyte effacement (LEE) that encodes a type three secretion system (T3SS), the afimbrial adhesin intimin and its translocated receptor, Tir, and several effector proteins. We previously showed that E. albertii strain 1551-2 invades several epithelial cell lineages by a process that is dependent on the intimin-Tir interaction. To understand the contribution of T3SS-dependent effectors present in E. albertii 1551-2 during the invasion process, we performed a genetic analysis of the LEE and non-LEE genes and evaluated the expression of the LEE operons in various stages of bacterial interaction with differentiated intestinal Caco-2 cells. The kinetics of the ability of the 1551-2 strain to colonize and form AE lesions was also investigated in epithelial HeLa cells. We showed that the LEE expression was constant during the early stages of infection but increased at least 4-fold during bacterial persistence in the intracellular compartment. An in silico analysis indicated the presence of a new tccP/espFU subtype, named tccP3. We found that the encoded protein colocalizes with Tir and polymerized F-actin during the infection process in vitro. Moreover, assays performed with Nck null cells demonstrated that the 1551-2 strain can trigger F-actin polymerization in an Nck-independent pathway, despite the fact that TccP3 is not required for this phenotype. Our study highlights the importance of the T3SS during the invasion process and for the maintenance of E. albertii 1551-2 inside the cells. In addition, this work may help to elucidate the versatility of the T3SS for AE pathogens, which are usually considered extracellular and rarely reach the intracellular environment.

Distribution of Major Pilin Subunit Genes Among Atypical Enteropathogenic Escherichia coli and Influence of Growth Media on Expression of the ecp Operon.

Atypical enteropathogenic Escherichia coli (aEPEC) strains are unable to produce the bundle-forming pilus (BFP), which is responsible for the localized adherence pattern, a characteristic of the pathogenicity of typical EPEC strains. The lack of BFP in aEPEC strains suggests that other fimbrial or non-fimbrial adhesins are involved in their adhesion to the host cells. The aim of this study was to investigate the distribution of major subunit fimbrial genes known to be important adherence factors produced by several E. coli pathotypes in a collection of 72 aEPEC strains. Our results demonstrate that a high percentage (94-100%) of aEPEC strains harbored ecpA, fimA, hcpA, and lpfA fimbrial genes. Other fimbrial genes including pilS, pilV, sfpA, daaC, papA, and sfa were detected at lower frequencies (1-8%). Genes encoding fimbrial subunits, which are characteristic of enteroaggregative E. coli or enterotoxigenic E. coli were not found. No correlation was found between fimbrial gene profiles and adherence phenotypes. Since all aEPEC strains contained ecpA, the major pilin gene of the E. coli common pilus (ECP), a subset of ecpA+ strains was analyzed for transcription of ecpRABCDE and production of ECP upon growth in three different culture conditions at 37°C. Transcription of ecpRABCDE occurred in all conditions; however, ECP production was medium dependent. In all, the data suggest that aEPEC strains are highly heterogeneous in terms of their fimbrial gene profiles. Despite lacking BFP production, other mechanisms of cell adherence exist in aEPEC strains to ensure host colonization, e.g., mediated by other prevalent pili such as ECP. Moreover, the production of ECP by aEPEC strains might be influenced by yet unknown post-transcriptional factors.

Phylogenetic distribution of tir-cytoskeleton coupling protein (tccP and tccP2) genes in atypical enteropathogenic Escherichia coli.

Enteropathogenic Escherichia coli (EPEC) strains employ the type III secretion system (T3SS) effector Tir to induce actin cytoskeletal rearrangements. While some EPEC require tyrosine phosphorylation (Y-P) of Tir to trigger actin assembling, certain strains whose Tir is not tyrosine phosphorylated utilize the T3SS effector Tir-cytoskeleton coupling protein (TccP/TccP2) for efficient actin polymerization. The presence of tccP/tccP2 in typical EPEC belonging to distinct evolutionary lineages is well established but, in contrast, little is known about the distribution of these genes in atypical EPEC (aEPEC) showing distinct phylogenetic background. In this study, we screened 72 pathogenic aEPEC for the presence of tccP/tccP2 genes, and further characterized positive strains regarding tir type, phylogroups and production of TccP/TccP2. The tccP and/or tccP2 genes were detected in 45.8% of the strains, with a predominance of tccP2 allele. Most of these strains carried tirY-P, suggesting that can trigger actin polymerization using both Tir tyrosine phosphorylation and TccP/TccP2 pathways. aEPEC strains carrying tccP or tccP2 were significantly associated to phylogroups E and B1, respectively. We also observed a strain-to-strain variation regarding TccP/TccP2 production. Our results demonstrate a wide distribution of tccP/tccP2 genes among pathogenic aEPEC strains, as well associations between specific alleles and phylogenetic backgrounds.

Lambs are an important source of atypical enteropathogenic Escherichia coli in southern Brazil.

Food-producing animals can harbor Escherichia coli strains with potential to cause diseases in humans. In this study, the presence of enteropathogenic E. coli (EPEC) was investigated in fecal samples from 130 healthy sheep (92 lambs and 38 adults) raised for meat in southern Brazil. EPEC was detected in 19.2% of the sheep examined, but only lambs were found to be positive. A total of 25 isolates was characterized and designated atypical EPEC (aEPEC) as tested negative for bfpA gene and BFP production. The presence of virulence markers linked to human disease as ehxA, paa, and lpfAO113 was observed in 60%, 24%, and 88% of the isolates, respectively. Of the 11 serotypes identified, eight were described among human pathogenic strains, while three (O1:H8, O11:H21 and O125:H19) were not previously detected in aEPEC. Associations between intimin subtypes and phylogroups were observed, including eae-θ2/A, eae-ß1/B1, eae-α2/B2 and eae-γ1/D. Although PFGE typing of 16 aEPEC isolates resulted in 14 unique pulsetypes suggesting a genetic diversity, specific clones were found to be distributed in some flocks. In conclusion, potentially pathogenic aEPEC strains are present in sheep raised for meat, particularly in lambs, which can better contribute to dissemination of these bacteria than adult animals.

Flagellin and GroEL mediates in vitro binding of an atypical enteropathogenic Escherichia coli to cellular fibronectin.

BACKGROUND: Enteropathogenic Escherichia coli (EPEC) is distinguished mainly by the presence of EPEC adherence factor plasmid (pEAF) in typical EPEC (tEPEC) and its absence in atypical EPEC (aEPEC). The initial adherence to the intestinal mucosa is complex and mediated by adhesins other than bundle-forming pilus, which is not produced by aEPEC. Extracellular matrix (ECM) proteins of eukaryotic cells are commonly recognized by bacterial adhesins. Therefore, binding to ECM proteins may facilitate colonization, invasion and/or signaling by intestinal pathogens. Previous studies from our group demonstrated that aEPEC O26:H11 (strain BA2103) showed high binding activity to fibronectin, not shared by its counterpart, aEPEC O26:HNM. RESULTS: In the present study, using mass spectrometry after fibronectin-associated immunoprecipitation, two proteins, flagellin (50 kDa) and GroEL (52 kDa), were identified and BA2103 binding ability to fibronectin was inhibited in the presence of anti-H11 and anti-GroEL sera, but not by either naïve rabbit or other unrelated sera. It was also observed that the presence of purified flagellin inhibits adhesion of BA2103 to cellular fibronectin in a dose-dependent manner. Additionally, BA2103 GroEL is similar to the same protein of uropathogenic E. coli. CONCLUSIONS: Our results suggest that flagellin may play a role in the in vitro interaction of BA2103 with cellular fibronectin, and GroEL can be an accessory protein in this process.

Presence of Shiga toxin 2e-producing Escherichia coli and atypical enteropathogenic E. coli in an asymptomatic child.

INTRODUCTION: Escherichia coli causes gastroenteritis in humans and animals. CASE PRESENTATION: In this study, both Shiga toxin-producing E. coli (STEC) and atypical enteropathogenic E. coli (EPEC) strains were identified in a stool sample from a healthy child, and they were serotyped as Shiga toxin-producing E. coli (STEC) ONT : H19 and atypical enteropathogenic E. coli (EPEC) O37 : H45. CONCLUSION: This is the first report, to our knowledge, of a concomitant presence of diarrhoeagenic E. coli (DEC) strains in an asymptomatic child. None of the microorganisms was able to produce diarrhoea, maybe because they were transient bacteria or because of the good immune status of the child. Attention should be paid to this result and it could be of interest in vaccine prospects.