The mammalian gastrointestinal (GI) tract provides a complex and competitive environment for the microbiota1. Successful colonization by pathogens depends on scavenging nutrients, sensing chemical signals, competing with the resident bacteria, and precisely regulating expression of virulence genes2. The GI pathogen enterohemorrhagic E.coli (EHEC) relies on inter-kingdom chemical sensing systems to regulate virulence gene expression3–4. 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 (HK), and FusR the response regulator (RR). 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 intestine5. Bacteroides thetaiotaomicron (B.theta) produces multiple fucosidases that cleave fucose from host glycans, resulting in high fucose availability in the gut lumen6. During growth in mucin, B.theta contributes to EHEC virulence by cleaving fucose from mucin, thereby activating the FusKR signaling cascade, modulating EHEC’s virulence gene expression. Our findings suggest that EHEC uses fucose, a host-derived signal made available by the microbiota, to modulate EHEC pathogenicity and metabolism.

Single-molecule real-time (SMRT) DNA sequencing allows the systematic detection of chemical modifications such as methylation but has not previously been applied on a genome-wide scale. We used this approach to detect 49,311 putative 6-methyladenine (m6A) residues and 1,407 putative 5-methylcytosine (m5C) residues in the genome of a pathogenic Escherichia coli strain. We obtained strand-specific information for methylation sites and a quantitative assessment of the frequency of methylation at each modified position. We deduced the sequence motifs recognized by the methyltransferase enzymes present in this strain without prior knowledge of their specificity. Furthermore, we found that deletion of a phage-encoded methyltransferase-endonuclease (restriction-modification; RM) system induced global transcriptional changes and led to gene amplification, suggesting that the role of RM systems extends beyond protecting host genomes from foreign DNA.

SummaryThe human pathogens enteropathogenic and enterohemorrhagic Escherichia coli (EPEC and EHEC) share a unique mechanism of colonization that results from the concerted action of effector proteins translocated into the host cell by a type III secretion system (T3SS). EPEC and EHEC not only induce characteristic attaching and effacing (A/E) lesions, but also subvert multiple host cell signalling pathways during infection. Our understanding of the mechanisms by which A/E pathogens hijack host cell signalling has advanced dramatically in recent months with the identification of novel activities for many effectors. In addition to further characterization of established effectors (Tir, EspH and Map), new effectors have emerged as important mediators of virulence through activities such as mimicry of Rho guanine nucleotide exchange factors (Map and EspM), inhibition of apoptosis (NleH and NleD), interference with inflammatory signalling pathways (NleB, NleC, NleE and NleH) and phagocytosis (EspF, EspH and EspJ). The findings have highlighted the multifunctional nature of the effectors and their ability to participate in redundant, synergistic or antagonistic relationships, acting in a co-ordinated spatial and temporal manner on different host organelles and cellular pathways during infection.

Activity-based protein profiling (ABPP) is a chemoproteomic tool for detecting active enzymes in complex biological systems. We used ABPP to identify secreted bacterial and host serine hydrolases that are active in animals infected with the cholera pathogen Vibrio cholerae. Four V. cholerae proteases were consistently active in infected rabbits, and one, VC0157 (renamed IvaP), was also active in human cholera stool. Inactivation of IvaP influenced the activity of other secreted V. cholerae and rabbit enzymes in vivo, while genetic disruption of all four proteases increased the abundance and binding of an intestinal lectin—intelectin—to V. cholerae in infected rabbits. Intelectin also bound to other enteric bacterial pathogens, suggesting it may constitute a previously unrecognized mechanism of bacterial surveillance in the intestine that is inhibited by pathogen-secreted proteases. Our work demonstrates the power of activity-based proteomics to reveal host-pathogen enzymatic dialogue in an animal model of infection.

Beyond its role in host defense, bacterial DNA methylation also plays important roles in the regulation of gene expression, virulence and antibiotic resistance. Bacterial cells in a clonal population can generate epigenetic heterogeneity to increase population-level phenotypic plasticity. Single molecule, real-time (SMRT) sequencing enables the detection of N6-methyladenine and N4-methylcytosine, two major types of DNA modifications comprising the bacterial methylome. However, existing SMRT sequencing-based methods for studying bacterial methylomes rely on a population-level consensus that lacks the single-cell resolution required to observe epigenetic heterogeneity. Here, we present SMALR (single-molecule modification analysis of long reads), a novel framework for single molecule-level detection and phasing of DNA methylation. Using seven bacterial strains, we show that SMALR yields significantly improved resolution and reveals distinct types of epigenetic heterogeneity. SMALR is a powerful new tool that enables de novo detection of epigenetic heterogeneity and empowers investigation of its functions in bacterial populations.

SummaryIn this work we discover that a specific recognition of the N-terminal lectin domain of FimH adhesin by the usher FimD is essential for the biogenesis of type 1 pili in Escherichia coli. These filamentous organelles are assembled by the chaperone-usher pathway, in which binary complexes between fimbrial subunits and the periplasmic chaperone FimC are recognized by the outer membrane protein FimD (the usher). FimH adhesin initiates fimbriae polymerization and is the first subunit incorporated in the filament. Accordingly, FimD shows higher affinity for the FimC/FimH complex although the structural basis of this specificity is unknown. We have analysed the assembly into fimbria, and the interaction with FimD in vivo, of FimH variants in which the N-terminal lectin domain of FimH was deleted or substituted by different immunoglobulin (Ig) domains, or in which these Ig domains were fused to the N-terminus of full-length FimH. From these data, along with the analysis of a FimH mutant with a single amino acid change (G16D) in the N-terminal lectin domain, we conclude that the lectin domain of FimH is recognized by FimD usher as an essential step for type 1 pilus biogenesis.

We report that the N terminus of the type III secretion system translocator proteins EspB, EspD, and EspA mediate protein secretion and translocation from wild-type enteropathogenic Escherichia coli and hypersecretion from sepL and sepD mutants. EspA containing the translocation signal of Map and Tir containing the secretion signal of EspA are biologically active.Deployment of a type III secretion system (T3SS) is an infection strategy used by many human pathogens (reviewed in references 15 and 24). The T3SS is composed of a cytosolic ATPase, inner and outer membrane rings, and an extracellular needle. In Yersinia, LcrV forms a distal needle tip structure (25), while in enteropathogenic Escherichia coli (EPEC) and enterohemorrhagic E. coli (EHEC), the needle is extended by long EspA filaments (9,19). The substrates of the T3SS are translocator and effector proteins. Translocator proteins of the Yersinia YopB and YopD family form a translocation pore in the host cell plasma membrane that enables effector protein translocation (reviewed in reference 24). Once translocated, the effectors affect host cell functions, interfering with signaling pathways and a broad range of physiological processes (reviewed in references 14 and 16).

Here we show that the type III secretion gatekeeper protein SepL resembles an aberrant effector protein in binding to a class 1 type III secretion chaperone (Orf12, here renamed CesL). We also show that short N-terminal fragments (<70 amino acids) from SepL are capable of targeting fusion proteins for secretion and translocation.