H-NST Induces LEE Expression and the Formation of Attaching and Effacing Lesions in Enterohemorrhagic Escherichia coli

Background

Enteropathogenic E. coli (EPEC) and enterohemorrhagic E. coli are important causes of morbidity and mortality worldwide. These enteric pathogens contain a type III secretion system (T3SS) responsible for the attaching and effacing (A/E) lesion phenotype. The T3SS is encoded by the locus of enterocyte effacement (LEE) pathogenicity island. The H-NS-mediated repression of LEE expression is counteracted by Ler, the major activator of virulence gene expression in A/E pathogens. A regulator present in EPEC, H-NST, positively affects expression of H-NS regulon members in E. coli K-12, although the effect of H-NST on LEE expression and virulence of A/E pathogens has yet-to-be determined.

Results

We examine the effect of H-NST on LEE expression and A/E lesion formation on intestinal epithelial cells. We find that H-NST positively affects the levels of LEE-encoded proteins independently of ler and induces A/E lesion formation. We demonstrate H-NST binding to regulatory regions of LEE1 and LEE3, the first report of DNA-binding by H-NST. We characterize H-NST mutants substituted at conserved residues including Ala16 and residues Arg60 and Arg63, which are part of a potential DNA-binding domain. The single mutants A16V, A16L, R60Q and the double mutant R60Q/R63Q exhibit a decreased effect on LEE expression and A/E lesion formation. DNA mobility shift assays reveal that these residues are important for H-NST to bind regulatory LEE DNA targets. H-NST positively affects Ler binding to LEE DNA in the presence of H-NS, and thereby potentially helps Ler displace H-NS bound to DNA.

Conclusions

H-NST induces LEE expression and A/E lesion formation likely by counteracting H-NS-mediated repression. We demonstrate that H-NST binds to DNA and identify arginine residues that are functionally important for DNA-binding. Our study suggests that H-NST provides an additional means for A/E pathogens to alleviate repression of virulence gene expression by H-NS to promote virulence capabilities.     

Evidence for Direct Protein-Protein Interaction between Members of the Enterobacterial Hha/YmoA and H-NS Families of Proteins

Escherichia coli nucleoid-associated H-NS protein interacts with the Hha protein, a member of a new family of global modulators that also includes the YmoA protein from Yersinia enterocolitica. This interaction has been found to be involved in the regulation of the expression of the toxin alpha-hemolysin. In this study, we further characterize the interaction between H-NS and Hha. We show that the presence of DNA in preparations of copurified His-Hha and H-NS is not directly implicated in the interaction between the proteins. The precise molecular mass of the H-NS protein retained by Hha, obtained by mass spectrometry analysis, does not show any posttranslational modification other than removal of the N-terminal Met residue. We constructed an H-NS-His recombinant protein and found that, as expected, it interacts with Hha. We used a Ni(2+)-nitrilotriacetic acid agarose method for affinity chromatography copurification of proteins to identify the H-NS protein of Y. enterocolitica. We constructed a six-His-YmoA recombinant protein derived from YmoA, the homologue of Hha in Y. enterocolitica, and found that it interacts with Y. enterocolitica H-NS. We also cloned and sequenced the hns gene of this microorganism. In the course of these experiments we found that His-YmoA can also retain H-NS from E. coli. We also found that the hns gene of Y. enterocolitica can complement an hns mutation of E. coli. Finally, we describe for the first time systematic characterization of missense mutant alleles of hha and truncated Hha' proteins, and we report a striking and previously unnoticed similarity of the Hha family of proteins to the oligomerization domain of the H-NS proteins.     

Predicting the effect of ions on the conformation of the H-NS dimerization domain

The histone-like nucleoid structuring protein (H-NS) is a DNA-organizing protein in bacteria. It contains a DNA-binding domain and a dimerization domain, connected by a flexible linker region. Dimerization occurs through the formation of a helical bundle, including a coiled-coil interaction motif. Two conformations have been resolved, for different sequences of Escherichia coli H-NS, resulting in an antiparallel coiled-coil for the shorter wild-type sequence, and a parallel coiled-coil for the longer C21S mutant. Because H-NS functions as a thermo- and osmosensor, these conformations may both be functionally relevant. Molecular simulation can complement experiments by modeling the dynamical time evolution of biomolecular systems in atomistic detail. We performed a molecular-dynamics study of the H-NS dimerization domain, showing that the parallel complex is sensitive to changes in salt conditions: it is unstable in absence of NaCl, but stable at physiological salt concentrations. In contrast, the stability of the antiparallel complex is not salt-dependent. The stability of the parallel complex also appears to be affected by mutation of the critical but nonconserved cysteine residue at position 21, whereas the antiparallel complex is not. Together, our simulations suggest that osmoregulation could be mediated by changes in the ratio of parallel- and antiparallel-oriented H-NS dimers.     

The structural and functional organization of H-NS-like proteins is evolutionarily conserved in Gram-negative bacteria

The structural gene of the H-NS protein, a global regulator of bacterial metabolism, has been identified in the group of enterobacteria as well as in closely related bacteria, such as Erwinia chrysanthemi and Haemophilus influenzae . Isolated outside these groups, the BpH3 protein of Bordetella pertussis exhibits a low amino acid conservation with H-NS, particularly in the N-terminal domain. To obtain information on the structure, function and/or evolution of H-NS, we searched for other H-NS-related proteins in the latest databases. We found that HvrA, a trans -activator protein in Rhodobacter capsulatus , has a low but significant similarity with H-NS and H-NS-like proteins. This Gram-negative bacterium is phylogenetically distant from Escherichia coli . Using theoretical analysis (e.g. secondary structure prediction and DNA binding domain modelling) of the amino acid sequence of H-NS, StpA (an H-NS-like protein in E. coli ), BpH3 and HvrA and by in vivo and in vitro experiments (e.g. complementation of various H-NS-related phenotypes and competitive gel shift assay), we present evidence that these proteins belong to the same class of DNA binding proteins. In silico analysis suggests that this family also includes SPB in R. sphaeroides , XrvA in Xanthomonas oryzae and VicH in Vibrio cholerae . These results demonstrate that proteins structurally and functionally related to H-NS are widespread in Gram-negative bacteria.     

Twelve species of the nucleoid-associated protein from Escherichia coli. Sequence recognition specificity and DNA binding affinity.

The genome of Escherichia coli is composed of a single molecule of circular DNA with the length of about 47,000 kilobase pairs, which is associated with about 10 major DNA-binding proteins, altogether forming the nucleoid. We expressed and purified 12 species of the DNA-binding protein, i.e. CbpA (curved DNA-binding protein A), CbpB or Rob (curved DNA-binding protein B or right arm of the replication origin binding protein), DnaA (DNA-binding protein A), Dps (DNA-binding protein from starved cells), Fis (factor for inversion stimulation), Hfq (host factor for phage Q(beta)), H-NS (histone-like nucleoid structuring protein), HU (heat-unstable nucleoid protein), IciA (inhibitor of chromosome initiation A), IHF (integration host factor), Lrp (leucine-responsive regulatory protein), and StpA (suppressor of td(-) phenotype A). The sequence specificity of DNA binding was determined for all the purified nucleoid proteins using gel-mobility shift assays. Five proteins (CbpB, DnaA, Fis, IHF, and Lrp) were found to bind to specific DNA sequences, while the remaining seven proteins (CbpA, Dps, Hfq, H-NS, HU, IciA, and StpA) showed apparently sequence-nonspecific DNA binding activities. Four proteins, CbpA, Hfq, H-NS, and IciA, showed the binding preference for the curved DNA. From the apparent dissociation constant (K(d)) determined using the sequence-specific or nonspecific DNA probes, the order of DNA binding affinity were determined to be: HU > IHF > Lrp > CbpB(Rob) > Fis > H-NS > StpA > CbpA > IciA > Hfq/Dps, ranging from 25 nM (HU binding to the non-curved DNA) to 250 nM (Hfq binding to the non-curved DNA), under the assay conditions employed.     

Molecular characterization of the EhaG and UpaG trimeric autotransporter proteins from pathogenic Escherichia coli

Trimeric autotransporter proteins (TAAs) are important virulence factors of many Gram-negative bacterial pathogens. A common feature of most TAAs is the ability to mediate adherence to eukaryotic cells or extracellular matrix (ECM) proteins via a cell surface-exposed passenger domain. Here we describe the characterization of EhaG, a TAA identified from enterohemorrhagic Escherichia coli (EHEC) O157:H7. EhaG is a positional orthologue of the recently characterized UpaG TAA from uropathogenic E. coli (UPEC). Similarly to UpaG, EhaG localized at the bacterial cell surface and promoted cell aggregation, biofilm formation, and adherence to a range of ECM proteins. However, the two orthologues display differential cellular binding: EhaG mediates specific adhesion to colorectal epithelial cells while UpaG promotes specific binding to bladder epithelial cells. The EhaG and UpaG TAAs contain extensive sequence divergence in their respective passenger domains that could account for these differences. Indeed, sequence analyses of UpaG and EhaG homologues from several E. coli genomes revealed grouping of the proteins in clades almost exclusively represented by distinct E. coli pathotypes. The expression of EhaG (in EHEC) and UpaG (in UPEC) was also investigated and shown to be significantly enhanced in an hns isogenic mutant, suggesting that H-NS acts as a negative regulator of both TAAs. Thus, while the EhaG and UpaG TAAs contain some conserved binding and regulatory features, they also possess important differences that correlate with the distinct pathogenic lifestyles of EHEC and UPEC.     

Functional replacement of the oligomerization domain of H-NS by the Hha protein of Escherichia coli

Members of the H-NS family of proteins play a relevant role as modulators of gene expression in gram-negative bacteria. Interaction of these proteins with members of the Hha/YmoA family of proteins has been previously reported. It has been hypothesized that the latter proteins are functionally equivalent to the N-terminal domain of H-NS-like proteins. In this report we test this assumption by replacing the N-terminal domain of Escherichia coli H-NS by Hha. It has been possible to obtain a functional protein that can compensate for some of the hns-induced phenotypes. These results highlight the relevance of H-NS-Hha interactions to generate heterooligomeric complexes that modulate gene expression in gram-negative bacteria.     

EscA is a crucial component of the type III secretion system of enteropathogenic Escherichia coli

The virulence of many Gram-negative pathogens is associated with type III secretion systems (T3SSs), which deliver virulence effector proteins into the cytoplasm of host cells. Components of enteropathogenic Escherichia coli (EPEC) T3SS are encoded within the locus of enterocyte effacement (LEE). While most LEE-encoded T3SS proteins in EPEC have assigned names and functions, a few of them remain poorly characterized. Here, we studied a small LEE-encoded protein, Orf15, that shows no homology to other T3SS/flagellar proteins and is only present in attaching and effacing pathogens, including enterohemorrhagic E. coli and Citrobacter rodentium. Our findings demonstrated that it is essential for type III secretion (T3S) and that it is localized to the periplasm and associated with the inner membrane. Membrane association was driven by the N-terminal 19 amino acid residues, which were also shown to be essential for T3S. Consistent with its localization, Orf15 was found to interact with the EPEC T3SS outer membrane ring component, EscC, which was previously shown to be embedded within the outer membrane and protruding into the periplasmic space. Interestingly, we found that the predicted coiled-coil structure of Orf15 is critical for the protein's function. Overall, our findings suggest that Orf15 is a structural protein that contributes to the structural integrity of the T3S complex, and therefore we propose to rename it EscA.     

Structural changes of Escherichia coli ferric uptake regulator during metal-dependent dimerization and activation explored by NMR and X-ray crystallography

Ferric uptake regulator (Fur) is a global bacterial regulator that uses iron as a cofactor to bind to specific DNA sequences. Escherichia coli Fur is usually isolated as a homodimer with two metal sites per subunit. Metal binding to the iron site induces protein activation; however the exact role of the structural zinc site is still unknown. Structural studies of three different forms of the Escherichia coli Fur protein (nonactivated dimer, monomer, and truncated Fur-(1-82)) were performed. Dimerization of the oxidized monomer was followed by NMR in the presence of a reductant (dithiothreitol) and Zn(II). Reduction of the disulfide bridges causes only local structure variations, whereas zinc addition to reduced Fur induces protein dimerization. This demonstrates for the first time the essential role of zinc in the stabilization of the quaternary structure. The secondary structures of the mono- and dimeric forms are almost conserved in the N-terminal DNA-binding domain, except for the first helix, which is not present in the nonactivated dimer. In contrast, the C-terminal dimerization domain is well structured in the dimer but appears flexible in the monomer. This is also confirmed by heteronuclear Overhauser effect data. The crystal structure at 1.8A resolution of a truncated protein (Fur-(1-82)) is described and found to be identical to the N-terminal domain in the monomeric and in the metal-activated state. Altogether, these data allow us to propose an activation mechanism for E. coli Fur involving the folding/unfolding of the N-terminal helix.     

Crystal structure of the N-terminal dimerisation domain of VicH, the H-NS-like protein of Vibrio cholerae

The histone-like nucleoid structuring (H-NS) protein is a global modulator of gene expression in Gram-negative bacteria. VicH, the H-NS protein of Vibrio cholerae, regulates the expression of certain major virulence determinants implicated in the pathogenesis of cholera. We present here the 2.5A crystal structure of the N-terminal oligomerisation domain of VicH (VicH_Nt). VicH_Nt adopts the same fold and dimeric assembly as the NMR structure of Escherichia coli H-NS_Nt, thus validating this fold against conflicting data. The structural similarity of V.cholerae VicH_Nt and E.coli H-NS_Nt, despite differences in origin, system of expression, experimental conditions and techniques used, indicates that the fold determined in our studies is robust to experimental conditions. Structural analysis and homology modelling were carried out to further elucidate the molecular basis of the functional polyvalence of the N-terminal domain. Our analysis of members of the H-NS superfamily supports the suggestion that the oligomerisation function of H-NS_Nt is conserved even in more distantly related proteins.     

Isolation and characterization of vicH, encoding a new pleiotropic regulator in Vibrio cholerae

During the last decade, the hns gene and its product, the H-NS protein, have been extensively studied in Escherichia coli. H-NS-like proteins seem to be widespread in gram-negative bacteria. However, unlike in E. coli and in Salmonella enterica serovar Typhimurium, little is known about their role in the physiology of those organisms. In this report, we describe the isolation of vicH, an hns-like gene in Vibrio cholerae, the etiological agent of cholera. This gene was isolated from a V. cholerae genomic library by complementation of different phenotypes associated with an hns mutation in E. coli. It encodes a 135-amino-acid protein showing approximately 50% identity with both H-NS and StpA in E. coli. Despite a low amino acid conservation in the N-terminal part, VicH is able to cross-react with anti-H-NS antibodies and to form oligomers in vitro. The vicH gene is expressed as a single gene from two promoters in tandem and is induced by cold shock. A V. cholerae wild-type strain expressing a vicHDelta92 gene lacking its 3' end shows pleiotropic alterations with regard to mucoidy and salicin metabolism. Moreover, this strain is unable to swarm on semisolid medium. Similarly, overexpression of the vicH wild-type gene results in an alteration of swarming behavior. This suggests that VicH could be involved in the virulence process in V. cholerae, in particular by affecting flagellum biosynthesis.