Highly conserved type 1 pili promote enterotoxigenic E. coli pathogen-host interactions

Enterotoxigenic Escherichia coli (ETEC), defined by their elaboration of heat-labile (LT) and/or heat-stable (ST) enterotoxins, are a common cause of diarrheal illness in developing countries. Efficient delivery of these toxins requires ETEC to engage target host enterocytes. This engagement is accomplished using a variety of pathovar-specific and conserved E. coli adhesin molecules as well as plasmid encoded colonization factors. Some of these adhesins undergo significant transcriptional modulation as ETEC encounter intestinal epithelia, perhaps suggesting that they cooperatively facilitate interaction with the host. Among genes significantly upregulated on cell contact are those encoding type 1 pili. We therefore investigated the role played by these pili in facilitating ETEC adhesion, and toxin delivery to model intestinal epithelia. We demonstrate that type 1 pili, encoded in the E. coli core genome, play an essential role in ETEC virulence, acting in concert with plasmid-encoded pathovar specific colonization factor (CF) fimbriae to promote optimal bacterial adhesion to cultured intestinal epithelium (CIE) and to epithelial monolayers differentiated from human small intestinal stem cells. Type 1 pili are tipped with the FimH adhesin which recognizes mannose with stereochemical specificity. Thus, enhanced production of highly mannosylated proteins on intestinal epithelia promoted FimH-mediated ETEC adhesion, while conversely, interruption of FimH lectin-epithelial interactions with soluble mannose, anti-FimH antibodies or mutagenesis of fimH effectively blocked ETEC adhesion. Moreover, fimH mutants were significantly impaired in delivery of both heat-stable and heat-labile toxins to the target epithelial cells in vitro, and these mutants were substantially less virulent in rabbit ileal loop assays, a classical model of ETEC pathogenesis. Collectively, our data suggest that these highly conserved pili play an essential role in virulence of these diverse pathogens

Maturation of GABAergic Inhibition Promotes Strengthening of Temporally Coherent Inputs among Convergent Pathways

Spike-timing-dependent plasticity (STDP), a form of Hebbian plasticity, is inherently stabilizing. Whether and how GABAergic inhibition influences STDP is not well understood. Using a model neuron driven by converging inputs modifiable by STDP, we determined that a sufficient level of inhibition was critical to ensure that temporal coherence (correlation among presynaptic spike times) of synaptic inputs, rather than initial strength or number of inputs within a pathway, controlled postsynaptic spike timing. Inhibition exerted this effect by preferentially reducing synaptic efficacy, the ability of inputs to evoke postsynaptic action potentials, of the less coherent inputs. In visual cortical slices, inhibition potently reduced synaptic efficacy at ages during but not before the critical period of ocular dominance (OD) plasticity. Whole-cell recordings revealed that the amplitude of unitary IPSCs from parvalbumin positive (Pv+) interneurons to pyramidal neurons increased during the critical period, while the synaptic decay time-constant decreased. In addition, intrinsic properties of Pv+ interneurons matured, resulting in an increase in instantaneous firing rate. Our results suggest that maturation of inhibition in visual cortex ensures that the temporally coherent inputs (e.g. those from the open eye during monocular deprivation) control postsynaptic spike times of binocular neurons, a prerequisite for Hebbian mechanisms to induce OD plasticity

Role of electrostatic repulsion in controlling pH-dependent conformational changes of viral fusion proteins

Viral fusion proteins undergo dramatic conformational transitions during membrane fusion. For viruses that enter through the endosome, these conformational rearrangements are typically pH sensitive. Here, we provide a comprehensive review of the molecular interactions that govern pH-dependent rearrangements and introduce a paradigm for electrostatic residue pairings that regulate progress through the viral fusion coordinate. Analysis of structural data demonstrates a significant role for side-chain protonation in triggering conformational change. To characterize this behavior, we identify two distinct residue pairings, which we define as Histidine-Cation (HisCat) and Anion-Anion (AniAni) interactions. These side-chain pairings destabilize a particular conformation via electrostatic repulsion through side-chain protonation. Furthermore, two energetic control mechanisms, thermodynamic and kinetic, regulate these structural transitions. This review expands on the current literature by identification of these residue clusters, discussion of data demonstrating their function, and speculation of how these residue pairings contribute to the energetic controls

Type 1 pili mediated interactions enhance toxin delivery.

<p><b>a.</b> Quantification of intracellular cGMP in infected cells. Cells were infected with WT ETEC in the absence or presence mannose sugar or with <i>fimH</i> or <i>fimA</i> mutants. Cells infected with <i>estH/estP</i> mutants which lack production of both heat stable toxins ST-H (ST-1b) and ST-P (ST-1a), represent basal level of cGMP in cells. <b>b.</b> Quantification of the amount of intracellular cAMP in infected cells. Cells were infected with WT ETEC in the absence or presence mannose sugar or with <i>fimH</i> or <i>fimA</i> mutants. Cells infected with <i>eltAB</i>, LT mutants, represent basal level of cAMP in cells. <b>c.</b> LT secretion by different mutants. Each bar represent mean with SEM (error bar) of 2 experiments consisting of 5 replicates per experiment for each strain. All P values were calculated by nonparametric Mann-Whitney test. *** p<0.0001.</p

Type 1 pili are required for virulence in the rabbit ileal loop assay.

<p>Type 1 pili are required for optimal bacterial engagement of rabbit intestinal epithelia. <b>a.</b> sections of rabbit ileum in which attached bacteria (green) are identified with anti-O78 (top panel) or anti-FimH (bottom panel). Nuclei are stained with DAPI (blue) and membranes are stained with CellMask (red). <b>b.</b> bacteria adherent to the ileal mucosal surface following infection with wild type ETEC H10407 or <i>fimH</i> and <i>fimA</i> mutants. <b>c.</b> Type 1 pili are required for toxicity in the rabbit ileal loop assay. Shown in the graph is the amount of fluid accumulation in each loop infected with WT or <i>fimH</i> or <i>fimA</i> mutants 18 h post inoculation. Loops infected with <i>eltAB</i> mutants or mock infected (PBS) were used as controls. Data represent the summary of experiments from 7 different rabbits (n = 7). Inset image shows infected ileal loops from one representative experiment. Each loop in the inset image is labeled with the infecting bacterial strain or with the negative control (PBS).</p

Properties of human enteroid-derived small intestinal monolayers.

<p><b>a.</b> Hematoxylin and eosin staining, DIC, and UEA1 lectin immunofluorescence confocal microscopic images showing formation of continuous monolayers. The apical surface is detected in the bottom image with FITC conjugated UEA1 lectin. <b>b.</b> UEA1 and anti-villin 1 antibody immunofluorescence in Laser Scanning Confocal Microscopy (LSCM) images from sections of polarized small intestinal enteroid monolayers. Nuclei (DAPI) are shown in blue. <b>c.</b> LSCM of sections showing MUC2 immunofluorescence (red) in probable goblet cell and co-localization with UEA1 in the merged image. <b>d.</b> Chromogranin A positive cells (red). <b>e.</b> Transmission electron microscopy (3000x) of polarized small intestinal enteroid monolayer sections showing a goblet cell flanked by enterocytes with distinct microvilli on the apical surface. Transwell filters in sections are indicated by *.</p

Type 1 pili are required for optimal adhesion to small intestinal epithelia.

<p><b>a.</b> Representative laser scanning confocal microscopy (LSCM) images of sections prepared from human small intestinal enteroid-derived polarized monolayers infected with WT H10407 (red, anti-O78). Surface staining with the UEA1 lectin is shown in green. Nuclei are stained in blue (DAPI). <b>b.</b> Three dimensional reconstruction of LSCM z stacks of ETEC H10407 infected polarized monolayers showing the distribution of zonula occludens-1 (ZO-1, green) at the apical surface. Bacteria were visualized with anti-O78 (red) and nuclei are stained with DAPI (blue). <b>c.</b> Transmission electron microscopy images of ETEC H10407 adhering to microvilli on the surface of small intestinal monolayers (magnification 7500x and 15000x for left and right images respectively). <b>d.</b> LCSM images of wild type (wt) versus <i>fimH</i> mutant bacteria adherent to the surface of small intestinal enteroids Bacteria were visualized with anti-O78 (green) and cell membranes with CellMask (red), nuclei (DAPI, blue). <b>e.</b> Quantitative analysis of ETEC adhesion to enteroid-derived monolayers represented in panel <b>d</b>. Each dot plot represents adhesion data obtained using ileal cell derived from two individual subjects. Horizontal lines represent geometric means of data combined from 2 independent experiments. P values were calculated by nonparametric Mann-Whitney testing.</p

FimH adhesin of ETEC interacts with intestinal epithelial cells.

<p><b>a.</b> Confocal microscopy images show binding of the biotinylated FimH lectin domain (FimHLD) or FimHLD:Q133K to the apical surface cultured intestinal epithelium (CIE). Biotinylated FimHLD was detected with streptavidin-conjugated fluorescent nanocrystals (Qdot, green); plasma membranes were stained with CellMask (red) and nuclei with DAPI (blue). Image at right shows three dimensional reconstruction of z stacks of CIE following interaction with FimHLD or the mutant protein. <b>b.</b> Quantitative analysis of FimHLD binding to CIE represented in panels <b>a</b> using Volocity three-dimensional (3D) image analysis software (version 6.2; PerkinElmer, Inc.). P value was calculated using nonparametric Mann-Whitney testing. <b>c.</b> Immunoelectron microscopy images of CIE infected with ETEC H10407. Left panel, microvilli structure at the apical surface of the CIE; right panels show immunogold labeling of FimH localized to the ETEC-host interacting surface.</p

Enhanced presentation of mannosylated glycoproteins increases FimH binding and ETEC adhesion.

<p><b>a.</b> Confocal microscopy images detecting FimHLD binding to the kifunensine treated CIE. Biotinylated FimHLD was detected with streptavidin-conjugated fluorescent nanocrystals (Qdot, green) and nuclei with DAPI (blue). <b>b.</b> Quantitative analysis of FimHLD binding to CIE represented in panels <b>a</b> using Volocity three-dimensional (3D) image analysis software (version 6.2; PerkinElmer, Inc.). Data represent mean ± standard deviation of results of 3 independent experiments each with triplicate wells per concentration tested (n = 9). <b>c.</b> Confocal microscopic images showing ETEC adhesion to kifunensine treated CIE. The CIE grown on trans-well filters were treated with kifunensine and infected with WT ETEC or <i>fimH</i> mutants. One hour post infection wells were processed for microscopic examination. Bacteria (green), cell membrane (red), DAPI (blue). <b>d.</b> Quantitative analysis was done by counting number of bacteria present per focus area. Horizontal dashed lines represent geometric mean of 25 total data points combined from 2 replicate experiments. P values were calculated by nonparametric Mann-Whitney test. *** indicates p<0.0001.</p

Type 1 pili expression promotes optimal adhesion of ETEC to intestinal epithelia.

<p><b>a.</b> Transmission electron micrograph of ETEC H10407 expressing type 1 pili. The FimH tip adhesin was detected using α-FimH antibody and gold secondary antibody conjugate. <b>b</b>. Flow cytometric analysis of type 1 pili expression by ETEC H10407 and <i>fimH</i> mutants. <b>c.</b> Assessment of type 1 pili function using yeast agglutination assays. Negative yeast agglutination reflected the loss of type 1 pili activity. <b>d.</b> FimA immunoblot of type 1 pili extracts from static culture of WT ETEC, <i>fimH</i> mutants and mutants complemented with wild type <i>fimH</i> gene (p<i>fimH</i>). The <i>fimH</i> mutant complemented with a plasmid encoding a Q133K substitution in FimH is included as a negative control. <b>e.</b> Confocal microscopic images showing adhesion of WT ETEC, <i>fimH</i> mutants or complemented mutants to polarized cultured intestinal epithelia. Bacteria (anti-O78, green), cell membrane (CellMask, red), nuclei (DAPI, blue). <b>f.</b> Quantitative analysis was done by counting number of bacteria per focus area. Horizontal dashed lines represent geometric means of 3 combined individual experiments. P values were calculated by nonparametric Mann-Whitney test. *** indicates p<0.0001.</p