Clostridium difficile toxins facilitate bacterial colonization by modulating the fence and gate function of colonic epithelium

The contribution of Clostridium difficile toxin A and B (TcdA and TcdB) to cellular intoxication has been extensively studied, but their impact on bacterial colonization remains unclear. By setting-up two- and three-dimensional in vitro models of polarized gut epithelium, we investigated how C. difficile infection is affected by host cell polarity and whether TcdA and TcdB contribute to such events. Indeed, we observed that C. difficile adhesion and penetration of the epithelial barrier is substantially enhanced in poorly polarized or EGTA-treated cells, indicating that bacteria bind preferentially to the basolateral cell surface. In this context, we demonstrated that sub-lethal concentrations of C. difficile TcdA are able to alter cell polarity by causing redistribution of plasma membrane components between distinct surface domains. Taken together, the data suggest that toxin-mediated modulation of host cell organization may account for the capacity of this opportunistic pathogen to gain access to basolateral receptors leading to a successful colonization of the colonic mucosa

Stable iron isotopes and microbial mediation in red pigmentation of the Rosso Ammonitico (mid-late Jurassic, Verona area, Italy).

The iron (Fe) isotopic composition of 17 Jurassic limestones from the Rosso Ammonitico of Verona (Italy) have been analyzed by Multiple-Collector Inductively Coupled Plasma Mass Spectrometry (MC-ICP-MS). Such analysis allowed for the recognition of a clear iron isotopic fractionation (mean -0.8 per thousand, ranging between -1.52 to -0.06 per thousand) on a millimeter-centimeter scale between the red and grey facies of the studied formation. After gentle acid leaching, measurements of the Fe isotopic compositions gave delta(56)Fe values that were systematically lower in the red facies residues (median: -0.84 per thousand, range: -1.46 to +0.26 per thousand) compared to the grey facies residues (median: -0.08 per thousand, range: -0.34 to +0.23 per thousand). In addition, the red facies residues were characterized by a lighter delta(56)Fe signal relative to their corresponding leachates. These Fe isotopic fractionations could be a sensitive fingerprint of a biotic process; systematic isotopic differences between the red and grey facies residues, which consist of hematite and X-ray amorphous iron hydroxides, respectively, are hypothesized to have resulted from the oxidizing activity of iron bacteria and fungi in the red facies. The grey Fe isotopic data match the Fe isotopic signature of the terrestrial baseline established for igneous rocks and low-C(org) clastic sedimentary rocks. The Fe isotopic compositions of the grey laminations are consistent with the influx of detrital iron minerals and lack of microbial redox processes at the water-interface during deposition. Total Fe concentration measurements were performed by Inductively Coupled Plasma Atomic Emission Spectroscopy (ICP-AES) (confirmed by concentration estimations obtained by MC-ICP-MS analyses of microdrilled samples) on five samples, and resultant values range between 0.30% (mean) in the grey facies and 1.31% (mean) in the red facies. No correlation was observed between bulk Fe content and pigmentation or between bulk Fe content and Fe isotopic compositions. The rapid transformation of the original iron oxyhydroxides to hematite could have preserved the original isotopic composition if it had occurred at about the same temperature. This paper supports the use of Fe isotopes as sensitive tracers of biological activities recorded in old sedimentary sequences that contain microfossils of iron bacteria and fungi. However, a careful interpretation of the iron isotopic fractionation in terms of biotic versus abiotic processes requires supporting data or direct observations to characterize the biological, (geo)chemical, or physical context in relation to the geologic setting. This will become even more pertinent when Fe isotopic studies are expanded to the interplanetary realm.Journal ArticleResearch Support, Non-U.S. Gov'tinfo:eu-repo/semantics/publishe

Neisseria meningitidis subverts the polarized organization and intracellular trafficking of host cells to cross the epithelial barrier

Translocation of the nasopharyngeal barrier by Neisseria meningitidis occurs via an intracellular microtubule-dependent pathway and represents a crucial step in its pathogenesis. Despite this fact, the interaction of invasive meningococci with host subcellular compartments and the resulting impact on their organization and function have not been investigated. The influence of serogroup B strain MC58 on host cell polarity and intracellular trafficking system was assessed by confocal microscopy visualization of different plasma membrane-associated components (such as E-cadherin, ZO-1 and transferrin receptor) and evaluation of the transferrin uptake and recycling in infected Calu-3 monolayers. Additionally, the association of N. meningitidis with different endosomal compartments was evaluated through the concomitant staining of bacteria and markers specific for Rab11, Rab22a, Rab25 and Rab3 followed by confocal microscopy imaging. Subversion of the host cell architecture and intracellular trafficking system, denoted by mis-targeting of cell plasma membrane components and perturbations of transferrin transport, was shown to occur in response to N. meningitidis infection. Notably, the appearance of all of these events seems to positively correlate with the efficiency of N. meningitidis to cross the epithelial barrier. Our data reveal for the first time that N. meningitidis is able to modulate the host cell architecture and function, which might serve as a strategy of this pathogen for overcoming the nasopharyngeal barrier without affecting the monolayer integrity

Schematic representation of the contribution of SslE to <i>E.coli</i> pathogenesis.

<p>Gut mucus forms two layers, an inner firm mucus layer devoid of bacteria, and an outer layer that is not sterile and is a major habitat for commensal bacteria. <i>E. coli</i> can penetrate this barrier through the SslE-mediated enzymatic degradation of the mucus, targeting epithelial cells. This interaction will eventually lead to IL-8 release and neutrophils recruitment.</p

Kinetics of transepithelial electrical resistance in HT29-MTX cells over a 21 day period of differentiation.

<p>(A) TEER values were measured at different time points throughout a 21 day period of differentiation. (B) RT-PCR analysis of MUC mRNAs. After 21 days of differentiation, HT29-MTX mRNA was isolated, and cDNA was used to compare the level of MUC gene expression. Data are represented as relative fold increase of MUC mRNA in differentiated mucus-producing cells <i>versus</i> non-differentiated (control) cells. Control cells were assigned a value of 1.0. Levels of the different transcripts were normalized to β-actin, used as a house keeping gene. Error bars represent the SD.</p

<i>E. coli</i> growth rate in association with HT29-MTX cells.

<p>Using the transwell system, three sets of non-differentiated cells (A) and three sets of differentiated-mucus—producing- cells (B) were infected with WT and <i>sslE</i> KO bacteria for 2 hrs. Medium was removed to eliminate non-adhering bacteria and two sets of wells were used to do a total association assay, while the other wells were further incubated for 24 and 48 hrs at air liquid interface. At the end of the incubation period a total association assay was performed. The data presented are means ± standard deviations for 3 replicate experiments (n = 9).*P≤0.05. Error bars, SD.</p

SslE induces IL-8 secretion and stimulates neutrophil chemotaxis.

<p>(A) IL-8 levels in supernatants from the apical compartments of polarized HT29-MTX cells infected with IHE3034 WT and IHE3034Δ<i>SslE</i> KO bacteria. Data show mean chemokine concentrations in culture supernatants representative of three independent experiments *P≤0.05. Error bars, SD. (B) To measure neutrophil chemotaxis, bottom chambers of transwell supports were filled with supernatants deriving from HT29-MTX infected cells. Neutrophils were added to the upper chambers. After 1 h at 37°C, cells that had migrated toward the lower compartments were quantified by flow cytometry. DMEM has been used as a negative control and recombinant IL-8 as a positive control. The graph represents a typical experiment out of three performed with similar results. **** P ≤ 0.0001. Error bars, SD.</p

Modulation of SslE gene expression upon interaction with intestinal epithelial cells.

<p>(A) SslE transcription level in bacteria adhering to differentiated cells and in planktonic organisms. (B) SslE transcription level upon the interaction with differentiated or not differentiated HT29-MTX cells. Starting mRNA copy number of the unknown samples was determined using the comparative ΔΔ_{<b><i>CT</i></b>} method, and levels of the different transcripts were normalized to 16S rRNA, used as a housekeeping gene. Error bars represent the SD. **, P ≤ 0.01. n = 3.</p