Subversion of Mucosal Barrier Polarity by Pseudomonas Aeruginosa

The lumenal surfaces of human body are lined by a monolayer of epithelia that together with mucus secreting cells and specialized immune cells form the mucosal barrier. This barrier is one of the most fundamental components of the innate immune system, protecting organisms from the vast environmental microbiota. The mucosal epithelium is comprised of polarized epithelial cells with distinct apical and basolateral surfaces that are defined by unique set of protein and lipid composition and are separated by tight junctions. The apical surface serves as a barrier to the outside world and is specialized for the exchange of materials with the lumen. The basolateral surface is adapted for interaction with other cells and for exchange with the bloodstream. A wide network of proteins and lipids regulates the formation and maintenance of the epithelium polarity. Many human pathogens have evolved virulence mechanisms that target this network and interfere with epithelial polarity to enhance binding to the apical surface, enter into cells, and/or cross the mucosal barrier. This review highlights recent advances in our understanding of how Pseudomonas aeruginosa, an important opportunistic human pathogen that preferentially infects damaged epithelial tissues, exploits the epithelial cell polarization machinery to enhance infection

RNAi Screen Reveals an Abl Kinase-Dependent Host Cell Pathway Involved in Pseudomonas aeruginosa Internalization

Internalization of the pathogenic bacterium Pseudomonas aeruginosa by non-phagocytic cells is promoted by rearrangements of the actin cytoskeleton, but the host pathways usurped by this bacterium are not clearly understood. We used RNAi-mediated gene inactivation of ∼80 genes known to regulate the actin cytoskeleton in Drosophila S2 cells to identify host molecules essential for entry of P. aeruginosa. This work revealed Abl tyrosine kinase, the adaptor protein Crk, the small GTPases Rac1 and Cdc42, and p21-activated kinase as components of a host signaling pathway that leads to internalization of P. aeruginosa. Using a variety of complementary approaches, we validated the role of this pathway in mammalian cells. Remarkably, ExoS and ExoT, type III secreted toxins of P. aeruginosa, target this pathway by interfering with GTPase function and, in the case of ExoT, by abrogating P. aeruginosa–induced Abl-dependent Crk phosphorylation. Altogether, this work reveals that P. aeruginosa utilizes the Abl pathway for entering host cells and reveals unexpected complexity by which the P. aeruginosa type III secretion system modulates this internalization pathway. Our results furthermore demonstrate the applicability of using RNAi screens to identify host signaling cascades usurped by microbial pathogens that may be potential targets for novel therapies directed against treatment of antibiotic-resistant infections

Gene Expression Profiles of Chlamydophila pneumoniae during the Developmental Cycle and Iron Depletion–Mediated Persistence

The obligate intracellular, gram-negative bacterium Chlamydophila pneumoniae (Cpn) has impact as a human pathogen. Little is known about changes in the Cpn transcriptome during its biphasic developmental cycle (the acute infection) and persistence. The latter stage has been linked to chronic diseases. To analyze Cpn CWL029 gene expression, we designed a pathogen-specific oligo microarray and optimized the extraction method for pathogen RNA. Throughout the acute infection, ratio expression profiles for each gene were generated using 48 h post infection as a reference. Based on these profiles, significantly expressed genes were separated into 12 expression clusters using self-organizing map clustering and manual sorting into the “early”, “mid”, “late”, and “tardy” cluster classes. The latter two were differentiated because the “tardy” class showed steadily increasing expression at the end of the cycle. The transcriptome of the Cpn elementary body (EB) and published EB proteomics data were compared to the cluster profile of the acute infection. We found an intriguing association between “late” genes and genes coding for EB proteins, whereas “tardy” genes were mainly associated with genes coding for EB mRNA. It has been published that iron depletion leads to Cpn persistence. We compared the gene expression profiles during iron depletion–mediated persistence with the expression clusters of the acute infection. This led to the finding that establishment of iron depletion–mediated persistence is more likely a mid-cycle arrest in development rather than a completely distinct gene expression pattern. Here, we describe the Cpn transcriptome during the acute infection, differentiating “late” genes, which correlate to EB proteins, and “tardy” genes, which lead to EB mRNA. Expression profiles during iron mediated–persistence led us to propose the hypothesis that the transcriptomic “clock” is arrested during acute mid-cycle

Par3 integrates Tiam1 and phosphatidylinositol 3-kinase signaling to change apical membrane identity

Pathogens can alter epithelial polarity by recruiting polarity proteins to the apical membrane, but how a change in protein localization is linked to polarity disruption is not clear. In this study, we used chemically induced dimerization to rapidly relocalize proteins from the cytosol to the apical surface. We demonstrate that forced apical localization of Par3, which is normally restricted to tight junctions, is sufficient to alter apical membrane identity through its interactions with phosphatidylinositol 3-kinase (PI3K) and the Rac1 guanine nucleotide exchange factor Tiam1. We further show that PI3K activity is required upstream of Rac1, and that simultaneously targeting PI3K and Tiam1 to the apical membrane has a synergistic effect on membrane remodeling. Thus, Par3 coordinates the action of PI3K and Tiam1 to define membrane identity, revealing a signaling mechanism that can be exploited by human mucosal pathogens

Pseudomonas aeruginosa exploits a PIP3-dependent pathway to transform apical into basolateral membrane

Pseudomonas aeruginosa, an important human pathogen, preferentially binds and enters injured cells from the basolateral (BL) surface. We previously demonstrated that activation of phosphatidylinositol 3-kinase (PI3K) and Akt are necessary and sufficient for P. aeruginosa entry from the apical (AP) surface and that AP addition of phosphatidylinositol 3,4,5-trisphosphate (PIP3) is sufficient to convert AP into BL membrane (Kierbel, A., A. Gassama-Diagne, K. Mostov, and J.N. Engel. 2005. Mol. Biol. Cell. 16:2577–2585; Gassama-Diagne, A., W. Yu, M. ter Beest, F. Martin-Belmonte, A. Kierbel, J. Engel, and K. Mostov. 2006. Nat. Cell Biol. 8:963–970). We now show that P. aeruginosa subverts this pathway to gain entry from the AP surface. In polarized monolayers, P. aeruginosa binds near cell–cell junctions without compromising them where it activates and recruits PI3K to the AP surface. Membrane protrusions enriched for PIP3 and actin accumulate at the AP surface at the site of bacterial binding. These protrusions lack AP membrane markers and are comprised of BL membrane constituents, which are trafficked there by transcytosis. The end result is that this bacterium transforms AP into BL membrane, creating a local microenvironment that facilitates its colonization and entry into the mucosal barrier

Sex-differential PTSD symptom trajectories across one year following suspected serious injury

Background Recent years have shown an increased application of prospective trajectory-oriented approaches to posttraumatic stress disorder (PTSD). Although women are generally considered at increased PTSD risk, sex and gender differences in PTSD symptom trajectories have not yet been extensively studied. Objective To perform an in-depth investigation of differences in PTSD symptom trajectories across one-year post-trauma between men and women, by interpreting the general trends of trajectories observed in sex-disaggregated samples, and comparing within-trajectory symptom course and prevalence rates. Method We included N = 554 participants (62.5% men, 37.5% women) from a multi-centre prospective cohort of emergency department patients with suspected severe injury. PTSD symptom severity was assessed at 1, 3, 6, and 12 months post-trauma, using the Clinician-Administered PTSD Scale for DSM-IV. Latent growth mixture modelling on longitudinal PTSD symptoms was performed within the sex-disaggregated and whole samples. Bayesian modelling with informative priors was applied for reliable model estimation, considering the imbalanced prevalence of the expected latent trajectories. Results In terms of general trends, the same trajectories were observed for men and women, i.e. resilient, recovery, chronic symptoms and delayed onset. Within-trajectory symptom courses were largely comparable, but resilient women had higher symptoms than resilient men. Sex differences in prevalence rates were observed for the recovery (higher in women) and delayed onset (higher in men) trajectories. Model fit for the sex-disaggregated samples was better than for the whole sample, indicating preferred application of sex-disaggregation. Analyses within the whole sample led to biased estimates of overall and sex-specific trajectory prevalence rates. Conclusions Sex-disaggregated trajectory analyses revealed limited sex differences in PTSD symptom trajectories within one-year post-trauma in terms of general trends, courses and prevalence rates. The observed biased trajectory prevalence rates in the whole sample emphasize the necessity to apply appropriate statistical techniques when conducting sex-sensitive research

Sex-differential PTSD symptom trajectories across one year following suspected serious injury

Background: Recent years have shown an increased application of prospective trajectory-oriented approaches to posttraumatic stress disorder (PTSD). Although women are generally considered at increased PTSD risk, sex and gender differences in PTSD symptom trajectories have not yet been extensively studied. Objective: To perform an in-depth investigation of differences in PTSD symptom trajectories across one-year post-trauma between men and women, by interpreting the general trends of trajectories observed in sex-disaggregated samples, and comparing within-trajectory symptom course and prevalence rates. Method: We included N = 554 participants (62.5% men, 37.5% women) from a multi-centre prospective cohort of emergency department patients with suspected severe injury. PTSD symptom severity was assessed at 1, 3, 6, and 12 months post-trauma, using the Clinician-Administered PTSD Scale for DSM-IV. Latent growth mixture modelling on longitudinal PTSD symptoms was performed within the sex-disaggregated whole samples. Bayesian modelling with informative priors was applied for reliable model estimation, considering the imbalanced prevalence of the expected latent trajectories. Results: In terms of general trends, the same trajectories were observed for men and women, i.e. resilient, recovery, chronic symptoms and delayed onset. Within-trajectory symptom courses were largely comparable, but resilient women had higher symptoms than resilient men. Sex differences in prevalence rates were observed for the recovery (higher in women) and delayed onset (higher in men) trajectories. Model fit for the sex-disaggregated samples was better than for the whole sample, indicating preferred application of sex-disaggregation. Analyses within the whole sample led to biased estimates of overall and sex-specific trajectory prevalence rates. Conclusions: Sex-disaggregated trajectory analyses revealed limited sex differences in PTSD symptom trajectories within one-year post-trauma in terms of general trends, courses and prevalence rates. The observed biased trajectory prevalence rates in the whole sample emphasize the necessity to apply appropriate statistical techniques when conducting sex-sensitive research

Prenatal Exposure to Environmental Phenols: Concentrations in Amniotic Fluid and Variability in Urinary Concentrations during Pregnancy

Background: Maternal urinary biomarkers are often used to assess fetal exposure to phenols and their precursors. Their effectiveness as a measure of exposure in epidemiological studies depends on their variability during pregnancy and their ability to accurately predict fetal exposure

Pseudomonas aeruginosa Pili and Flagella Mediate Distinct Binding and Signaling Events at the Apical and Basolateral Surface of Airway Epithelium

Pseudomonas aeruginosa, an important opportunistic pathogen of man, exploits numerous factors for initial attachment to the host, an event required to establish bacterial infection. In this paper, we rigorously explore the role of two major bacterial adhesins, type IV pili (Tfp) and flagella, in bacterial adherence to distinct host receptors at the apical (AP) and basolateral (BL) surfaces of polarized lung epithelial cells and induction of subsequent host signaling and pathogenic events. Using an isogenic mutant of P. aeruginosa that lacks flagella or utilizing beads coated with purified Tfp, we establish that Tfp are necessary and sufficient for maximal binding to host N-glycans at the AP surface of polarized epithelium. In contrast, experiments utilizing a P. aeruginosa isogenic mutant that lacks Tfp or using beads coated with purified flagella demonstrate that flagella are necessary and sufficient for maximal binding to heparan sulfate (HS) chains of heparan sulfate proteoglycans (HSPGs) at the BL surface of polarized epithelium. Using two different cell-free systems, we demonstrate that Tfp-coated beads show highest binding affinity to complex N-glycan chains coated onto plastic plates and preferentially aggregate with beads coated with N-glycans, but not with single sugars or HS. In contrast, flagella-coated beads bind to or aggregate preferentially with HS or HSPGs, but demonstrate little binding to N-glycans. We further show that Tfp-mediated binding to host N-glycans results in activation of phosphatidylinositol 3-kinase (PI3K)/Akt pathway and bacterial entry at the AP surface. At the BL surface, flagella-mediated binding to HS activates the epidermal growth factor receptor (EGFR), adaptor protein Shc, and PI3K/Akt, and induces bacterial entry. Remarkably, flagella-coated beads alone can activate EGFR and Shc. Together, this work provides new insights into the intricate interactions between P. aeruginosa and lung epithelium that may be potentially useful in the development of novel treatments for P. aeruginosa infections