Splicing of histone deacetylase 7 modulates smooth muscle cell proliferation and neointima formation through nuclear β-catenin translocation

Vascular smooth muscle cell (SMC) proliferation has an indispensable role in the pathogenesis of vascular disease, but the mechanism is not fully elucidated. The epigenetic enzyme histone deacetylase 7 (HDAC7) is involved in endothelial homeostasis and SMC differentiation and could have a role in SMC proliferation. In this study, we sought to examine the effect of 2 HDAC7 isoforms on SMC proliferation and neointima formation.

Mesenchymal stem cells enhance survival and bacterial clearance in murine Escherichia coli pneumonia

Rationale: Bacterial pneumonia is the most common infectious cause of death worldwide and treatment is increasingly hampered by antibiotic resistance. Mesenchymal stem cells (MSCs) have been demonstrated to provide protection against acute inflammatory lung injury; however, their potential therapeutic role in the setting of bacterial pneumonia has not been well studied.

Objective: This study focused on testing the therapeutic and mechanistic effects of MSCs in a mouse model of Gram-negative pneumonia.

Methods and results: Syngeneic MSCs from wild-type mice were isolated and administered via the intratracheal route to mice 4 h after the mice were infected with Escherichia coli. 3T3 fibroblasts and phosphate-buffered saline (PBS) were used as controls for all in vivo experiments. Survival, lung injury, bacterial counts and indices of inflammation were measured in each treatment group. Treatment with wild-type MSCs improved 48 h survival (MSC, 55%; 3T3, 8%; PBS, 0%; p<0.05 for MSC vs 3T3 and PBS groups) and lung injury compared with control mice. In addition, wild-type MSCs enhanced bacterial clearance from the alveolar space as early as 4 h after administration, an effect that was not observed with the other treatment groups. The antibacterial effect with MSCs was due, in part, to their upregulation of the antibacterial protein lipocalin 2.

Conclusions: Treatment with MSCs enhanced survival and bacterial clearance in a mouse model of Gram-negative pneumonia. The bacterial clearance effect was due, in part, to the upregulation of lipocalin 2 production by MSCs

Impact of cigarette smoke exposure on host-bacterial pathogen interactions

The human respiratory tract of individuals with normal lung function maintains a fine-tuned balance, being asymptomatically colonised by the normal microbiota in the upper airways and sterile in the lower tract. This equilibrium may be disrupted by the exposure to insults such as cigarette smoke. In the respiratory tract, the complex and noxious nature of inhaled cigarette smoke alters host-microorganisminteraction dynamics at all anatomical levels, causing infections in many cases. Moreover, continuous exposure to cigarette smoke itself causes deleterious effects on the host that can trigger the development of chronic respiratory diseases, such as chronic obstructive pulmonary disease (COPD) and lung cancer. COPD is an irreversible airflow obstruction associated with emphysema, fibrosis, mucus hypersecretion and persistent colonisation of the lower airways by opportunistic pathogens. COPD patients keep a stable (without exacerbation) but progressively worsening condition and suffer periodic exacerbations caused, in most cases, by infections. Although smoking and smoking-associated diseases are associated with a high risk of infection, most therapies aim to reduce inflammatory parameters, but do not necessarily take into account the presence of persistent colonisers. The effect of cigarette smoke on host-pathogen interaction dynamics in the respiratory tract, together with current and novel therapies, is discussed. Copyright©ERS 2012.

Role of Bacterial Surface Structures on the Interaction of Klebsiella pneumoniae with Phagocytes

Phagocytosis is a key process of the immune system. The human pathogen Klebsiella pneumoniae is a well known example of a pathogen highly resistant to phagocytosis. A wealth of evidence demonstrates that the capsule polysaccharide (CPS) plays a crucial role in resistance to phagocytosis. The amoeba Dictyostelium discoideum shares with mammalian macrophages the ability to phagocytose and kill bacteria. The fact that K. pneumoniae is ubiquitous in nature and, therefore, should avoid predation by amoebae, poses the question whether K. pneumoniae employs similar means to counteract amoebae and mammalian phagocytes. Here we developed an assay to evaluate K. pneumoniae-D. discoideum interaction. The richness of the growth medium affected the threshold at which the cps mutant was permissive for Dictyostelium and only at lower nutrient concentrations the cps mutant was susceptible to predation by amoebae. Given the critical role of bacterial surface elements on host-pathogen interactions, we explored the possible contribution of the lipopolysaccharide (LPS) and outer membrane proteins (OMPs) to combat phagoyctosis by D. discoideum. We uncover that, in addition to the CPS, the LPS O-polysaccharide and the first core sugar participate in Klebsiella resistance to predation by D. discoideum. K. pneumoniae LPS lipid A decorations are also necessary to avoid predation by amoebae although PagP-dependent palmitoylation plays a more important role than the lipid A modification with aminoarabinose. Mutants lacking OMPs OmpA or OmpK36 were also permissive for D. discoideium growth. Except the LPS O-polysaccharide mutants, all mutants were more susceptible to phagocytosis by mouse alveolar macrophages. Finally, we found a correlation between virulence, using the pneumonia mouse model, and resistance to phagocytosis. Altogether, this work reveals novel K. pneumoniae determinants involved in resistance to phagocytosis and supports the notion that Dictyostelium amoebae might be useful as host model to measure K. pneumoniae virulence and not only phagocytosis. © 2013 March et al.

Klebsiella pneumoniae targets an EGF receptor-dependent pathway to subvert inflammation

The NF-kB transcriptional factor plays a key role governing the activation of immune responses. Klebsiella pneumoniae is an important cause of community-acquired and nosocomial pneumonia. Evidence indicates that K. pneumoniae infections are characterized by lacking an early in?ammatory response. Recently, we have demonstrated that Klebsiella antagonizes the activation of NF-kB via the deubiquitinase CYLD. In this work, by applying a high-throughput siRNA gain-of-function screen interrogating the human kinome, we identi?ed 17 kinases that when targeted by siRNA restored IL-1b-dependent NF-kB translocation in infected cells. Further characterization revealed that K. pneumoniae activates an EGF receptor (EGFR)- phosphatidylinositol 3-OH kinase (PI3K)–AKT–PAK4–ERK–GSK3b signalling pathway to attenuate the cytokine-dependent nuclear translocation of NF-kB. Our data also revealed that CYLD is a downstream effector of K. pneumoniae-induced EGFR–
PI3K–AKT–PAK4–ERK–GSK3b signalling pathway. Our efforts to identify the bacterial factor(s) responsible for EGFR activation demonstrate that a capsule (CPS) mutant did not activate EGFR hence
suggesting that CPS could mediate the activation of EGFR. Supporting this notion, puri?ed CPS did activate EGFR as well as the EGFR-dependent PI3K–AKT–PAK4–ERK–GSK3b signalling pathway. CPS-mediated EGFR activation was dependent on a TLR4–MyD88–c-SRC-dependent pathway. Several promising drugs have been developed to antagonize this cascade. We propose that agents targeting this signalling pathway might provide selective alternatives for the management of K. pneumoniae pneumonias.

Capsule polysaccharide is a bacterial decoy for antimicrobial peptides

Antimicrobial peptides (APs) are important host weapons against infections. Nearly all APs are cationic and their microbicidal action is initiated through interactions with the anionic bacterial surface. It is known that pathogens have developed countermeasures to resist these agents by reducing the negative charge of membranes, by active efflux and by proteolytic degradation. Here we uncover a new strategy of resistance based on the neutralization of the bactericidal activity of APs by anionic bacterial capsule polysaccharide (CPS). Purified CPSs from Klebsiella pneumoniae K2, Streptococcus pneumoniae serotype 3 and Pseudomonas aeruginosa increased the resistance to polymyxin B of an unencapsulated K. pneumoniae mutant. Furthermore, these CPSs increased the MICs of polymyxin B and human neutrophil alpha-defensin 1 (HNP-1) for unencapsulated K. pneumoniae, Escherichia coli and P. aeruginosa PAO1. Polymyxin B or HNP-1 released CPS from capsulated K. pneumoniae, S. pneumoniae serotype 3 and P. aeruginosa overexpressing CPS. Moreover, this material also reduced the bactericidal activity of APs. We postulate that APs may trigger in vivo the release of CPS, which in turn will protect bacteria against APs. We found that anionic CPSs, but not cationic or uncharged ones, blocked the bactericidal activity of APs by binding them, thereby reducing the amount of peptides reaching the bacterial surface. Supporting this, polycations inhibited such interaction and the bactericidal activity was restored. We postulate that trapping of APs by anionic CPSs is an additional selective virulence trait of these molecules, which could be considered as bacterial decoys for APs.

The uptake of a Klebsiella pneumoniae capsule polysaccharide mutant triggers an inflammatory response by human airway epithelial cells

The means by which airway epithelial cells sense a bacterial infection and which intracellular signalling pathways are activated upon infection are poorly understood. A549 cells and human primary airway cells (NHBE) were used to investigate the response to infection with Klebsiella pneumoniae. Infection of A549 and NHBE with K. pneumoniae 52K10, a capsule polysaccharide (CPS) mutant, increased the surface levels of ICAM-1 and caused the release of IL-8. By contrast, the wild-type strain did not elicit these responses. Consistent with a functional role for these responses, there was a correlation between ICAM-1 levels and the number of adherent leukocytes on the epithelial cell surface. In addition, treatment of neutrophils with IL-8 enhanced their ability to kill K. pneumoniae. Strain 52K10 was internalized by A549 cells more efficiently than the wild-type, and when infections with 52K10 were performed in the presence of cytochalasin D the inflammatory response was abrogated. These findings suggest that cellular activation is mediated by bacterial internalization and that CPS prevents the activation through the blockage of bacterial adhesion and uptake. Collectively, the results indicate that bacterial internalization by airway epithelial cells could be the triggering signal for the activation of the innate immune system of the airway. Infection of A549 cells by 52K10 was shown to trigger the nuclear translocation of NF-kappaB. Evidence is presented showing that 52K10 activated IL-8 production through Toll-like receptor (TLR) 2 and TLR4 pathways and that A549 cells could use soluble CD14 as TLR co-receptor.

Capsule polysaccharide mediates bacterial resistance to antimicrobial peptides

The innate immune system plays a critical role in the defense of areas exposed to microorganisms. There is an increasing body of evidence indicating that antimicrobial peptides and proteins (APs) are one of the most important weapons of this system and that they make up the protective front for the respiratory tract. On the other hand, it is known that pathogenic organisms have developed countermeasures to resist these agents such as reducing the net negative charge of the bacterial membranes. Here we report the characterization of a novel mechanism of resistance to APs that is dependent on the bacterial capsule polysaccharide (CPS). Klebsiella pneumoniae CPS mutant was more sensitive than the wild type to human neutrophil defensin 1, beta-defensin 1, lactoferrin, protamine sulfate, and polymyxin B. K. pneumoniae lipopolysaccharide O antigen did not play an important role in AP resistance, and CPS was the only factor conferring protection against polymyxin B in strains lacking O antigen. In addition, we found a significant correlation between the amount of CPS expressed by a given strain and the resistance to polymyxin B. We also showed that K. pneumoniae CPS mutant bound more polymyxin B than the wild-type strain with a concomitant increased in the self-promoted pathway. Taken together, our results suggest that CPS protects bacteria by limiting the interaction of APs with the surface. Finally, we report that K. pneumoniae increased the amount of CPS and upregulated cps transcription when grown in the presence of polymyxin B and lactoferrin.

NO-loaded Zn2+-exchanged zeolite materials: A potential bifunctional anti-bacterial strategy

Nitric oxide (NO) is important for the regulation of a number of diverse biological processes, including vascular tone, neurotransmission, inflammatory cell responsiveness, defence against invading pathogens and wound healing. Transition metal exchanged zeolites are nanoporous materials with high-capacity storage properties for gases such as NO. The NO stores are liberated upon contact with aqueous environments, thereby making them ideal candidates for use in biological and clinical settings. Here, we demonstrate the NO release capacity and powerful bactericidal properties of a novel NO-storing Zn2+-exchanged zeolite material at a 50 wt.% composition in a polytetrafluoroethylene polymer. Further to our published data showing the anti-thrombotic effects of a similar NO-loaded zeolite, this study demonstrates the antibacterial properties of NO-releasing zeolites against clinically relevant strains of bacteria, namely Gram-negative Pseudomonas aeruginosa and Gram-positive methicillin-sensitive and methicillin-resistant Staphylococcus aureus and Clostridium difficile. Thus our study highlights the potential of NO-loaded zeolites as biocompatible medical device coatings with anti-infective properties. (C) 2009 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved.

A tale of two chitons: Is habitat specialisation linked to distinct associated bacterial communities?

Although most chitons (Mollusca: Polyplacophora) are shallow-water molluscs, diverse species also occur in deep-sea habitats. We investigated the feeding strategies of two species, Leptochiton boucheti and Nierstraszella lineata, recovered on sunken wood sampled in the western Pacific, close to the Vanuatu Islands. The two species display distinctly different associations with bacterial partners. Leptochiton boucheti harbours Mollicutes in regions of its gut epithelium and has no abundant bacterium associated with its gill. Nierstraszella lineata displays no dense gut-associated bacteria, but harbours bacterial filaments attached to its gill epithelium, related to the Deltaproteobacteria symbionts found in gills of the wood-eating limpet Pectinodonta sp. Stable carbon and nitrogen isotope signatures and an absence of cellulolytic activity give evidence against a direct wood-feeding diet; both species are secondary consumers within the wood food web. We suggest that the distinct associations with bacterial partners are linked to niche specialisations of the two species. Nierstraszella lineata is in a taxonomic family restricted to sunken wood and is possibly adapted to more anoxic conditions thanks to its gill-associated bacteria. Leptochiton boucheti is phylogenetically more proximate to an ancestral form not specialised on wood and may itself be more of a generalist; this observation is congruent with its association with Mollicutes, a bacterial clade comprising gut-associated bacteria occurring in several metazoan phyla.

Distribution and Translocation of Selenium from Soil to Grain and Its Speciation in Paddy Rice (Oryza sativa L.)

Selenium, an essential micronutrient for humans, is insufficient in dietary intake for millions of people worldwide. Rice as the most popular staple food in the world is one of the dominant selenium (Se) sources for people. The distribution and translocation of Se from soil to grain were investigated in a Se-rich environment in this study. The Se levels in soils ranged widely from 0.5 to 47.7 mg kg(-1). Selenium concentration in rice bran was 1.94 times higher than that in corresponding polished rice. The total Se concentrations in the rice fractions were in the following order: straw > bran > whole grain > polished rice > husk. Significant linear relationships between different rice fractions were observed with each other, and Se in the soil has a linear relationship with different rice fractions as well. Se concentration in rice can easily be predicted by soil Se concentrations or any rice fractions and vice versa according to their linear relationships. In all rice samples for Se speciation, SeMet was the major Se species, followed by MeSeCys and SeCys. The average percentage for SeMet (82.9%) and MeSeCys (6.2%) was similar in the range of total Se from 2.2 to 8.4 mg kg(-1) tested. The percentage of SeCys decreased from 6.3 to 2.8%, although its concentration elevated with the increase in total Se in rice. This could be due to the fact that SeCys is the precursor for the formation of other organic Se compounds. The information obtained may have considerable significance for assessing translocation and accumulation of Se in plant.

An arsenic-contaminated field trial to assess the uptake and translocation of arsenic by genotypes of rice

Compared to other cereals, rice has particular strong As accumulation. Therefore, it is very important to understand As uptake and translocation among different genotypes. A field study in Chenzhou city, Hunan province of China, was employed to evaluate the effect of arsenic-contaminated soil on uptake and distribution in 34 genotypes of rice (including unpolished rice, husk, shoot, and root). The soil As concentrations ranged from 52.49 to 83.86 mg kg-1, with mean As concentration 64.44 mg kg-1. The mean As concentrations in rice plant tissues were different among the 34 rice genotypes. The highest As concentrations were accumulated in rice root (196.27-385.98 mg kg-1 dry weight), while the lowest was in unpolished rice (0.31-0.52 mg kg-1 dry weight). The distribution of As in rice tissue and paddy soil are as follows root » soil > shoot > husk > unpolished rice. The ranges of concentrations of inorganic As in all of unpolished rice were from 0.26 to 0.52 mg kg-1 dry weight. In particular, the percentage of inorganic As in the total As was more than 67 %, indicating that the inorganic As was the predominant species in unpolished rice. The daily dietary intakes of inorganic As in unpolished rice ranged from 0.10 to 0.21 mg for an adult, and from 0.075 to 0.15 mg for a child. Comparison with tolerable daily intakes established by FAO/WHO, inorganic As in most of unpolished rice samples exceeded the recommended intake values. The 34 genotypes of rice were classified into four clusters using a criteria value of rescaled distance between 5 and 10. Among the 34 genotypes, the genotypes II you 416 (II416) with the lowest enrichment of As and the lowest daily dietary intakes of inorganic As could be selected as the main cultivar in As-contaminated field.

Chemical Communication of Antibiotic Resistance by a Highly Resistant Subpopulation of Bacterial Cells

The overall antibiotic resistance of a bacterial population results from the combination of a wide range of susceptibilities displayed by subsets of bacterial cells. Bacterial heteroresistance to antibiotics has been documented for several opportunistic Gram-negative bacteria, but the mechanism of heteroresistance is unclear. We use Burkholderia cenocepacia as a model opportunistic bacterium to investigate the implications of heterogeneity in the response to the antimicrobial peptide polymyxin B (PmB) and also other bactericidal antibiotics. Here, we report that B. cenocepacia is heteroresistant to PmB. Population analysis profiling also identified B. cenocepacia subpopulations arising from a seemingly homogenous culture that are resistant to higher levels of polymyxin B than the rest of the cells in the culture, and can protect the more sensitive cells from killing, as well as sensitive bacteria from other species, such as Pseudomonas aeruginosa and Escherichia coli. Communication of resistance depended on upregulation of putrescine synthesis and YceI, a widely conserved low-molecular weight secreted protein. Deletion of genes for the synthesis of putrescine and YceI abrogate protection, while pharmacologic inhibition of putrescine synthesis reduced resistance to polymyxin B. Polyamines and YceI were also required for heteroresistance of B. cenocepacia to various bactericidal antibiotics. We propose that putrescine and YceI resemble "danger" infochemicals whose increased production by a bacterial subpopulation, becoming more resistant to bactericidal antibiotics, communicates higher level of resistance to more sensitive members of the population of the same or different species.