RNase 7 Contributes to the Cutaneous Defense against Enterococcus faecium

Background

Human skin is able to mount a fast response against invading microorganisms by the release of antimicrobial proteins such as the ribonuclease RNase 7. Because RNase 7 exhibits high activity against Enterococcus faecium the aim of this study was to further explore the role of RNase 7 in the cutaneous innate defense system against E. faecium.

Methodology/Principal Findings

Absolute quantification using real-time PCR and ELISA revealed that primary keratinocytes expressed high levels of RNase 7. Immunohistochemistry showed RNase 7 expression in all epidermal layers of the skin with an intensification in the upper more differentiated layers. Furthermore, RNase 7 was secreted by keratinocytes in vitro and in vivo in a site-dependent way. RNase 7 was still active against E. faecium at low pH (5.5) or high NaCl (150 mM) concentration and the bactericidal activity of RNase 7 against E. faecium required no ribonuclease activity as shown by recombinant RNase 7 lacking enzymatic activity. To further explore the role of RNase 7 in cutaneous defense against E. faecium, we investigated whether RNase 7 contributes to the E. faecium killing activity of skin extracts derived from stratum corneum. Treatment of the skin extract with an RNase 7 specific antibody, which neutralizes the antimicrobial activity of RNase 7, diminished its E. faecium killing activity.

Conclusions/Significance

Our data indicate that RNase 7 contributes to the E. faecium-killing activity of skin extracts and suggest an important role for RNase 7 in the protection of human skin against E. faecium colonization.     

Cyclin G Functions as a Positive Regulator of Growth and Metabolism in Drosophila

In multicellular organisms, growth and proliferation is adjusted to nutritional conditions by a complex signaling network. The Insulin receptor/target of rapamycin (InR/TOR) signaling cascade plays a pivotal role in nutrient dependent growth regulation in Drosophila and mammals alike. Here we identify Cyclin G (CycG) as a regulator of growth and metabolism in Drosophila. CycG mutants have a reduced body size and weight and show signs of starvation accompanied by a disturbed fat metabolism. InR/TOR signaling activity is impaired in cycG mutants, combined with a reduced phosphorylation status of the kinase Akt1 and the downstream factors S6-kinase and eukaryotic translation initiation factor 4E binding protein (4E-BP). Moreover, the expression and accumulation of Drosophila insulin like peptides (dILPs) is disturbed in cycG mutant brains. Using a reporter assay, we show that the activity of one of the first effectors of InR signaling, Phosphoinositide 3-kinase (PI3K92E), is unaffected in cycG mutants. However, the metabolic defects and weight loss in cycG mutants were rescued by overexpression of Akt1 specifically in the fat body and by mutants in widerborst (wdb), the B''-subunit of the phosphatase PP2A, known to downregulate Akt1 by dephosphorylation. Together, our data suggest that CycG acts at the level of Akt1 to regulate growth and metabolism via PP2A in Drosophila.     

RIAM Activates Integrins by Linking Talin to Ras GTPase Membrane-targeting Sequences

Rap1 small GTPases interact with Rap1-GTP-interacting adaptor molecule (RIAM), a member of the MRL (Mig-10/RIAM/Lamellipodin) protein family, to promote talin-dependent integrin activation. Here, we show that MRL proteins function as scaffolds that connect the membrane targeting sequences in Ras GTPases to talin, thereby recruiting talin to the plasma membrane and activating integrins. The MRL proteins bound directly to talin via short, N-terminal sequences predicted to form amphipathic helices. RIAM-induced integrin activation required both its capacity to bind to Rap1 and to talin. Moreover, we constructed a minimized 50-residue Rap-RIAM module containing the talin binding site of RIAM joined to the membrane-targeting sequence of Rap1A. This minimized Rap-RIAM module was sufficient to target talin to the plasma membrane and to mediate integrin activation, even in the absence of Rap1 activity. We identified a short talin binding sequence in Lamellipodin (Lpd), another MRL protein; talin binding Lpd sequence joined to a Rap1 membrane-targeting sequence is sufficient to recruit talin and activate integrins. These data establish the mechanism whereby MRL proteins interact with both talin and Ras GTPases to activate integrins.Increased affinity (“activation”) of cellular integrins is central to physiological events such as cell migration, assembly of the extracellular matrix, the immune response, and hemostasis (1). Each integrin comprises a type I transmembrane α and β subunit, each of which has a large extracellular domain, a single transmembrane domain, and a cytoplasmic domain (tail). Talin binds to most integrin β cytoplasmic domains and the binding of talin to the integrin β tail initiates integrin activation (2–4). A small, PTB-like domain of talin mediates activation via a two-site interaction with integrin β tails (5), and this PTB domain is functionally masked in the intact talin molecule (6). A central question in integrin biology is how the talin-integrin interaction is regulated to control integrin activation; recent work has implicated Ras GTPases as critical signaling modules in this process (7).Ras proteins are small monomeric GTPases that cycle between the GTP-bound active form and the GDP-bound inactive form. Guanine nucleotide exchange factors (GEFs) promote Ras activity by exchanging bound GDP for GTP, whereas GTPase-activating proteins (GAPs)³ enhance the hydrolysis of Ras-bound GTP to GDP (for review, see Ref. 8). The Ras subfamily members Rap1A and Rap1B stimulate integrin activation (9, 10). For example, expression of constitutively active Rap1 activates integrin αMβ2 in macrophage, and inhibition of Rap1 abrogated integrin activation induced by inflammatory agonists (11–13). Murine T-cells expressing constitutively active Rap1 manifest enhanced integrin dependent cell adhesion (14). In platelets, Rap1 is rapidly activated by platelet agonists (15, 16). A knock-out of Rap1B (17) or of the Rap1GEF, RasGRP2 (18), resulted in impairment of αIIbβ3-dependent platelet aggregation, highlighting the importance of Rap1 in platelet aggregation in vivo. Thus, Rap1 GTPases play important roles in the activation of several integrins in multiple biological contexts.Several Rap1 effectors have been implicated in integrin activation (19–21). Rap1-GTP-interacting adaptor molecule (RIAM) is a Rap1 effector that is a member of the MRL (Mig-10/RIAM/Lamellipodin) family of adaptor proteins (20). RIAM contains Ras association (RA) and pleckstrin homology (PH) domains and proline-rich regions, which are defining features of the MRL protein family. In Jurkat cells, RIAM overexpression induces β1 and β2 integrin-mediated cell adhesion, and RIAM knockdown abolishes Rap1-dependent cell adhesion (20), indicating RIAM is a downstream regulator of Rap1-dependent signaling. RIAM regulates actin dynamics as RIAM expression induces cell spreading; conversely, its depletion reduces cellular F-actin content (20). Whereas RIAM is greatly enriched in hematopoietic cells, Lamellipodin (Lpd) is a paralogue present in fibroblasts and other somatic cells (22).Recently we used forward, reverse, and synthetic genetics to engineer and order an integrin activation pathway in Chinese hamster ovary cells expressing a prototype activable integrin, platelet αIIbβ3. We found that Rap1 induced formation of an “integrin activation complex” containing RIAM and talin (23). Here, we have established the mechanism whereby Ras GTPases cooperate with MRL family proteins, RIAM and Lpd, to regulate integrin activation. We find that MRL proteins function as scaffolds that connect the membrane targeting sequences in Ras GTPases to talin, thereby recruiting talin to integrins at the plasma membrane.     

Biohybrid Polymer-Antimicrobial Peptide Medium against Enterococcus faecalis

Antimicrobial peptides (AMPs) are conserved evolutionary components of the innate immune system that are being tested as alternatives to antibiotics. Slow release of AMPs using biodegradable polymers can be advantageous in maintaining high peptide levels for topical treatment, especially in the oral environment in which dosage retention is challenged by drug dilution with saliva flow and by drug inactivation by salivary enzymatic activity. Enterococcus faecalis is a multidrug resistant nosocomial pathogen and a persistent pathogen in root canal infections. In this study, four ultra-short lipopeptides (C16-KGGK, C16-KLLK, C16-KAAK and C16-KKK) and an amphipathic α-helical antimicrobial peptide (Amp-1D) were tested against E. faecalis. The antibacterial effect was determined against planktonic bacteria and bacteria grown in biofilm. Of the five tested AMPs, C16-KGGK was the most effective. Next C16-KGGK was formulated with one of two polymers poly (lactic acid co castor oil) (DLLA) or ricinoleic acid-based poly (ester-anhydride) P(SA-RA). Peptide-synthetic polymer conjugates, also referred to as biohybrid mediums were tested for antibacterial activity against E. faecalis grown in suspension and in biofilms. The new formulations exhibited strong and improved anti- E. faecalis activity.     

Identification and Characterization of Vancomycin-resistant Enterococcus species Frequently Isolated from Laboratory Mice

To determine the prevalence of drug resistant bacteria colonizing laboratory mice, we isolated and characterized vancomycin-resistant Enterococcus species (VRE) from commercially available mice. A total of 24 VRE isolates were obtained from 19 of 21 mouse strains supplied by 4 commercial breeding companies. Of these, 19 isolates of E. gallinarum and 5 isolates of E. casseliflavus possessing the vanC1 and vanC2/3 genes intrinsically, exhibited intermediate resistance to vancomycin respectively. In addition, these isolates also exhibited diverse resistant patterns to erythromycin, tetracycline, and ciprofloxacin, whereas the use of antibiotics had not been undertaken in mouse strains tested in this study. Although 6 virulence-associated genes (ace, asa, cylA, efaA, esp, and gelE) and secretion of gelatinase and hemolysin were not detected in all isolates, 23 of 24 isolates including the isolates of E. casselifalvus secreted ATP into culture supernatants. Since secretion of ATP by bacteria resident in the intestinal tract modulates the local immune responses, the prevalence of ATP-secreting VRE in mice therefore needs to be considered in animal experiments that alter the gut microflora by use of antibiotics.     

Citrate Metabolism by Enterococcus faecalis FAIR-E 229

Citrate metabolism by Enterococcus faecalis FAIR-E 229 was studied in various growth media containing citrate either in the presence of glucose or lactose or as the sole carbon source. In skim milk (130 mM lactose, 8 mM citrate), cometabolism of citrate and lactose was observed from the first stages of the growth phase. Lactose was stoichiometrically converted into lactate, while citrate was converted into acetate, formate, and ethanol. When de Man-Rogosa-Sharpe (MRS) broth containing lactose (28 mM) instead of glucose was used, E. faecalis FAIR-E 229 catabolized only the carbohydrate. Lactate was the major end product, and small amounts of ethanol were also detected. Increasing concentrations of citrate (10, 40, 70, and 100 mM) added to MRS broth enhanced both the maximum growth rate of E. faecalis FAIR-E 229 and glucose catabolism, although citrate itself was not catabolized. Glucose was converted stoichiometrically into lactate, while small amounts of ethanol were produced as well. Finally, when increasing initial concentrations of citrate (10, 40, 70, and 100 mM) were used as the sole carbon sources in MRS broth without glucose, the main end products were acetate and formate. Small amounts of lactate, ethanol, and acetoin were also detected. This work strongly supports the suggestion that enterococcal strains have the metabolic potential to metabolize citrate and therefore to actively contribute to the flavor development of fermented dairy products.     

Immunometabolism: Cellular Metabolism Turns Immune Regulator

Immune cells are highly dynamic in terms of their growth, proliferation, and effector functions as they respond to immunological challenges. Different immune cells can adopt distinct metabolic configurations that allow the cell to balance its requirements for energy, molecular biosynthesis, and longevity. However, in addition to facilitating immune cell responses, it is now becoming clear that cellular metabolism has direct roles in regulating immune cell function. This review article describes the distinct metabolic signatures of key immune cells, explains how these metabolic setups facilitate immune function, and discusses the emerging evidence that intracellular metabolism has an integral role in controlling immune responses.     

Antimicrobial Action of Oleanolic Acid on Listeria monocytogenes,Enterococcus faecium,and Enterococcus faecalis

This study investigated the antimicrobial action of oleanolic acid against Listeria monocytogenes, Enterococcus faecium, and Enterococcus faecalis. To determine the cytotoxicity of oleanolic acid, HEp-2 cells were incubated with oleanolic acid at 37^oC. MICs (minimal inhibition concentrations) for L. monocytogenes, E. faecium, and E. faecalis were determined using two-fold microdilutions of oleanolic acid, and bacterial cell viability was then assessed by exposing the bacteria to oleanolic acid at 2 × MIC. To investigate the mode of antimicrobial action of oleanolic acid, we measured leakage of compounds absorbing at 280 nm, along with propidium iodide uptake. Scanning electron microscope (SEM) images were also analysed. The viability of HEp-2 cells decreased (P < 0.05) at oleanolic acid concentrations greater than 128 μg mL^-1. The MICs were 16-32 μg mL^-1 for L. monocytogenes and 32-64 μg mL^-1 for E. faecium and E. faecalis, and bacterial cell viability decreased (P < 0.05) about 3-4 log CFU mL^-1 after exposure to 2 × MIC of oleanolic acid. Leakage of 280 nm absorbing materials and propidium iodide uptake was higher in oleanolic acid –treated cells than in the control. The cell membrane was damaged in oleanolic acid-treated cells, but the control group had intact cell membrane in SEM images. The results indicate that oleanolic acid can kill L. monocytogenes, E. faecium, and E. faecalis by destroying the bacterial cell membrane.     

Salinomycin Activates AMP-Activated Protein Kinase-Dependent Autophagy in Cultured Osteoblastoma Cells: A Negative Regulator against Cell Apoptosis

Background

The malignant osteoblastoma has poor prognosis, thus the search for novel and more efficient chemo-agents against this disease is urgent. Salinomycin induces broad anti-cancer effects both in vivo and in vitro, however, its role in osteoblastoma is still not clear.

Key Findings

Salinomycin induced both apoptosis and autophagy in cultured U2OS and MG-63 osteoblastoma cells. Inhibition of autophagy by 3-methyladenine (3-MA), or by RNA interference (RNAi) of light chain 3B (LC3B), enhanced salinomycin-induced cytotoxicity and apoptosis. Salinomycin induced a profound AMP-activated protein kinase (AMPK) activation, which was required for autophagy induction. AMPK inhibition by compound C, or by AMPKα RNAi prevented salinomycin-induced autophagy activation, while facilitating cancer cell death and apoptosis. On the other hand, the AMPK agonist AICAR promoted autophagy activation in U2OS cells. Salinomycin-induced AMPK activation was dependent on reactive oxygen species (ROS) production in osteoblastoma cells. Antioxidant n-acetyl cysteine (NAC) significantly inhibited salinomycin-induced AMPK activation and autophagy induction.

Conclusions

Salinomycin activates AMPK-dependent autophagy in osteoblastoma cells, which serves as a negative regulator against cell apoptosis. AMPK-autophagy inhibition might be a novel strategy to sensitize salinomycin’s effect in cancer cells.     

Abscisic Acid Activates the Murine Microglial Cell Line N9 through the Second Messenger Cyclic ADP-ribose

Abscisic acid (ABA) is a phytohormone regulating important functions in higher plants, notably responses to abiotic stress. Recently, chemical or physical stimulation of human granulocytes was shown to induce production and release of endogenous ABA, which activates specific cell functions. Here we provide evidence that ABA stimulates several functional activities of the murine microglial cell line N9 (NO and tumor necrosis factor-α production, cell migration) through the second messenger cyclic ADP-ribose and an increase of intracellular calcium. ABA production and release occur in N9 cells stimulated with bacterial lipopolysaccharide, phorbol myristate acetate, the chemoattractant peptide f-MLP, or β-amyloid, the primary plaque component in Alzheimer disease. Finally, ABA priming stimulates N9 cell migration toward β-amyloid. These results indicate that ABA is a pro-inflammatory hormone inducing autocrine microglial activation, potentially representing a new target for anti-inflammatory therapies aimed at limiting microglia-induced tissue damage in the central nervous system.Microglial cells are the monocyte/macrophage equivalent of the central nervous system and represent the first line of defense in the brain, by removing infectious agents and damaged cells (1). Microglia can also release a variety of trophic factors and cytokines able to regulate the communication between neuronal and other glial cells and can contribute to tissue repair and neuroprotection (2–4). Pathologic microglial activation, however, confers neurotoxic properties to these cells, thereby causing neuronal degeneration (5). Excessive activation of microglia, under conditions of chronic inflammation, can contribute to the pathogenesis of neurodegenerative diseases, such as multiple sclerosis and Alzheimer and Parkinson diseases, by producing and releasing a number of potentially cytotoxic substances, including pro-inflammatory cytokines and NO (4, 6–8). Therefore, identification of the molecular mechanisms underlying microglial activation might lead to the development of new anti-inflammatory drugs for the treatment of these diseases.Abscisic acid (ABA)² is a plant hormone regulating important biological functions in higher plants, including response to abiotic stress, control of stomatal closure, regulation of seed dormancy, and germination (9). Recently, ABA was shown to behave as an endogenous pro-inflammatory hormone in human granulocytes (10), stimulating several functional activities of these cells (migration, phagocytosis, reactive oxygen species, and NO production) through a signaling cascade that involves a protein kinase A-mediated ADP-ribosyl cyclase phosphorylation and consequent overproduction of the universal Ca²⁺ mobilizer cyclic ADP-ribose (cADPR) (11). This mechanism leads to an increase of the intracellular Ca²⁺ concentration, which is ultimately responsible for granulocyte activation (10).The facts that microglial cells play a defensive role in the central nervous system similar to that of granulocytes in other tissues and that cADPR has been described as the second messenger involved in the activation of microglia induced by lipopolysaccharide (LPS) (12) prompted us to investigate the effect of ABA in these cells.Indeed, exogenous ABA, at concentrations ranging from 250 nm to 20 μm, elicits functional activation of murine N9 cells, stimulating TNF-α release and cell migration through activation of the ADP-ribosyl cyclase CD38 and overproduction of cADPR. Moreover, N9 cells produce and release ABA when stimulated with LPS, amyloid β-peptide (βA), phorbol myristate acetate (PMA), or the chemoattractant peptide f-MLP. These results indicate that ABA behaves as an endogenous, pro-inflammatory hormone in murine microglia and provide a new target for future investigations into the role of this hormone in inflammatory and degenerative diseases of the central nervous system accompanied by microglial activation.