Legionella pneumophila exploits PI(4)P to anchor secreted effector proteins to the replicative vacuole

The causative agent of Legionnaires' disease, Legionella pneumophila, employs the intracellular multiplication (Icm)/defective organelle trafficking (Dot) type IV secretion system (T4SS) to upregulate phagocytosis and to establish a replicative vacuole in amoebae and macrophages. Legionella-containing vacuoles (LCVs) do not fuse with endosomes but recruit early secretory vesicles. Here we analyze the role of host cell phosphoinositide (PI) metabolism during uptake and intracellular replication of L. pneumophila. Genetic and pharmacological evidence suggests that class I phosphatidylinositol(3) kinases (PI3Ks) are dispensable for phagocytosis of wild-type L. pneumophila but inhibit intracellular replication of the bacteria and participate in the modulation of the LCV. Uptake and degradation of an icmT mutant strain lacking a functional Icm/Dot transporter was promoted by PI3Ks. We identified Icm/Dot-secreted proteins which specifically bind to phosphatidylinositol(4) phosphate (PI(4)P) in vitro and preferentially localize to LCVs in the absence of functional PI3Ks. PI(4)P was found to be present on LCVs using as a probe either an antibody against PI(4)P or the PH domain of the PI(4)P-binding protein FAPP1 (phosphatidylinositol(4) phosphate adaptor protein-1). Moreover, the presence of PI(4)P on LCVs required a functional Icm/Dot T4SS. Our results indicate that L. pneumophila modulates host cell PI metabolism and exploits the Golgi lipid second messenger PI(4)P to anchor secreted effector proteins to the LCV.     

Cutting edge: A critical functional role for IL-23 in psoriasis

Interleukin-23 is a key cytokine involved in the generation of Th17 effector cells. Clinical efficacy of an anti-p40 mAb blocking both IL-12 and IL-23 and disease association with single nucleotide polymorphisms in the IL23R gene raise the question of a functional role of IL-23 in psoriasis. In this study, we provide a comprehensive analysis of IL-23 and its receptor in psoriasis and demonstrate its functional importance in a disease-relevant model system. The expression of IL-23 and its receptor was increased in the tissues of patients with psoriasis. Injection of a mAb specifically neutralizing human IL-23 showed IL-23-dependent inhibition of psoriasis development comparable to the use of anti-TNF blockers in a clinically relevant xenotransplant mouse model of psoriasis. Together, our results identify a critical functional role for IL-23 in psoriasis and provide the rationale for new treatment strategies in chronic epithelial inflammatory disorders.     

The Legionella pneumophila phosphatidylinositol-4 phosphate-binding type IV substrate SidC recruits endoplasmic reticulum vesicles to a replication-permissive vacuole

Legionella pneumophila, the causative agent of Legionnaires' disease, uses the intracellular multiplication/defective organelle trafficking (Icm/Dot) type IV secretion system to establish within amoebae and macrophages an endoplasmic reticulum (ER)-derived replication-permissive compartment, the Legionella-containing vacuole (LCV). The Icm/Dot substrate SidC and its paralogue SdcA anchor to LCVs via phosphatidylinositol-4 phosphate [PtdIns(4)P]. Here we identify the unique 20 kDa PtdIns(4)P-binding domain of SidC, which upon heterologous expression in Dictyostelium binds to LCVs and thus is useful as a PtdIns(4)P-specific probe. LCVs harbouring L. pneumophilaDeltasidC-sdcA mutant bacteria recruit ER and ER-derived vesicles less efficiently and carry endosomal but not lysosomal markers. The phenotypes are complemented by supplying sidC on a plasmid. L. pneumophilaDeltasidC-sdcA grows at wild-type rate in calnexin-negative LCVs, suggesting that communication with the ER is dispensable for establishing a replicative compartment. The amount of SidC and calnexin is directly proportional on isolated LCVs, and in a cell-free system, the recruitment of calnexin-positive vesicles to LCVs harbouring DeltasidC-sdcA mutant bacteria is impaired. Beads coated with purified SidC or its 70 kDa N-terminal fragment recruit ER vesicles in Dictyostelium and macrophage lysates. Our results establish SidC as an L. pneumophila effector protein, which anchors to PtdIns(4)P on LCVs and recruits ER vesicles to a replication-permissive vacuole.     

Decomposition and stabilisation of Norway spruce needle-derived material in Alpine soils using a <Superscript>13</Superscript>C-labelling approach in the field

Only scarce information is available on how organic C is incorporated into the soil during the decay and how (micro) climate influences this process. Therefore, we investigated the effect of exposure and elevation on the organic litter decomposition and C-stabilisation in acidic soils of an Alpine environment. An experiment with artificially ¹³C labelled Norway spruce needles was carried out at north- and south-exposed sites between 1200 and 2400 m a.s.l. in the Italian Alps using mesocosms. After 1 year, the ¹³C recoveries of the bulk soil were 18.6% at the north-facing slopes and 31.5% at the south-facing slopes. A density fractionation into a light (LF; ≤1.6 g cm^?3) and a heavy fraction (HF; >1.6 g cm^?3) of the soil helped to identify how the applied substrate was stabilised. At the northern slope, 10.5% of the substrate was recovered in the LF and 8.1% in the HF and at the south-facing slope 22.8% in the LF and 8.1% in the HF. The overall ¹³C recovery was higher at the south-facing sites due to restricted water availability. Although the climate is humid in the whole area, soil moisture availability becomes more important at south-facing sites due to higher evapotranspiration. However, at sites >1700 m a.s.l, the situation changed, as the northern slope had higher recovery rates. At such altitudes, temperature effects are more dominant. This highlights the importance of locally strongly varying edaphic factors when investigating the carbon cycle.     

Environmental predators as models for bacterial pathogenesis

Environmental bacteria are constantly threatened by bacterivorous predators such as free-living protozoa and nematodes. In the course of their coevolution with environmental predators, some bacteria developed sophisticated defence mechanisms, including the secretion of toxins, or the capacity to avoid lysosomal killing and to replicate intracellularly within protozoa. To analyse the interactions with bacterial pathogens on a molecular, cellular or organismic level, protozoa and other non-mammalian hosts are increasingly used. These include amoebae, as well as genetically tractable hosts, such as the social amoeba Dictyostelium discoideum, the nematode Caenorhabditis elegans and the fruit fly Drosophila melanogaster. Using these hosts, the virulence mechanisms of opportunistic pathogenic bacteria such as Legionella, Mycobacterium, Pseudomonas or Vibrio were found to be not only relevant for the interactions of the bacteria with protozoa, nematodes and insect phagocytes, but also with mammalian hosts including humans. Thus, non-mammalian model hosts provide valuable insight into the pathogenesis of environmental bacteria.     

Pathogen trafficking pathways and host phosphoinositide metabolism

Phosphoinositide (PI) glycerolipids are key regulators of eukaryotic signal transduction, cytoskeleton architecture and membrane dynamics. The host cell PI metabolism is targeted by intracellular bacterial pathogens, which evolved intricate strategies to modulate uptake processes and vesicle trafficking pathways. Upon entering eukaryotic host cells, pathogenic bacteria replicate in distinct vacuoles or in the host cytoplasm. Vacuolar pathogens manipulate PI levels to mimic or modify membranes of subcellular compartments and thereby establish their replicative niche. Legionella pneumophila , Brucella abortus , Mycobacterium tuberculosis and Salmonella enterica translocate effector proteins into the host cell, some of which anchor to the vacuolar membrane via PIs or enzymatically turnover PIs. Cytoplasmic pathogens target PI metabolism at the plasma membrane, thus modulating their uptake and antiapoptotic signalling pathways. Employing this strategy, Shigella flexneri directly injects a PI-modifying effector protein, while Listeria monocytogenes exploits PI metabolism indirectly by binding to transmembrane receptors. Thus, regardless of the intracellular lifestyle of the pathogen, PI metabolism is critically involved in the interactions with host cells.     

Adjuvant trials of aromatase inhibitors: determining the future landscape of adjuvant endocrine therapy

This review will discuss the role of aromatase inhibitors (AIs) in the adjuvant setting, and will summarize major strategies behind individual adjuvant trials using aromatase inhibitors. Studies with the third generation AIs including anastrozole, letrozole and exemestane, have shown better outcome and improved therapeutic ratio over second line hormonal approaches (i.e. progestins or aminoglutethimide) and, more recently, over tamoxifen also. These promising results have led recently to testing of AIs in the adjuvant setting for postmenopausal patients. Most trials now in progress are evaluating the role of new AIs versus tamoxifen (T) given×5 years, which in most institutions is currently the standard hormonal adjuvant therapy for breast cancer. Three adjuvant approaches are being tested. First is the use of AI+T×5 years in combination versus each agent alone, as reflected in the recently completed ATAC trial. Second is a sequential approach T first×2–3 years followed by AIs×2–3 years, or the other way round; and third, T×5 years followed by AIs for additional 5 years (i.e. total duration of adjuvant hormones of 10 years). Many patients in the above trials will survive their first cancer. Hence, the non-oncological outcomes known to be affected by hormones are of rising importance. Therefore, the assessment of lipids as surrogates for cardiovascular morbidity, and of bone mineral status, as a marker for osteoporosis/bone fractures, is an important component of these trials. Also discussed in this review are proposals for future studies of AIs with focus on hormone resistance, such as early alteration of multiple hormonal agents or their intermittent use, the impact of the new generation of SERMs or ‘pure’ antiestrogens on activity of AIs, and the rising importance of AIs interacting with biologicals, cytokines or hormone modulators.     

Alpha1beta1 integrin is crucial for accumulation of epidermal T cells and the development of psoriasis

Psoriasis is a common T cell-mediated autoimmune inflammatory disease. We show that blocking the interaction of alpha1beta1 integrin (VLA-1) with collagen prevented accumulation of epidermal T cells and immunopathology of psoriasis. Alpha1beta1 integrin, a major collagen-binding surface receptor, was exclusively expressed by epidermal but not dermal T cells. Alpha1beta1-positive T cells showed characteristic surface markers of effector memory cells and contained high levels of interferon-gamma but not interleukin-4. Blockade of alpha1beta1 inhibited migration of T cells into the epidermis in a clinically relevant xenotransplantation model. This was paralleled by a complete inhibition of psoriasis development, comparable to that caused by tumor necrosis factor-alpha blockers. These results define a crucial role for alpha1beta1 in controlling the accumulation of epidermal type 1 polarized effector memory T cells in a common human immunopathology and provide the basis for new strategies in psoriasis treatment focusing on T cell-extracellular matrix interactions.