Reactive Oxygen Species Localization Programs Inflammation to Clear Microbes of Different Size.

How the number of immune cells recruited to sites of infection is determined and adjusted to differences in the cellular stoichiometry between host and pathogen is unknown. Here, we have uncovered a role for reactive oxygen species (ROS) as sensors of microbe size. By sensing the differential localization of ROS generated in response to microbes of different size, neutrophils tuned their interleukin (IL)-1ß expression via the selective oxidation of NF-κB, in order to implement distinct inflammatory programs. Small microbes triggered ROS intracellularly, suppressing IL-1ß expression to limit neutrophil recruitment as each phagocyte eliminated numerous pathogens. In contrast, large microbes triggered ROS extracellularly, amplifying IL-1ß expression to recruit numerous neutrophils forming cooperative clusters. Defects in ROS-mediated microbe size sensing resulted in large neutrophil infiltrates and clusters in response to small microbes that contribute to inflammatory disease. These findings highlight the impact of ROS localization on signal transduction.

Leucine-rich repeat kinase 2 at a glance.

Leucine-rich repeat kinase 2 (LRRK2) is a multidomain scaffolding protein with dual guanosine triphosphatase (GTPase) and kinase enzymatic activities, providing this protein with the capacity to regulate a multitude of signalling pathways and act as a key mediator of diverse cellular processes. Much of the interest in LRRK2 derives from mutations in the LRRK2 gene being the most common genetic cause of Parkinson's disease, and from the association of the LRRK2 locus with a number of other human diseases, including inflammatory bowel disease. Therefore, the LRRK2 research field has focused on the link between LRRK2 and pathology, with the aim of uncovering the underlying mechanisms and, ultimately, finding novel therapies and treatments to combat them. From the biochemical and cellular functions of LRRK2, to its relevance to distinct disease mechanisms, this Cell Science at a Glance article and the accompanying poster deliver a snapshot of our current understanding of LRRK2 function, dysfunction and links to disease.

LRRK2 activation controls the repair of damaged endomembranes in macrophages.

Cells respond to endolysosome damage by either repairing the damage or targeting damaged endolysosomes for degradation via lysophagy. However, the signals regulating the decision for repair or lysophagy are poorly characterised. Here, we show that the Parkinson's disease (PD)-related kinase LRRK2 is activated in macrophages by pathogen- or sterile-induced endomembrane damage. LRRK2 recruits the Rab GTPase Rab8A to damaged endolysosomes as well as the ESCRT-III component CHMP4B, thereby favouring ESCRT-mediated repair. Conversely, in the absence of LRRK2 and Rab8A, damaged endolysosomes are targeted to lysophagy. These observations are recapitulated in macrophages from PD patients where pathogenic LRRK2 gain-of-function mutations result in the accumulation of endolysosomes which are positive for the membrane damage marker Galectin-3. Altogether, this work indicates that LRRK2 regulates endolysosomal homeostasis by controlling the balance between membrane repair and organelle replacement, uncovering an unexpected function for LRRK2, and providing a new link between membrane damage and PD.

Transmembrane redox control and proteolysis of PdeC, a novel type of c-di-GMP phosphodiesterase.

The nucleotide second messenger c-di-GMP nearly ubiquitously promotes bacterial biofilm formation, with enzymes that synthesize and degrade c-di-GMP being controlled by diverse N-terminal sensor domains. Here, we describe a novel class of widely occurring c-di-GMP phosphodiesterases (PDE) that feature a periplasmic "CSS domain" with two highly conserved cysteines that is flanked by two transmembrane regions (TM1 and TM2) and followed by a cytoplasmic EAL domain with PDE activity. Using PdeC, one of the five CSS domain PDEs of Escherichia coli K-12, we show that DsbA/DsbB-promoted disulfide bond formation in the CSS domain reduces PDE activity. By contrast, the free thiol form is enzymatically highly active, with the TM2 region promoting dimerization. Moreover, this form is processed by periplasmic proteases DegP and DegQ, yielding a highly active TM2 + EAL fragment that is slowly removed by further proteolysis. Similar redox control and proteolysis was also observed for a second CSS domain PDE, PdeB. At the physiological level, CSS domain PDEs modulate production and supracellular architecture of extracellular matrix polymers in the deeper layers of mature E. coli biofilms.

LRRK2 is a negative regulator of Mycobacterium tuberculosis phagosome maturation in macrophages.

Mutations in the leucine-rich repeat kinase 2 (LRRK2) are associated with Parkinson's disease, chronic inflammation and mycobacterial infections. Although there is evidence supporting the idea that LRRK2 has an immune function, the cellular function of this kinase is still largely unknown. By using genetic, pharmacological and proteomics approaches, we show that LRRK2 kinase activity negatively regulates phagosome maturation via the recruitment of the Class III phosphatidylinositol-3 kinase complex and Rubicon to the phagosome in macrophages. Moreover, inhibition of LRRK2 kinase activity in mouse and human macrophages enhanced Mycobacterium tuberculosis phagosome maturation and mycobacterial control independently of autophagy. In vivo, LRRK2 deficiency in mice resulted in a significant decrease in M. tuberculosis burdens early during the infection. Collectively, our findings provide a molecular mechanism explaining genetic evidence linking LRRK2 to mycobacterial diseases and establish an LRRK2-dependent cellular pathway that controls M. tuberculosis replication by regulating phagosome maturation.

The emerging role of LRRK2 in tauopathies.

Parkinson's disease (PD) is conventionally described as an α-synuclein aggregation disorder, defined by Lewy bodies and neurites, and mutations in leucine-rich repeat kinase 2 (LRRK2) are the most common autosomal dominant cause of PD. However, LRRK2 mutations may be associated with diverse pathologies in patients with Parkinson's syndrome including tau pathology resembling progressive supranuclear palsy (PSP). The recent discovery that variation at the LRRK2 locus is associated with the progression of PSP highlights the potential importance of LRRK2 in tauopathies. Here, we review the emerging evidence and discuss the potential impact of LRRK2 dysfunction on tau aggregation, lysosomal function, and endocytosis and exocytosis.

From structure to ætiology: a new window on the biology of leucine-rich repeat kinase 2 and Parkinson's disease.

Since the discovery of mutations in leucine-rich repeat kinase 2 (LRRK2) as an underlying genetic cause for the development of Parkinson's disease (PD) in 2004 (Neuron 44, 601-607; Neuron 44, 595-600), and subsequent efforts to develop LRRK2 kinase inhibitors as a therapy for Parkinson's (Expert Opin. Ther. Targets 21, 751-753), elucidating the atomic resolution structure of LRRK2 has been a major goal of research into this protein. At over 250 kDa, the large size and complicated domain organisation of LRRK2 has made this a highly challenging target for structural biologists, however, a number of recent studies using both in vitro and in situ approaches (Nature 588, 344-349; Cell 182, 1508-1518.e1516; Cell 184, 3519-3527.e3510) have provided important new insights into LRRK2 structure and the complexes formed by this protein.

Calcineurin Orchestrates Lateral Transfer of Aspergillus fumigatus during Macrophage Cell Death.

RATIONALE: Pulmonary aspergillosis is a lethal mold infection in the immunocompromised host. Understanding initial control of infection and how this is altered in the immunocompromised host are key goals for comprehension of the pathogenesis of pulmonary aspergillosis. OBJECTIVES: To characterize the outcome of human macrophage infection with Aspergillus fumigatus and how this is altered in transplant recipients on calcineurin inhibitor immunosuppressants. METHODS: We defined the outcome of human macrophage infection with A. fumigatus, as well as the impact of calcineurin inhibitors, through a combination of single-cell fluorescence imaging, transcriptomics, proteomics, and in vivo studies. MEASUREMENTS AND MAIN RESULTS: Macrophage phagocytosis of A. fumigatus enabled control of 90% of fungal germination. However, fungal germination in the late phagosome led to macrophage necrosis. During programmed necroptosis, we observed frequent cell-cell transfer of A. fumigatus between macrophages, which assists subsequent control of germination in recipient macrophages. Lateral transfer occurred through actin-dependent exocytosis of the late endosome in a vasodilator-stimulated phosphoprotein envelope. Its relevance to the control of fungal germination was also shown by direct visualization in our zebrafish aspergillosis model in vivo. The calcineurin inhibitor FK506 (tacrolimus) reduced cell death and lateral transfer in vitro by 50%. This resulted in uncontrolled fungal germination in macrophages and also resulted in hyphal escape. CONCLUSIONS: These observations identify programmed, necrosis-dependent lateral transfer of A. fumigatus between macrophages as an important host strategy for controlling fungal germination. This process is critically dependent on calcineurin. Our studies provide fundamental insights into the pathogenesis of pulmonary aspergillosis in the immunocompromised host.

The pH-responsive PacC transcription factor of Aspergillus fumigatus governs epithelial entry and tissue invasion during pulmonary aspergillosis.

Destruction of the pulmonary epithelium is a major feature of lung diseases caused by the mould pathogen Aspergillus fumigatus. Although it is widely postulated that tissue invasion is governed by fungal proteases, A. fumigatus mutants lacking individual or multiple enzymes remain fully invasive, suggesting a concomitant requirement for other pathogenic activities during host invasion. In this study we discovered, and exploited, a novel, tissue non-invasive, phenotype in A. fumigatus mutants lacking the pH-responsive transcription factor PacC. Our study revealed a novel mode of epithelial entry, occurring in a cell wall-dependent manner prior to protease production, and via the Dectin-1 ß-glucan receptor. ΔpacC mutants are defective in both contact-mediated epithelial entry and protease expression, and significantly attenuated for pathogenicity in leukopenic mice. We combined murine infection modelling, in vivo transcriptomics, and in vitro infections of human alveolar epithelia, to delineate two major, and sequentially acting, PacC-dependent processes impacting epithelial integrity in vitro and tissue invasion in the whole animal. We demonstrate that A. fumigatus spores and germlings are internalised by epithelial cells in a contact-, actin-, cell wall- and Dectin-1 dependent manner and ΔpacC mutants, which aberrantly remodel the cell wall during germinative growth, are unable to gain entry into epithelial cells, both in vitro and in vivo. We further show that PacC acts as a global transcriptional regulator of secreted molecules during growth in the leukopenic mammalian lung, and profile the full cohort of secreted gene products expressed during invasive infection. Our study reveals a combinatorial mode of tissue entry dependent upon sequential, and mechanistically distinct, perturbations of the pulmonary epithelium and demonstrates, for the first time a protective role for Dectin-1 blockade in epithelial defences. Infecting ΔpacC mutants are hypersensitive to cell wall-active antifungal agents highlighting the value of PacC signalling as a target for antifungal therapy.

Common genetic risk for Parkinson's disease and dysfunction of the endo-lysosomal system.

Parkinson's disease is a progressive neurological disorder, characterized by prominent movement dysfunction. The past two decades have seen a rapid expansion of our understanding of the genetic basis of Parkinson's, initially through the identification of monogenic forms and, more recently, through genome-wide association studies identifying common risk variants. Intriguingly, a number of cellular pathways have emerged from these analysis as playing central roles in the aetiopathogenesis of Parkinson's. In this review, the impact of data deriving from genome-wide analyses for Parkinson's upon our functional understanding of the disease will be examined, with a particular focus on examples of endo-lysosomal and mitochondrial dysfunction. The challenges of moving from a genetic to a functional understanding of common risk variants for Parkinson's will be discussed, with a final consideration of the current state of the genetic architecture of the disorder. This article is part of a discussion meeting issue 'Understanding the endo-lysosomal network in neurodegeneration'.

A Novel Variant in the WRN Gene Detected in a Case of Early-Onset Severe Insulin Resistance Displaying Some but Not All Hallmarks of Progeroid Werner Syndrome.

OBJECTIVE: Determining the cause of severe insulin resistance and early-onset diabetes in the case of a young woman in which a wide range of differential diagnoses did not apply. RESEARCH DESIGN AND METHODS: Diagnostic workup including medical history, physical examination, specialist consultations, imaging methods, laboratory assessment, and genetic testing carried out by next-generation panel sequencing. RESULTS: After ruling out several differential diagnoses, genetic testing revealed a previously unknown homozygous variant within the canonical splice site of intron 4 in the WRN gene classified as pathogenic. Thus, although not all cardinal clinical criteria according to existing guidelines had been met, the phenotype of our patient was attributed to Werner syndrome (WS), an autosomal-recessive inherited progeroid syndrome. CONCLUSIONS: WS, although rare, must be considered as a differential diagnosis in cases of severe insulin resistance. Moreover, recognized clinical criteria of WS may not lead to diagnosis in all cases.

Knowledge Mapping for Climate Change and Food- and Waterborne Diseases.

The authors extracted from the PubMed and ScienceDirect bibliographic databases all articles published between 1998 and 2009 that were relevant to climate change and food- and waterborne diseases. Any material within each article that provided information about a relevant pathogen and its relationship with climate and climate change was summarized as a key fact, entered into a relational knowledge base, and tagged with the terminology (predefined terms) used in the field. These terms were organized, quantified, and mapped according to predefined hierarchical categories. For noncholera Vibrio sp. and Cryptosporidium sp., data on climatic and environmental influences (52% and 49% of the total number of key facts, respectively) pertained to specific weather phenomena (as opposed to climate change phenomena) and environmental determinants, whereas information on the potential effects of food-related determinants that might be related to climate or climate change were virtually absent. This proportion was lower for the other pathogens studied (Campylobacter sp. 40%, Salmonella sp. 27%, Norovirus 25%, Listeria sp. 8%), but they all displayed a distinct concentration of information on general food-and water-related determinants or effects, albeit with little detail. Almost no information was available concerning the potential effects of changes in climatic variables on the pathogens evaluated, such as changes in air or water temperature, precipitation, humidity, UV radiation, wind, cloud coverage, sunshine hours, or seasonality. Frequency profiles revealed an abundance of data on weather and food-specific determinants, but also exposed extensive data deficiencies, particularly with regard to the potential effects of climate change on the pathogens evaluated. A reprioritization of public health research is warranted to ensure that funding is dedicated to explicitly studying the effects of changes in climate variables on food- and waterborne diseases.

Climate Change Impact Assessment of Food- and Waterborne Diseases.

The PubMed and ScienceDirect bibliographic databases were searched for the period of 1998-2009 to evaluate the impact of climatic and environmental determinants on food- and waterborne diseases. The authors assessed 1,642 short and concise sentences (key facts), which were extracted from 722 relevant articles and stored in a climate change knowledge base. Key facts pertaining to temperature, precipitation, water, and food for 6 selected pathogens were scrutinized, evaluated, and compiled according to exposure pathways. These key facts (corresponding to approximately 50,000 words) were mapped to 275 terminology terms identified in the literature, which generated 6,341 connections. These relationships were plotted on semantic network maps to examine the interconnections between variables. The risk of campylobacteriosis is associated with mean weekly temperatures, although this link is shown more strongly in the literature relating to salmonellosis. Irregular and severe rain events are associated with Cryptosporidium sp. outbreaks, while noncholera Vibrio sp. displays increased growth rates in coastal waters during hot summers. In contrast, for Norovirus and Listeria sp. the association with climatic variables was relatively weak, but much stronger for food determinants. Electronic data mining to assess the impact of climate change on food- and waterborne diseases assured a methodical appraisal of the field. This climate change knowledge base can support national climate change vulnerability, impact, and adaptation assessments and facilitate the management of future threats from infectious diseases. In the light of diminishing resources for public health this approach can help balance different climate change adaptation options.

Mosquito nets in a rural area of Western Kenya: ownership, use and quality.

BACKGROUND: Insecticide-treated nets (ITNs) are regarded as one of the most effective strategies to prevent malaria in Africa. This study analyses the use and quality of nets owned by households in an area of high net coverage. METHODS: A structured questionnaire on ownership and use of nets was administered to the households of individuals sampled from a local health centre in south Kisii district, Kenya. A physical inspection of all the nets in the households was done and their conditions recorded on spot check forms designed for that purpose. RESULTS: Of the 670 households surveyed, 95% owned at least one net. Only 59% of household residents slept under a net during the night prior to the survey. 77% of those who slept under a net used an insecticide-treated net (ITN) or long-lasting insecticide-treated nets (LLIN). Out of 1,627 nets in the survey households, 40% were deemed to be of poor quality because of holes. Compared to other age groups, children aged 5-14 years were most likely to have slept under nets of poor quality (odds ratio 1.41; p = 0.007). CONCLUSIONS: Although net ownership was high following increased delivery of ITNs, continuous promotion of effective maintenance and routine use is needed and efforts to replace damaged nets must be implemented.

Local c-di-GMP Signaling in the Control of Synthesis of the E. coli Biofilm Exopolysaccharide pEtN-Cellulose.

In many bacteria, the biofilm-promoting second messenger c-di-GMP is produced and degraded by multiple diguanylate cyclases (DGC) and phosphodiesterases (PDE), respectively. High target specificity of some of these enzymes has led to theoretical concepts of "local" c-di-GMP signaling. In Escherichia coli K-12, which has 12 DGCs and 13 PDEs, a single DGC, DgcC, is specifically required for the biosynthesis of the biofilm exopolysaccharide pEtN-cellulose without affecting the cellular c-di-GMP pool, but the mechanistic basis of this target specificity has remained obscure. DGC activity of membrane-associated DgcC, which is demonstrated in vitro in nanodiscs, is shown to be necessary and sufficient to specifically activate cellulose biosynthesis in vivo. DgcC and a particular PDE, PdeK (encoded right next to the cellulose operon), directly interact with cellulose synthase subunit BcsB and with each other, thus establishing physical proximity between cellulose synthase and a local source and sink of c-di-GMP. This arrangement provides a localized, yet open source of c-di-GMP right next to cellulose synthase subunit BcsA, which needs allosteric activation by c-di-GMP. Through mathematical modeling and simulation, we demonstrate that BcsA binding from the low cytosolic c-di-GMP pool in E. coli is negligible, whereas a single c-di-GMP molecule that is produced and released in direct proximity to cellulose synthase increases the probability of c-di-GMP binding to BcsA several hundred-fold. This local c-di-GMP signaling could provide a blueprint for target-specific second messenger signaling also in other bacteria where multiple second messenger producing and degrading enzymes exist.

LRRK2 in Infection: Friend or Foe?

In the field of Parkinson's disease (PD) research, leucine-rich repeat kinase 2 (LRRK2) remains one of the most enigmatic kinases. LRRK2 pathogenic mutations result in increased kinase activity, making LRRK2 an attractive therapeutic target for PD. For over 10 years, the identification of a bona fide substrate and a physiological function for LRRK2 has been elusive, and only recently, Rab GTPases were identified as substrates for LRRK2 kinase activity. Additionally, LRRK2 gene expression data shows that LRRK2 is expressed at low levels in neurons and at high levels in cells of the immune system. These findings shifted research efforts from neuronal toxicity of LRRK2 mutations to the function of LRRK2 in both vesicle trafficking and the immune system, which has resulted in novel insights into the role of LRRK2 during infection and immunity. In this Perspective, we summarize the latest findings highlighting LRRK2 as a central regulator of vesicular trafficking, infection, immunity, and inflammation, speculating how LRRK2 function could influence neuronal pathology in PD.

Assessment of a groundwater contamination with vinyl chloride (VC) and precursor volatile organic compounds (VOC) by use of a geographical information system (GIS).

Regarding the health effects of volatile organic compounds (VOC) and their decomposition products (particularly vinyl chloride (VC)) under chronic low-dose exposure, VOC groundwater contaminations are seen to be an ongoing public health issue. This article presents results of a long-term investigation surveying VOC and VC groundwater contamination upstream of a large groundwater works in Cologne, Germany. For 10 years a contaminated aquifer has been monitored for different VOC and for VC. In total, 255 samples have been taken to assess both the 3-dimensional distribution and the temporal dynamics of the contaminants. VOC and VC precursor substances have been measured by means of pentane-liquid-liquid-extraction, GC and ECD, VC by means of derivatisation to 1,2 dibromochloroethane, GC, ECD, and by purge and trap technique and GC-MS-coupling. For spatial analysis all test results and additional hydrogeological attribute data have been transferred to a GIS. The spatial VOC distribution has been assessed by use of kriging interpolation indicating a decrease of the initial contaminants in time. A cluster analysis allowed to distinguish several independent contaminations within the large contamination area. The VC contamination was increasing. Anaerobic microbial dechlorination of VOC and subsequent VC accumulation were seen to be as credible from several indications (VC presence, downstream change of tetra/trichloroethylene-ratio and anaerobic conditions in the aquifer, high Fe(2+)- and Mn(2+)-concentrations). There was no statistically significant vertical differentiation of VOC and VC concentrations. The VOC load within the different water protection zones of the waterworks could be assessed.

A comparison of efficiencies of microbiological pollution removal in six sewage treatment plants with different treatment systems.

Six sewage treatment plants (STP) were investigated over a 12-month period in order to measure the microbiological load of untreated municipal wastewater and to evaluate the removal efficiencies of different treatment systems. The STP investigated can be classified into three categories: bigger plants with tertiary treatment, smaller plants with enhanced secondary treatment, and very small compact facilities. The plants studied had a considerable quantitative impact on the hydrology of the catchment area; consequently, it was anticipated that the microbiological load of the effluent would also be significant. Eighty samples were taken from the influent and effluent of the STP, regardless of weather conditions, and several bacterial and two parasitological parameters were analysed. The average microbiological reduction of each STP was dependent on its capacity and treatment procedures and varied between 1.9 and 3.5log10. Small compact facilities had a significantly lower removal efficiency (2.0+/-1log10) and discharged treated wastewater with a poorer microbiological quality compared to larger plants with tertiary treatment or with enhanced secondary treatment (2.8log10). Final sand filtration and extensive intermediate settling considerably improved the overall microbiological removal efficiency. During the study period, the microbiological water quality of the receiving water course was not significantly impaired by the discharge of any of the investigated plants; however, the compact facilities showed critical treatment deficiencies. In particular, the reduction of Giardia cysts was insufficient (<1.5log10) compared to that of the bigger plants (>3.0log10). In order to quantify the overall impact of microbiological loads on the receiving watercourse in this catchment area, it is also necessary to assess the pollution from combined sewer overflow basins and diffuse pollution. This will be considered in subsequent studies.

Risk factor analysis of diarrhoeal diseases in the Aral Sea area (Khorezm, Uzbekistan).

In the Aral Sea basin, human activities have resulted in the severe degradation of water and soil, which is considered to cause serious human health problems. This study investigated the risk factors: water, sanitation and related hygiene issues for diarrhoeal disease in Khorezm province, Uzbekistan. The risk factors were studied using a combination of quantitative and qualitative methods including water quality monitoring, standardised questionnaires and spot checks. Multiple linear regression analysis revealed that visible contamination of drinking water during storage and the absence of anal cleansing materials were significantly associated with the number of diarrhoeal episodes per household. Overall, the findings of the study show that the domestic domain plays a major role with regard to faecal-oral disease transmission in Khorezm, Uzbekistan. Unhealthy excreta disposal habits and unsafe drinking water storage practices have to be urgently tackled in order to break the faecal-oral transmission route.