24 research outputs found

    Dual RNA-seq of Nontypeable Haemophilus influenzae and Host Cell Transcriptomes Reveals Novel Insights into Host-Pathogen Cross Talk

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    ABSTRACT The ability to adhere and adapt to the human respiratory tract mucosa plays a pivotal role in the pathogenic lifestyle of nontypeable Haemophilus influenzae (NTHi). However, the temporal events associated with a successful colonization have not been fully characterized. In this study, by reconstituting the ciliated human bronchial epithelium in vitro, we monitored the global transcriptional changes in NTHi and infected mucosal epithelium simultaneously for up to 72 h by dual RNA sequencing. The initial stage of colonization was characterized by the binding of NTHi to ciliated cells. Temporal profiling of host mRNA signatures revealed significant dysregulation of the target cell cytoskeleton elicited by bacterial infection, with a profound effect on the intermediate filament network and junctional complexes. In response to environmental stimuli of the host epithelium, NTHi downregulated its central metabolism and increased the expression of transporters, indicating a change in the metabolic regime due to the availability of host substrates. Concurrently, the oxidative environment generated by infected cells instigated bacterial expression of stress-induced defense mechanisms, including the transport of exogenous glutathione and activation of the toxin-antitoxin system. The results of this analysis were validated by those of confocal microscopy, Western blotting, Bio-plex, and real-time quantitative reverse transcription-PCR (qRT-PCR). Notably, as part of our screening for novel signatures of infection, we identified a global profile of noncoding transcripts that are candidate small RNAs (sRNAs) regulated during human host infection in Haemophilus species. Our data, by providing a robust and comprehensive representation of the cross talk between the host and invading pathogen, provides important insights into NTHi pathogenesis and the development of efficacious preventive strategies. IMPORTANCE Simultaneous monitoring of infection-linked transcriptome alterations in an invading pathogen and its target host cells represents a key strategy for identifying regulatory responses that drive pathogenesis. In this study, we report the progressive events of NTHi colonization in a highly differentiated model of ciliated bronchial epithelium. Genome-wide transcriptome maps of NTHi during infection provided mechanistic insights into bacterial adaptive responses to the host niche, with modulation of the central metabolism as an important signature of the evolving milieu. Our data indicate that infected epithelia respond by substantial alteration of the cytoskeletal network and cytokine repertoire, revealing a dynamic cross talk that is responsible for the onset of inflammation. This work significantly enhances our understanding of the means by which NTHi promotes infection on human mucosae and reveals novel strategies exploited by this important pathogen to cause invasive disease

    WATZON: the Italian network of ecohydrology and critical zone observatories

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    The Italian initiative WATZON (WATer mixing in the critical ZONe) is a network of instrumented sites, bringing together six pre-existing long-term research observatories monitoring different compartments of the Critical Zone - the Earth's permeable near-surface layer from the tops of the trees to the bottom of the groundwater. These observatories cover different climatic and physiographic characteristics over the country, providing information over a climate and eco-hydrologic transect connecting the Mediterranean to the Alps. With specific initial scientific questions, monitoring strategies, databases, and modeling activities, the WATZON observatories and sites is well representative of the heterogeneity of the critical zone and of the scientific communities studying it. Despite this diversity, all WATZON sites share a common eco-hydrologic monitoring and modelling program with three main objectives: 1) assessing the description of water mixing process across the critical zone by using integrated high-resolution isotopic, geophysical and hydrometeorological measurements from point to catchment scale, under different physiographic conditions and climate forcing; 2) testing water exchange mechanisms between subsurface reservoirs and vegetation, and assessing ecohydrological dynamics in different environments by coupling the high-resolution data set from different critical zone study sites of the initiative with advanced ecohydrological models at multiple spatial scales; 3) developing a process-based conceptual framework of ecohydrological processes in the critical zone to translate scientific knowledge into evidence to support policy and management decisions concerning water and land use in forested and agricultural ecosystems. This work provides an overview of the WATZON network, its objectives, scientific questions, and data management, with a specific focus on existing initiatives for linking data and models based on WATZON data

    Assessing root water uptake transit time by simulating isotope transport in Hydrus-1D

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    Stable isotopes (2H and 18O) are common natural tracers for the investigation of water transport in the soil-plant-atmosphere continuum. Isotopic data can be coupled with soil water content data to inversely estimate soil hydraulic and transport parameters. The calibration of a hydrological model by inverse modelling is a prerequisite to determine the temporal origin of xylem water taken by roots. In this study, we used isotopic data to calibrate Hydrus-1D via inverse modelling to simulate one-dimensional water flow and isotope transport in a controlled soil-plant-atmosphere system. We propose the following protocol i) to estimate root water uptake transit time of irrigation water, and ii) to assess the sensitivity of the transit time distribution to the variation in the water available for root uptake. The dataset was obtained from an isotope-tracing experiment carried out between May and July 2018 on an olive tree placed in a pot inside a glasshouse. Meteorological variables and sap flow were monitored at 5-minute intervals, whereas shallow soil moisture (0-6 cm depth) was measured manually with an impedance probe at the daily timescale. The olive tree was irrigated with water of known isotopic composition. The pot surface was covered by a plastic sheet to avoid evaporation. Soil at different depths, twigs, wood cores and root samples were collected weekly for isotopic analyses. Water from soil and the xylem tissues was extracted by cryogenic vacuum distillation. Based on the results of a previous study carried out on the same dataset, we considered that no isotopic fractionation occurred during the water uptake and the transport within the olive tree. We used soil water content and δ18O data at different soil depths to optimize flow (soil hydraulic and root water uptake parameters) and transport (longitudinal dispersivity) parameters. Numerical simulations of isotope transport were validated with sap flow data (compared to actual transpiration) and δ18O in xylem water. Given that the timing of irrigation water for plant transpiration is fundamental for assessing the vulnerability of olive trees to drought, we will be proposing various scenarios based on different irrigation events to mimic drought periods. Based on these scenarios, we will be evaluating the sensitivity of the root water uptake transit time to the different water availability in the soil profile. Afterwards, the same protocol will be exploited to determine the root water uptake transit time for different tree species under various environmental conditions

    The Streptococcus pneumoniae Pilus-1 Displays a Biphasic Expression Pattern

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    The Streptococcus pneumoniae pilus-1 is encoded by pilus islet 1 (PI-1), which has three clonal variants (clade I, II and III) and is present in about 30% of clinical pneumococcal isolates. In vitro and in vivo assays have demonstrated that pilus-1 is involved in attachment to epithelial cells and virulence, as well as protection in mouse models of infection. Several reports suggest that pilus-1 expression is tightly regulated and involves the interplay of numerous genetic regulators, including the PI-1 positive regulator RlrA. In this report we provide evidence that pilus expression, when analyzed at the single-cell level in PI-1 positive strains, is biphasic. In fact, the strains present two phenotypically different sub-populations of bacteria, one that expresses the pilus, while the other does not. The proportions of these two phenotypes are variable among the strains tested and are not influenced by genotype, serotype, growth conditions, colony morphology or by the presence of antibodies directed toward the pilus components. Two sub-populations, enriched in pilus expressing or not expressing bacteria were obtained by means of colony selection and immuno-detection methods for five strains. PI-1 sequencing in the two sub-populations revealed the absence of mutations, thus indicating that the biphasic expression observed is not due to a genetic modification within PI-1. Microarray expression profile and western blot analyses on whole bacterial lysates performed comparing the two enriched sub-populations, revealed that pilus expression is regulated at the transcriptional level (on/off regulation), and that there are no other genes, in addition to those encoded by PI-1, concurrently regulated across the strains tested. Finally, we provide evidence that the over-expression of the RrlA positive regulator is sufficient to induce pilus expression in pilus-1 negative bacteria. Overall, the data presented here suggest that the observed biphasic pilus expression phenotype could be an example of bistability in pneumococcus

    Helicobacter pylori: A Brief History of a Still Lacking Vaccine

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    Helicobacter pylori colonizes the gastric mucosa of more than half of the human population worldwide. Soon after its discovery, the causative relationships between H. pylori infection and chronic atrophic gastritis, peptic ulcer and gastric mucosa-associated lymphoid tissue lymphoma were evidenced. Then, a significantly increased risk of developing gastric cancer was found to be associated with H. pylori infection. The efficacy of the treatment for H. pylori, based on a proton pump inhibitor plus antibiotics, has dropped below 80%, mainly due to antibiotic resistance. Vaccination would overcome antibiotic resistance and would lead to the eradication of this pathogen; however, in spite of almost twenty-five years of investigation on H. pylori vaccine candidates and good protective results obtained in animal models, no vaccine is currently licensed. This review focuses on the studies on the efficacy of those H. pylori vaccine candidates that underwent clinical trials. Efficacy trials have given unsatisfactory results, so far, with bacterial colonization remaining unaffected by vaccination. However, a vaccine able to counteract H. pylori-induced diseases, such as gastric cancer, even without providing sterilizing immunity, could be considered valuable

    Iron and pH Homeostasis Intersect at the Level of Fur Regulation in the Gastric Pathogen Helicobacter pylori

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    Helicobacter pylori persistently colonizes the stomach of the majority of the world's population and is a tremendous medical burden due to its causal role in diverse gastric maladies. Since the stomach is a constantly changing environment, successful colonization of H. pylori within this niche requires regulation of bacterial gene expression to cope with the environmental fluctuations. In H. pylori, the ferric uptake regulator (Fur) has been shown to play an intricate role in adaptation of the bacterium to two conditions known to oscillate within the gastric mucosa: iron limitation and low pH. To extend our knowledge of the process of regulation and adaptation in H. pylori, we show that Fur is required for efficient colonization of the Mongolian gerbil: the mutant strain exhibits a 100-fold increase in the 50% infectious dose, as well as a 100-fold defect in competitive colonization, when coinfected with wild-type bacteria. Furthermore, we used DNA microarrays to identify genes whose expression was altered in a Fur-deficient strain. We show that the Fur regulon of H. pylori consists of approximately 30 genes, most of which have been previously annotated as acid stress associated. Finally, we investigate the role of Fur in acid-responsive modulation of gene expression and show that a large number of genes are aberrantly expressed in the Fur mutant specifically upon acid exposure. This fact likely explains the requirement for this regulator for growth and colonization in the stomach

    The Alleles of the bft Gene Are Distributed Differently among Enterotoxigenic Bacteroides fragilis Strains from Human Sources and Can Be Present in Double Copies

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    Enterotoxigenic Bacteroides fragilis (ETBF) strains are associated with diarrheal disease in children. These strains produce a zinc metalloprotease enterotoxin, or fragilysin, that can be detected by a cytotoxicity assay with HT-29 cells. Recently, three different isoforms or variants of the enterotoxin gene, designated bft-1, bft-2, and bft-3, have been identified and sequenced. We used restriction fragment length polymorphism analysis of the PCR-amplified enterotoxin gene to detect the isoforms bft-1 and bft-2 or bft-3 borne by ETBF. By sequencing the portion of the bft gene corresponding to the mature toxin in some strains and applying allele-specific PCR for strains categorized as bft-2 or bft-3, we found in our collection two strains harboring bft-3, a variant that had been described for isolates from East Asia. Analysis of 66 ETBF strains from different sources showed that bft-1 is the most frequent allele, being present in 65% of isolates; it is largely predominant in isolates from feces of adults, while bft-2 is present in isolates from feces of children. This association is statistically significant (P, 0.0064). Sixteen strains were examined by Southern hybridization using, as probes, the bft and second metalloprotease genes, both included in a pathogenicity islet. Five strains were found to harbor double copies of both genes, suggesting that the whole islet was duplicated. Four of these strains, harboring bft-1 (three strains) or bft-2 (one strain), were found to produce a large amount of biologically active toxin, as determined by a cytotoxicity assay with HT-29 cells. The strains harboring bft-3, either in a single copy or in double copies, produced the smallest amount of toxin in our collection

    Helicobacter pylori Induces Apoptosis of Human Monocytes but Not Monocyte-Derived Dendritic Cells: Role of the cag Pathogenicity Island

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    Monocytes are circulating precursors of the dendritic cell subset, professional antigen-presenting cells with a unique ability to initiate the innate and adaptive immune response. In this study, we have investigated the effects of wild-type Helicobacter pylori strains and their isogenic mutants with mutations in known bacterial virulence factors on monocytes and monocyte-derived dendritic cells. We show that H. pylori strains induce apoptosis of human monocytes by a mechanism that is dependent on the expression of a functional cag pathogenicity island. This effect requires an intact injection organelle for direct contact between monocytes and the bacteria but also requires a still-unidentified effector that is different from VacA or CagA. The exposure of in vitro-generated monocyte-derived dendritic cells to H. pylori stimulates the release of inflammatory cytokines by a similar mechanism. Of note is that dendritic cells are resistant to H. pylori-induced apoptosis. These phenomena may play a critical role in the evasion of the immune response by H. pylori, contributing to the persistence of the infection
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