Regulation of Xylella fastidiosa virulence factors by c-di-GMP phosphodiesterases

Xylella fastidiosa is an important bacterial plant pathogen that colonizes the xylem of hundreds of plant species. X. fastidiosa cause Pierce's disease in grapevine by occlusion of the xylem by extensive bacterial colonization, extracellular polysaccharides and the formation of a biofilm. These traits are mediated in a cell-density manner by a cell-to-cell signaling system that transduces a diffusible signaling factor (DSF). This dissertation demonstrates that PD1994, PD1617 and RpfG regulate important traits for bacterial virulence such as cell-cell signaling, biofilm formation and cell aggregation. X. fastidiosa strains harboring mutations in pd1994 (which encodes for a defective GGDEF- EAL-domain protein) and in pd1617 (which encodes for a EAL-domain protein) have increased growth rate, increased biofilm formation, increased plant colonization and decreased cell aggregation. Gene expression analysis of the pd1994 mutant strain showed overexpression of rpfF, which is a DSF synthase, suggesting that PD1994 regulates DSF signaling by repressing rpfF expression. Additionally, the pd1994mutant showed overexpression of pd1617 and rpfG (with EAL and HD-GYP domains respectively, that may be responsible for c-di-GMP turnover), which suggested that this mutant may have low c-di-GMP levels and that PD1994 regulates c-di-GMP turnover by repression of RpfG activity and PD1617 gene expression. X. fastidiosa harboring a mutation on rpfG exhibited decreased biofilm formation while it had no effect in growth or cell aggregation. Together, these results suggest that PD1994, PD1617 and RpfG regulate the DSF regulatory network by controlling the turnover of the second messenger c-di-GMP

The Synergistic Effect of Nanocrystals Combined With Ultrasound in the Generation of Reactive Oxygen Species for Biomedical Applications

Reactive oxygen species (ROS) effects on living cells and tissues is multifaceted and their level or dose can considerably affect cell proliferation and viability. It is therefore necessary understand their role also designing ways able to regulate their amount inside cells, i.e., using engineered nanomaterials with either antioxidant properties or, for cancer therapy applications, capable to induce oxidative stress and cell death, through tunable ROS production. In this paper, we report on the use of single-crystalline zinc oxide (ZnO) round-shaped nanoparticles, yet ZnO nanocrystals (NCs) functionalized with amino-propyl groups (ZnO-NH2 NCs), combined with pulsed ultrasound (US). We show the synergistic effects produced by NC-assisted US which are able to produce different amount of ROS, as a result of inertial cavitation under the pulsed US exposure. Using Passive Cavitation Detection (PCD) and Electron Paramagnetic Resonance (EPR) spectroscopy, we systematically study which are the key parameters, monitoring, and influencing the amount of generated ROS measuring their concentration in water media and comparing all the results with pure water batches. We thus propose a ROS generation mechanism based on the selective application of US to the ZnO nanocrystals in water solutions. Ultrasound B-mode imaging is also applied, proving in respect to pure water, the enhanced ecographic signal generation of the aqueous solution containing ZnO-NH2 NCs when exposed to pulsed ultrasound. Furthermore, to evaluate the applicability of ZnO-NH2 NCs in the biomedical field, the ROS generation is studied by interposing different tissue mimicking materials, like phantoms and ex vivo tissues, between the US transducer and the sample well. As a whole, we clearly proof the enhanced capability to produce ROS and to control their amount when using ZnO-NH2 NCs in combination with pulsed ultrasound anticipating their applicability in the fields of biology and health care

Improving dispersal of therapeutic nanoparticles in the human body

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RNAi Screen of Endoplasmic Reticulum–Associated Host Factors Reveals a Role for IRE1α in Supporting Brucella Replication

Brucella species are facultative intracellular bacterial pathogens that cause brucellosis, a global zoonosis of profound importance. Although recent studies have demonstrated that Brucella spp. replicate within an intracellular compartment that contains endoplasmic reticulum (ER) resident proteins, the molecular mechanisms by which the pathogen secures this replicative niche remain obscure. Here, we address this issue by exploiting Drosophila S2 cells and RNA interference (RNAi) technology to develop a genetically tractable system that recapitulates critical aspects of mammalian cell infection. After validating this system by demonstrating a shared requirement for phosphoinositide 3-kinase (PI3K) activities in supporting Brucella infection in both host cell systems, we performed an RNAi screen of 240 genes, including 110 ER-associated genes, for molecules that mediate bacterial interactions with the ER. We uncovered 52 evolutionarily conserved host factors that, when depleted, inhibited or increased Brucella infection. Strikingly, 29 of these factors had not been previously suggested to support bacterial infection of host cells. The most intriguing of these was inositol-requiring enzyme 1 (IRE1), a transmembrane kinase that regulates the eukaryotic unfolded protein response (UPR). We employed IRE1α(−/−) murine embryonic fibroblasts (MEFs) to demonstrate a role for this protein in supporting Brucella infection of mammalian cells, and thereby, validated the utility of the Drosophila S2 cell system for uncovering novel Brucella host factors. Finally, we propose a model in which IRE1α, and other ER-associated genes uncovered in our screen, mediate Brucella replication by promoting autophagosome biogenesis

A Pathogen Secreted Protein as a Detection Marker for Citrus Huanglongbing.

The citrus industry is facing an unprecedented crisis due to Huanglongbing (HLB, aka citrus greening disease), a bacterial disease associated with the pathogen Candidatus Liberibacter asiaticus (CLas) that affects all commercial varieties. Transmitted by the Asian citrus psyllid (ACP), CLas colonizes citrus phloem, leading to reduced yield and fruit quality, and eventually tree decline and death. Since adequate curative measures are not available, a key step in HLB management is to restrict the spread of the disease by identifying infected trees and removing them in a timely manner. However, uneven distribution of CLas cells in infected trees and the long latency for disease symptom development makes sampling of trees for CLas detection challenging. Here, we report that a CLas secreted protein can be used as a biomarker for detecting HLB infected citrus. Proteins secreted from CLas cells can presumably move along the phloem, beyond the site of ACP inoculation and CLas colonized plant cells, thereby increasing the chance of detecting infected trees. We generated a polyclonal antibody that effectively binds to the secreted protein and developed serological assays that can successfully detect CLas infection. This work demonstrates that antibody-based diagnosis using a CLas secreted protein as the detection marker for infected trees offers a high-throughput and economic approach that complements the approved quantitative polymerase chain reaction-based methods to enhance HLB management programs

Polydopamine nanoparticles as an organic and biodegradable multitasking tool for neuroprotection and remote neuronal stimulation

Oxidative stress represents a common issue in most neurological diseases, causing severe impairments of neuronal cell physiological activity that ultimately lead to neuron loss of function and cellular death. In this work, lipid-coated polydopamine nanoparticles (L-PDNPs) are proposed both as antioxidant and neuroprotective agents, and as well as a photo-thermal conversion platform able to stimulate neuronal activity. L-PDNPs showed the ability to counteract reactive oxygen species (ROS) accumulation in differentiated SH-SY5Y, prevented mitochondrial ROS-induced dysfunctions, and stimulated neurite outgrowth. Moreover, for the first time in the literature, the photo-thermal conversion capacity of L-PDNPs was used to increase the intracellular temperature of neuron-like cells through near-infrared (NIR) laser stimulation, and this phenomenon was thoroughly investigated using a fluorescent temperature-sensitive dye and modeled from a mathematical point of view. It was also demonstrated that the increment in temperature caused by the NIR stimulation of L-PDNPs was able to produce a Ca2+ influx in differentiated SH-SY5Y, being, to the best of our knowledge, the first example of organic nanostructures used in such an approach. This work could pave the way to new and exciting applications of polydopamine-based and of other NIR-responsive antioxidant nanomaterials in neuronal research

Phytophthora-citrus interactions and management strategies: a review

Citrus production is declining worldwide due to several biotic and abiotic factors. The diseases caused by Phytophthora spp. present major economic risks since they are soil-borne and spread quickly if environmental conditions are favorable, or irrigation is poorly managed. Phytophthora species are present in all citrus-producing areas around the world causing significant losses in crop yield and affecting tree health. Bark infection, damping-off, root rot, gummosis, brown rot, and cortical root rot are among the typical symptoms caused by Phytophthora spp. The pathogenicity of Phytophthora spp. depends mainly on the specific interactions between the isolates and citrus cultivars. The use of molecular technologies has allowed the study of Phytophthora-citrus interactions, leading to the identification of several classes of effector proteins secreted by Phytophthora spp. that challenge plant homeostasis and contribute in different ways to disease development and the elucidation of defense mechanisms employed by the host plant. Comprehensive management is need of time to conquer Phytophthora spp. for better root health and citrus productivity

The structure and function of the global citrus rhizosphere microbiome

Citrus is a globally important, perennial fruit crop whose rhizosphere microbiome is thought to play an important role in promoting citrus growth and health. Here, we report a comprehensive analysis of the structural and functional composition of the citrus rhizosphere microbiome. We use both amplicon and deep shotgun metagenomic sequencing of bulk soil and rhizosphere samples collected across distinct biogeographical regions from six continents. Predominant taxa include Proteobacteria, Actinobacteria, Acidobacteria and Bacteroidetes. The core citrus rhizosphere microbiome comprises Pseudomonas, Agrobacterium, Cupriavidus, Bradyrhizobium, Rhizobium, Mesorhizobium, Burkholderia, Cellvibrio, Sphingomonas, Variovorax and Paraburkholderia, some of which are potential plant beneficial microbes. We also identify over-represented microbial functional traits mediating plant-microbe and microbe-microbe interactions, nutrition acquisition and plant growth promotion in citrus rhizosphere. The results provide valuable information to guide microbial isolation and culturing and, potentially, to harness the power of the microbiome to improve plant production and health