scholarly journals Role of Cyclic di-GMP in Xylella fastidiosa Biofilm Formation, Plant Virulence, and Insect Transmission

2010 ◽  
Vol 23 (10) ◽  
pp. 1356-1363 ◽  
Author(s):  
Subhadeep Chatterjee ◽  
Nabil Killiny ◽  
Rodrigo P. P. Almeida ◽  
Steven E. Lindow
Keyword(s):  
Cell Density ◽  
Intracellular Signaling ◽  
Biofilm Formation ◽  
Extracellular Enzymes ◽  
Xylella Fastidiosa ◽  
Amorphous Layer ◽  
Extracellular Polysaccharides ◽  
Wild Type ◽  
A Cell

Xylella fastidiosa must coordinately regulate a variety of traits contributing to biofilm formation, host plant and vector colonization, and transmission between plants. Traits such as production of extracellular polysaccharides (EPS), adhesins, extracellular enzymes, and pili are expressed in a cell-density-dependent fashion mediated by a cell-to-cell signaling system involving a fatty acid diffusible signaling factor (DSF). The expression of gene PD0279 (which has a GGDEF domain) is downregulated in the presence of DSF and may be involved in intracellular signaling by modulating the levels of cyclic di-GMP. PD0279, designated cyclic di-GMP synthase A (cgsA), is required for biofilm formation, plant virulence, and vector transmission. cgsA mutants exhibited a hyperadhesive phenotype in vitro and overexpressed gumJ, hxfA, hxfB, xadA, and fimA, which promote attachment of cells to surfaces and, hence, biofilm formation. The mutants were greatly reduced in virulence to grape albeit still transmissible by insect vectors, although at a reduced level compared with transmission rates of the wild-type strain, despite the fact that similar numbers of cells of the cgsA mutant were acquired by the insects from infected plants. High levels of EPS were measured in cgsA mutants compared with wild-type strains, and scanning electron microscopy analysis also revealed a thicker amorphous layer surrounding the mutants. Overexpression of cgsA in a cgsA-complemented mutant conferred the opposite phenotypes in vitro. These results suggest that decreases of cyclic di-GMP result from the accumulation of DSF as cell density increases, leading to a phenotypic transition from a planktonic state capable of colonizing host plants to an adhesive state that is insect transmissible.

scholarly journals The Exopolysaccharide of Xylella fastidiosa Is Essential for Biofilm Formation, Plant Virulence, and Vector Transmission

2013 ◽  
Vol 26 (9) ◽  
pp. 1044-1053 ◽  
Author(s):  
N. Killiny ◽  
R. Hernandez Martinez ◽  
C. Korsi Dumenyo ◽  
D. A. Cooksey ◽  
R. P. P. Almeida
Keyword(s):  
Biofilm Formation ◽  
Pathogenic Bacteria ◽  
Xylella Fastidiosa ◽  
Growth Characteristics ◽  
Gas Chromatography Mass Spectrometry ◽  
Wild Type ◽  
Mutant Strains ◽  
Plant Movement

Exopolysaccharides (EPS) synthesized by plant-pathogenic bacteria are generally essential for virulence. The role of EPS produced by the vector-transmitted bacterium Xylella fastidiosa was investigated by knocking out two genes implicated in the EPS biosynthesis, gumD and gumH. Mutant strains were affected in growth characteristics in vitro, including adhesion to surfaces and biofilm formation. In addition, different assays were used to demonstrate that the mutant strains produced significantly less EPS compared with the wild type. Furthermore, gas chromatography–mass spectrometry showed that both mutant strains did not produce oligosaccharides. Biologically, the mutants were deficient in movement within plants, resulting in an avirulent phenotype. Additionally, mutant strains were affected in transmission by insects: they were very poorly transmitted by and retained within vectors. The gene expression profile indicated upregulation of genes implicated in cell-to-cell signaling and adhesins while downregulation in genes was required for within-plant movement in EPS-deficient strains. These results suggest an essential role for EPS in X. fastidiosa interactions with both plants and insects.

scholarly journals Csp1, A Cold-Shock Protein Homolog in Xylella fastidiosa Influences Pili Formation, Stress Response, and Gene Expression

2021 ◽  
Author(s):  
Wei Wei ◽  
Lindsey Price Burbank ◽  
Teresa Sawyer
Keyword(s):  
Gene Expression ◽  
Nucleic Acid ◽  
Biofilm Formation ◽  
Cold Shock ◽  
Xylella Fastidiosa ◽  
Cold Shock Protein ◽  
Nucleic Acid Binding ◽  
Wild Type ◽  
Surface Attachment

Bacterial cold shock-domain proteins (CSPs) are conserved nucleic acid binding chaperones that play important roles in stress adaptation and pathogenesis. Csp1 is a temperature-independent cold shock protein homolog in Xylella fastidiosa, a bacterial plant pathogen of grapevine and other economically important crops. Csp1 contributes to stress tolerance and virulence in X. fastidiosa. However, besides general single stranded nucleic acid binding activity, little is known about the specific function(s) of this protein. To further investigate the role(s) of Csp1, we compared phenotypic differences between wild type and a csp1 deletion mutant (Δcsp1). We observed decreases in cellular aggregation and surface attachment with the Δcsp1 strain compared to the wild type. Transmission electron microscopy imaging revealed that Δcsp1 had reduced pili compared to the wild type and complemented strains. The Δcsp1 strain also showed reduced survival after long term growth, in vitro. Since Csp1 binds DNA and RNA, its influence on gene expression was also investigated. Long-read Nanopore RNA-Seq analysis of wild type and Δcsp1 revealed changes in expression of several genes important for attachment and biofilm formation in Δcsp1. One gene of intertest,pilA1, encodes a type IV pili subunit protein and was up regulated in Δcsp1. Deleting pilA1 increased surface attachment in vitro and reduced virulence in grapevines.X. fastidiosa virulence depends on bacterial attachment to host tissue and movement within and between xylem vessels. Our results show Csp1 may play a role in both virulence and stress tolerance by influencing expression of genes important for biofilm formation.

scholarly journals Bacterial and plant produced lipids can exacerbate the Olive Quick Decline Syndrome caused by Xylella

2019 ◽  
Author(s):  
Valeria Scala ◽  
Nicoletta Pucci ◽  
Manuel Salustri ◽  
Vanessa Modesti ◽  
Alessia L’Aurora ◽  
...  
Keyword(s):  
Immune Response ◽  
Cell Density ◽  
Biofilm Formation ◽  
Southern Italy ◽  
Xylella Fastidiosa ◽  
Bacterial Biofilm ◽  
Insect Vector ◽  
Wide Range ◽  
Olive Quick Decline Syndrome

AbstractXylella fastidiosa is an insect vector-transmitted bacterial plant pathogen associated with severe diseases in a wide range of plants. In last decades, X. fastidiosa was detected in several European countries. Among X. fastidiosa subspecies, here we study X. fastidiosa subsp. pauca associated with the Olive Quick Decline Syndrome (OQDS) causing severe losses in Southern Italy. First, we collected Olea europaea L. (cv. Ogliarola salentina) samples in groves located in infected zones and uninfected zones. Secondly, the untargeted LC-TOF analysis of the lipid profiles of OQDS positive (+) and negative (-) plants showed a significant clustering of OQDS+ samples apart from OQDS-ones. Thirdly, using HPLC-MS/MS targeted methods and chemometric analysis, we identified a shortlist of 10 lipids significantly different in the infected versus healthy samples. Last, we observed a clear impact on X. fastidiosa subsp. pauca growth and biofilm formation in vitro liquid cultures supplemented with these compounds.Considering that growth and biofilm formation are primary ways by which X. fastidiosa causes disease, our results demonstrate that lipids produced as part of the plant’s immune response can exacerbate the disease. This is reminiscent of an allergic reaction in animal systems, offering the depression of plant immune response as a potential strategy for OQDS treatment.Author summaryGlobal trade and climate change are re-shaping the distribution map of pandemic pathogens. One major emerging concern is Xylella fastidiosa, a tropical bacterium recently introduced into Europe from America. Its impact has been dramatic: in the last 5-years only, Olive Quick Decline Syndrome (OQDS) has caused thousands of 200 years old olive trees to be felled in the southern Italy. Xylella fastidiosa through a tight coordination of the adherent biofilm and the planktonic states, invades the host systemically. The planktonic phase is correlated to low cell density and vessel colonization. Increase in cell density triggers a quorum sensing system based on cis 2-enoic fatty acids—diffusible signalling factors (DSF) that promote stickiness and biofilm. Xylem vessels are occluded by the combined effect of bacterial biofilm and plant defences (e.g. tyloses). This study provides novel insight on how X. fastidiosa subsp. pauca biology relates to the Olive Quick Decline Syndrome. We found that some class of lipids increase their amount in the infected olive tree. These lipid entities, provided to X. fastidiosa subsp. pauca behave as hormone-like molecules: modulating the dual phase, e.g. planktonic versus biofilm. Probably, part of these lipids represents a reaction of the plant to the bacterial contamination.

Increased cell death in osteopontin-deficient cardiac fibroblasts occurs by a caspase-3-independent pathway

2004 ◽  
Vol 287 (4) ◽  
pp. H1730-H1739 ◽  
Author(s):  
Ron Zohar ◽  
Baoqian Zhu ◽  
Peter Liu ◽  
Jaro Sodek ◽  
C. A. McCulloch
Keyword(s):  
Cell Death ◽  
Caspase 3 ◽  
Cardiac Fibroblasts ◽  
Chromatin Condensation ◽  
Terminal Deoxynucleotidyl Transferase ◽  
Tunel Staining ◽  
Wild Type ◽  
Nuclear Fragmentation ◽  
A Cell

Reperfusion-induced oxidative injury to the myocardium promotes activation and proliferation of cardiac fibroblasts and repair by scar formation. Osteopontin (OPN) is a proinflammatory cytokine that is upregulated after reperfusion. To determine whether OPN enhances fibroblast survival after exposure to oxidants, cardiac fibroblasts from wild-type (WT) or OPN-null (OPN−/−) mice were treated in vitro with H2O2to model reperfusion injury. Within 1 h, membrane permeability to propidium iodide (PI) was increased from 5 to 60% in OPN−/−cells but was increased to only 20% in WT cells. In contrast, after 1–8 h of treatment with H2O2, the percent of terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling (TUNEL)-stained cells was more than twofold higher in WT than OPN−/−cells. Electron microscopy of WT cells treated with H2O2showed chromatin condensation, nuclear fragmentation, and cytoplasmic and nuclear shrinkage, which are consistent with apoptosis. In contrast, H2O2-treated OPN−/−cardiac fibroblasts exhibited cell and nuclear swelling and membrane disruption that are indicative of cell necrosis. Treatment of OPN−/−and WT cells with a cell-permeable caspase-3 inhibitor reduced the percentage of TUNEL staining by more than fourfold in WT cells but decreased staining in OPN−/−cells by ∼30%. Although the percentage of PI-permeable WT cells was reduced threefold, the percent of PI-permeable OPN−/−cells was not altered. Restoration of OPN expression in OPN−/−fibroblasts reduced the percentage of PI-permeable cells but not TUNEL staining after H2O2treatment. Thus H2O2-induced cell death in OPN-deficient cardiac fibroblasts is mediated by a caspase-3-independent, necrotic pathway. We suggest that the increased expression of OPN in the myocardium after reperfusion may promote fibrosis by protecting cardiac fibroblasts from cell death.

scholarly journals Abrogation of Neuraminidase Reduces Biofilm Formation, Capsule Biosynthesis, and Virulence of Porphyromonas gingivalis

2011 ◽  
Vol 80 (1) ◽  
pp. 3-13 ◽  
Author(s):  
Chen Li ◽  
Kurniyati ◽  
Bo Hu ◽  
Jiang Bian ◽  
Jianlan Sun ◽  
...  
Keyword(s):  
Porphyromonas Gingivalis ◽  
Biofilm Formation ◽  
Adult Population ◽  
Transcriptional Analysis ◽  
Wild Type ◽  
Content Type ◽  
Multiple Organs ◽  
Biochemical Analyses

ABSTRACTThe oral bacteriumPorphyromonas gingivalisis a key etiological agent of human periodontitis, a prevalent chronic disease that affects up to 80% of the adult population worldwide.P. gingivalisexhibits neuraminidase activity. However, the enzyme responsible for this activity, its biochemical features, and its role in the physiology and virulence ofP. gingivalisremain elusive. In this report, we found thatP. gingivalisencodes a neuraminidase, PG0352 (SiaPg). Transcriptional analysis showed thatPG0352is monocistronic and is regulated by a sigma70-like promoter. Biochemical analyses demonstrated that SiaPgis an exo-α-neuraminidase that cleaves glycosidic-linked sialic acids. Cryoelectron microscopy and tomography analyses revealed that thePG0352deletion mutant (ΔPG352) failed to produce an intact capsule layer. Compared to the wild type,in vitrostudies showed that ΔPG352 formed less biofilm and was less resistant to killing by the host complement.In vivostudies showed that while the wild type caused a spreading type of infection that affected multiple organs and all infected mice were killed, ΔPG352 only caused localized infection and all animals survived. Taken together, these results demonstrate that SiaPgis an important virulence factor that contributes to the biofilm formation, capsule biosynthesis, and pathogenicity ofP. gingivalis, and it can potentially serve as a new target for developing therapeutic agents againstP. gingivalisinfection.

scholarly journals CRISPR-mediated isogenic cell-SELEX approach for generating highly specific aptamers against native membrane proteins

2020 ◽  
Author(s):  
Jonah C. Rosch ◽  
Emma H. Neal ◽  
Daniel A. Balikov ◽  
Mohsin Rahim ◽  
Ethan S. Lippmann
Keyword(s):  
Membrane Proteins ◽  
Glucose Transporter ◽  
High Specificity ◽  
Epithelial Cell Line ◽  
Target Cells ◽  
Wild Type ◽  
Simultaneous Exposure ◽  
Affinity Reagents ◽  
A Cell

AbstractIntroductionThe generation of affinity reagents that bind native membrane proteins with high specificity remains challenging. Most in vitro selection paradigms utilize different cell types for positive and negative rounds of selection (where the positive selection is against a cell that expresses the desired membrane protein and the negative selection is against a cell that lacks the protein). However, this strategy can yield affinity reagents that bind unintended membrane proteins on the target cells. To address this issue, we developed a systematic evolution of ligands by exponential enrichment (SELEX) scheme that utilizes isogenic pairs of cells generated via CRISPR techniques.MethodsUsing a Caco-2 epithelial cell line with constitutive Cas9 expression, we knocked out the SLC2A1 gene (encoding the GLUT1 glucose transporter) via lipofection with synthetic gRNAs. Cell-SELEX rounds were carried out against wild-type and GLUT1-null cells using a single-strand DNA (ssDNA) library. Next-generation sequencing (NGS) was used to quantify enrichment of prospective binders to the wild-type cells.Results10 rounds of cell-SELEX were conducted via simultaneous exposure of ssDNA pools to wild-type and GLUT1-null Caco-2 cells under continuous perfusion. The top binders identified from NGS were validated by flow cytometry and immunostaining for their specificity to the GLUT1 receptor.ConclusionsOur data indicate that highly specific aptamers can be isolated with a SELEX strategy that utilizes isogenic cell lines. This approach should be broadly useful for generating affinity reagents that selectively bind to membrane proteins in their native conformations on the cell surface.

scholarly journals Role of the Streptococcus mutans irvA Gene in GbpC-Independent, Dextran-Dependent Aggregation and Biofilm Formation

2009 ◽  
Vol 75 (22) ◽  
pp. 7037-7043 ◽  
Author(s):  
Min Zhu ◽  
Dragana Ajdić ◽  
Yuan Liu ◽  
David Lynch ◽  
Justin Merritt ◽  
...  
Keyword(s):  
Streptococcus Mutans ◽  
Biofilm Formation ◽  
Growth Conditions ◽  
Parental Background ◽  
A Cell ◽  
Major Surface ◽  
Biofilm Development ◽  
Small Aggregation

ABSTRACT Dextran-dependent aggregation (DDAG) of Streptococcus mutans is an in vitro phenomenon that is believed to represent a property of the organism that is beneficial for sucrose-dependent biofilm development. GbpC, a cell surface glucan-binding protein, is responsible for DDAG in S. mutans when cultured under defined stressful conditions. Recent reports have described a putative transcriptional regulator gene, irvA, located just upstream of gbpC, that is normally repressed by the product of an adjacent gene, irvR. When repression of irvA is relieved, there is a resulting increase in the expression of GbpC and decreases in competence and synthesis of the antibiotic mutacin I. This study examined the role of irvA in DDAG and biofilm formation by engineering strains that overexpressed irvA (IrvA+) on an extrachromosomal plasmid. The IrvA+ strain displayed large aggregation particles that did not require stressful growth conditions. A novel finding was that overexpression of irvA in a gbpC mutant background retained a measure of DDAG, albeit very small aggregation particles. Biofilms formed by the IrvA+ strain in the parental background possessed larger-than-normal microcolonies. In a gbpC mutant background, the overexpression of irvA reversed the fragile biofilm phenotype normally associated with loss of GbpC. Real-time PCR and Northern blot analyses found that expression of gbpC did not change significantly in the IrvA+ strain but expression of spaP, encoding the major surface adhesin P1, increased significantly. Inactivation of spaP eliminated the small-particle DDAG. The results suggest that IrvA promotes DDAG not only by GbpC, but also via an increase in P1.

scholarly journals Serotonin 5-HT2C Receptor Cys23Ser Single Nucleotide Polymorphism Associates with Receptor Function and Localization In Vitro

2019 ◽  
Vol 9 (1) ◽  
Author(s):  
Michelle A. Land ◽  
Holly L. Chapman ◽  
Brionna D. Davis-Reyes ◽  
Daniel E. Felsing ◽  
John A. Allen ◽  
...  
Keyword(s):  
Single Nucleotide Polymorphism ◽  
Subcellular Localization ◽  
Cell Lines ◽  
Intracellular Signaling ◽  
Calcium Release ◽  
Wild Type ◽  
Nucleotide Polymorphism ◽  
Single Nucleotide ◽  
Recycling Pathway

Abstract A non-synonymous single nucleotide polymorphism of the human serotonin 5-HT2C receptor (5-HT2CR) gene that converts a cysteine to a serine at amino acid codon 23 (Cys23Ser) appears to impact 5-HT2CR pharmacology at a cellular and systems level. We hypothesized that the Cys23Ser alters 5-HT2CR intracellular signaling via changes in subcellular localization in vitro. Using cell lines stably expressing the wild-type Cys23 or the Ser23 variant, we show that 5-HT evokes intracellular calcium release with decreased potency and peak response in the Ser23 versus the Cys23 cell lines. Biochemical analyses demonstrated lower Ser23 5-HT2CR plasma membrane localization versus the Cys23 5-HT2CR. Subcellular localization studies demonstrated O-linked glycosylation of the Ser23 variant, but not the wild-type Cys23, may be a post-translational mechanism which alters its localization within the Golgi apparatus. Further, both the Cys23 and Ser23 5-HT2CR are present in the recycling pathway with the Ser23 variant having decreased colocalization with the early endosome versus the Cys23 allele. Agonism of the 5-HT2CR causes the Ser23 variant to exit the recycling pathway with no effect on the Cys23 allele. Taken together, the Ser23 variant exhibits a distinct pharmacological and subcellular localization profile versus the wild-type Cys23 allele, which could impact aspects of receptor pharmacology in individuals expressing the Cys23Ser SNP.

scholarly journals Copper Supplementation in Watering Solution Reaches the Xylem But Does Not Protect Tobacco Plants Against Xylella fastidiosa Infection

Plant Disease ◽  
2020 ◽  
Vol 104 (3) ◽  
pp. 724-730 ◽  
Author(s):  
Qing Ge ◽  
Paul A. Cobine ◽  
Leonardo De La Fuente
Keyword(s):  
Biofilm Formation ◽  
Inductively Coupled Plasma ◽  
Tap Water ◽  
Xylella Fastidiosa ◽  
In Vitro Study ◽  
Optical Emission ◽  
In Planta ◽  
Inductively Coupled ◽  
High Concentrations

Xylella fastidiosa is a xylem-limited plant pathogenic bacterium that causes disease in many crops worldwide. Copper (Cu) is an antimicrobial agent widely used on X. fastidiosa hosts to control other diseases. Although the effects of Cu for control of foliar pathogens are well known, it is less studied on xylem-colonizing pathogens. Previous results from our group showed that low concentrations of CuSO4 increased biofilm formation, whereas high concentrations inhibited biofilm formation and growth in vitro. In this study, we conducted in planta experiments to determine the influence of Cu in X. fastidiosa infection using tobacco as a model. X. fastidiosa-infected and noninfected plants were watered with tap water or with water supplemented with 4 mM or 8 mM of CuSO4. Symptom progression was assessed, and sap and leaf ionome analysis was performed by inductively coupled plasma with optical emission spectroscopy. Cu uptake was confirmed by increased concentrations of Cu in the sap of plants treated with CuSO4-amended water. Leaf scorch symptoms in Cu-supplemented plants showed a trend toward more severe at later time points. Quantification of total and viable X. fastidiosa in planta indicated that CuSO4-amended treatments did not inhibit but slightly increased the growth of X. fastidiosa. Cu in sap was in the range of concentrations that promote X. fastidiosa biofilm formation according to our previous in vitro study. Based on these results, we proposed that the plant Cu homeostasis machinery controls the level of Cu in the xylem, preventing it from becoming elevated to a level that would lead to bacterial inhibition.

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