scholarly journals Functional and Pharmacological Analyses of the Role of Penicillium digitatum Proteases on Virulence

2019 ◽  
Vol 7 (7) ◽  
pp. 198 ◽  
Author(s):  
Ana-Rosa Ballester ◽  
Mario López-Pérez ◽  
Beatriz de la Fuente ◽  
Luis González-Candelas
Keyword(s):  
Protease Inhibitors ◽  
Functional Characterization ◽  
Citrus Fruit ◽  
Significant Loss ◽  
Penicillium Digitatum ◽  
Extracellular Proteases ◽  
Climate Conditions ◽  
Virulence Gene Expression

Penicillium digitatum is the major postharvest pathogen of citrus fruit under Mediterranean climate conditions. Previous results have shown that proteases is the largest enzyme family induced by P. digitatum during fruit infection. In the present work, we addressed the study of the role of P. digitatum’s proteases in virulence following two complementary approaches. In the first approach, we undertook the functional characterization of the P. digitatum prtT gene, which codes for a putative transcription factor previously shown to regulate extracellular proteases in other filamentous fungi. Deletion of prtT caused a significant loss in secreted protease activity during in vitro growth assays. However, there was no effect on virulence. Gene expression of the two major secreted acid proteases was barely affected in the ΔprtT deletant during infection of citrus fruit. Hence, no conclusion could be drawn on the role of these secreted acidic proteases on the virulence of P. digitatum. In the second approach, we studied the effect of different protease inhibitors and chelators on virulence. Co-inoculation of citrus fruit with P. digitatum conidia and a cocktail of protease inhibitors resulted in almost a complete absence of disease development. Analysis of individual inhibitors revealed that the metalloprotease inhibitor, 1,10-phenanthroline, was responsible for the observed effect. The application of metal ions reverted the protective effect caused by the metallopeptidase inhibitor. These results may set the basis for the development of new alternative treatments to combat this important postharvest pathogen.

scholarly journals Functional and Pharmacological Analyses of the Role of Penicillium digitatum Proteases on Virulence

2019 ◽  
Author(s):  
Ana-Rosa Ballester ◽  
Mario López-Pérez ◽  
Beatriz de la Fuente ◽  
Luis González-Candelas
Keyword(s):  
Protease Inhibitors ◽  
Functional Characterization ◽  
Citrus Fruit ◽  
Significant Loss ◽  
Penicillium Digitatum ◽  
Extracellular Proteases ◽  
Climate Conditions ◽  
Virulence Gene Expression

Penicillium digitatum is the major postharvest pathogen of citrus fruit under Mediterranean climate conditions. In the present work, we have addressed the study of the role of P. digitatum’s proteases in virulence following two complementary approaches. In a first approach, we have undertaken the functional characterization of the P. digitatum prtT gene, which codes for a transcription factor previously shown to regulate extracellular proteases in other filamentous fungi. Deletion of prtT caused a significant loss in secreted protease activity during in vitro growth assays. However, there was no effect on virulence. Gene expression of the two major secreted acid proteases was barely affected in the ΔprtT deletant during infection of citrus fruit. Hence, no conclusion could be drawn on the role of these secreted acidic proteases on the virulence of P. digitatum. In a second approach, we have studied the effect of different protease inhibitors and chelators in virulence. Co-inoculation of citrus fruit with P. digitatum conidia and a cocktail of protease inhibitors resulted in almost a complete absence of disease development. Analysis of individual inhibitors revealed that the metalloprotease inhibitor 1,10-phenanthroline was responsible for the observed effect. The application of metal ions reverted the protective effect caused by the metallopeptidase inhibitor. These results may set the basis for the development of new alternative treatments to combat this important postharvest pathogen.

scholarly journals Developing Penicillium digitatum Management Strategies on Post-Harvest Citrus Fruits with Metabolic Components and Colonization of Bacillus subtilis L1-21

2022 ◽  
Vol 8 (1) ◽  
pp. 80
Author(s):  
Yongmei Li ◽  
Mengyuan Xia ◽  
Pengbo He ◽  
Qiaoming Yang ◽  
Yixin Wu ◽  
...  
Keyword(s):  
Bacillus Subtilis ◽  
Culture Filtrate ◽  
Management Strategies ◽  
Citrus Fruit ◽  
Inhibition Zone ◽  
Penicillium Digitatum ◽  
Antifungal Compounds ◽  
Fruit Peel ◽  
Post Harvest

Citrus is among the most important plants in the fruit industry severely infected with pathogens. Citrus green mold caused by Penicillium digitatum is one of the most devastating diseases during post-harvest stages of citrus fruit. In this study, a potential endophyte Bacillus subtilis L1-21, isolated from healthy citrus plants, was assessed for its biocontrol activity against the pathogen P. digitatum. Based on an in vitro crosstalk assay, we suggested that B. subtilis L1-21 inhibits the pathogen with an inhibition zone of 3.51 ± 0.08 cm. Biocontrol efficacy was highest for the fermented culture filtrate of B. subtilis L1-21. Additionally, using GC-MS analysis, 13 compounds were detected in the extract of this endophyte. The culture filtrate in Landy medium could enlarge and deform pathogen spores and prevent them from developing into normal mycelium. Accordingly, the Landy culture filtrate of B. subtilis L1-21 was stable in the temperature range of 4–90 °C and pH of 3–11. Further, MALDI-TOF-MS for B. subtilis L1-21 detected surfactin, fengycin, bacillaene and bacilysin as potential antifungal compounds. GFP-tagged B. subtilis L1-21 easily colonized in citrus fruit peel and pulp, suggesting its role in eliminating the fungal pathogen. Altogether, it is highly expected that the production of antifungal compounds, and the colonization potential of B. subtilis L1-21 are required against the post-harvest P. digitatum pathogen on citrus fruit.

scholarly journals A Novel Secreted Cysteine-Rich Anionic (Sca) Protein from the Citrus Postharvest Pathogen Penicillium digitatum Enhances Virulence and Modulates the Activity of the Antifungal Protein B (AfpB)

2020 ◽  
Vol 6 (4) ◽  
pp. 203
Author(s):  
Sandra Garrigues ◽  
Jose F. Marcos ◽  
Paloma Manzanares ◽  
Mónica Gandía
Keyword(s):  
Citrus Fruit ◽  
Penicillium Digitatum ◽  
Antifungal Protein ◽  
Penicillium Expansum ◽  
Wild Type ◽  
Phenotypic Differences ◽  
Antifungal Proteins ◽  
Ascomycete Fungi ◽  
In Vitro Antifungal Activity

Antifungal proteins (AFPs) from ascomycete fungi could help the development of antimycotics. However, little is known about their biological role or functional interactions with other fungal biomolecules. We previously reported that AfpB from the postharvest pathogen Penicillium digitatum cannot be detected in the parental fungus yet is abundantly produced biotechnologically. While aiming to detect AfpB, we identified a conserved and novel small Secreted Cysteine-rich Anionic (Sca) protein, encoded by the gene PDIG_23520 from P. digitatum CECT 20796. The sca gene is expressed during culture and early during citrus fruit infection. Both null mutant (Δsca) and Sca overproducer (Scaop) strains show no phenotypic differences from the wild type. Sca is not antimicrobial but potentiates P. digitatum growth when added in high amounts and enhances the in vitro antifungal activity of AfpB. The Scaop strain shows increased incidence of infection in citrus fruit, similar to the addition of purified Sca to the wild-type inoculum. Sca compensates and overcomes the protective effect of AfpB and the antifungal protein PeAfpA from the apple pathogen Penicillium expansum in fruit inoculations. Our study shows that Sca is a novel protein that enhances the growth and virulence of its parental fungus and modulates the activity of AFPs.

Porcine sst1 can physically interact with other somatostatin receptors, and its expression is regulated by metabolic/inflammatory sensors

2014 ◽  
Vol 306 (5) ◽  
pp. E483-E493 ◽  
Author(s):  
Manuel D. Gahete ◽  
Mario Durán-Prado ◽  
Elena Delgado-Niebla ◽  
Juan J. Garrido ◽  
Simon J. Rhodes ◽  
...  
Keyword(s):  
Functional Characterization ◽  
Somatostatin Receptors ◽  
Amino Terminal ◽  
Mediated Effects ◽  
Relevant Role ◽  
Amino Terminal Domain ◽  
G Protein Coupled

The majority of the biological actions attributed to somatostatin (SST) are thought to be mediated by SST receptor 2 (sst2), the most ubiquitous sst, and, to a lesser extent, by sst5. However, a growing body of evidence suggests a relevant role of sst1 in mediating SST actions in (patho)physiological situations (i.e., endometriosis, type 2 diabetes). Moreover, sst1 together with sst2 and sst5 is involved in the well-known actions of SST on pituitary somatotropes in pig and primates. Here, we cloned the porcine sst1 (psst1) and performed a structural and functional characterization using both primary and heterologous models. The psst1 sequence presents the majority of signature motifs shared among G protein-coupled receptors and, specifically, among ssts and exhibits a high homology with other mammalian sst1, with only minor differences in the amino-terminal domain, reinforcing the idea of an early evolutive divergence between mammalian and nonmammalian sst1s. psst1 is functional in terms of decreasing cAMP levels in response to SST when transfected in heterologous models. The psst1 receptor is expressed in several tissues, and analyses of gene cis elements predict regulation by multiple transcription factors and metabolic stimuli. Finally, psst1 is coexpressed with other sst subtypes in various tissues, and in vitro data demonstrate that psst1 can interact with itself forming homodimers and with other ssts forming heterodimers. These data highlight the functional importance of sst1 on the SST-mediated effects and its functional interaction with different ssts, which point out the necessity of exploring the consequences of such interactions.

scholarly journals An Improved Method for the Study of Apoptosis-Related Genes Using the Tet-On System

2011 ◽  
Vol 16 (3) ◽  
pp. 332-337 ◽  
Author(s):  
Marc W. Halterman
Keyword(s):  
Gene Expression ◽  
Caspase 3 ◽  
Functional Characterization ◽  
Continuous Exposure ◽  
Genetic Screens ◽  
Pulse Dose ◽  
Target Effect ◽  
Regulated Gene Expression

Inducible gene expression systems are particularly useful for the functional characterization of genes with putative toxic properties. In the course of studying the role of hypoxia-regulated gene expression on cell survival using the tetracycline-inducible (tet-on) system, the author noted that exposure to the inducing ligand doxycycline (dox) inhibited caspase-3 cleavage in control samples. To limit this confounding off-target effect, he devised an in vitro pulse dose, delayed-injury protocol testing both dox and a novel tetracycline analog 9-t-butyl doxycycline (9-TB). Although 9-TB induced higher transgene levels compared to matched concentrations of dox, continuous exposure to both drugs inhibited caspase-3 cleavage in hypoxic samples. Conversely, a 6-h pulse dose of 9-TB followed by a 40-h washout period prior to hypoxic challenge activated robust transgene expression and lessened the inhibitory effects on caspase-3 processing. It is anticipated that these protocol modifications will improve the performance of tet-regulated genetic screens, particularly in situations where cell death is used as a primary end point.

scholarly journals Saquinavir Inhibits the Malaria Parasite's Chloroquine Resistance Transporter

2012 ◽  
Vol 56 (5) ◽  
pp. 2283-2289 ◽  
Author(s):  
Rowena E. Martin ◽  
Alice S. Butterworth ◽  
Donald L. Gardiner ◽  
Kiaran Kirk ◽  
James S. McCarthy ◽  
...  
Keyword(s):  
Plasmodium Falciparum ◽  
Heterologous Expression ◽  
Protease Inhibitors ◽  
Chloroquine Resistance ◽  
Content Type ◽  
Chloroquine Resistance Transporter ◽  
Resistant Phenotype ◽  
Transgenic Parasites

ABSTRACTThe antiretroviral protease inhibitors (APIs) ritonavir, saquinavir, and lopinavir, used to treat HIV infection, inhibit the growth ofPlasmodium falciparumat clinically relevant concentrations. Moreover, it has been reported that these APIs potentiate the activity of chloroquine (CQ) against this parasitein vitro. The mechanism underlying this effect is not understood, but the degree of chemosensitization varies between the different APIs and, with the exception of ritonavir, appears to be dependent on the parasite exhibiting a CQ-resistant phenotype. Here we report a study of the role of theP. falciparumchloroquine resistance transporter (PfCRT) in the interaction between CQ and APIs, using transgenic parasites expressing different PfCRT alleles and using theXenopus laevisoocyte system for the heterologous expression of PfCRT. Our data demonstrate that saquinavir behaves as a CQ resistance reverser and that this explains, at least in part, its ability to enhance the effects of CQ in CQ-resistantP. falciparumparasites.

scholarly journals Trypsin increases pseudorabies virus production through activation of the ERK signalling pathway

2006 ◽  
Vol 87 (5) ◽  
pp. 1109-1112 ◽  
Author(s):  
Béatrice Riteau ◽  
Christiane de Vaureix ◽  
François Lefèvre
Keyword(s):  
Pseudorabies Virus ◽  
Virus Production ◽  
Signalling Pathway ◽  
Herpesvirus Infection ◽  
Extracellular Proteases ◽  
Extracellular Signal ◽  
Inflammatory Processes ◽  
Infected Cells

Extracellular proteases that are expressed in primary and secondary foci of viral infection are potentially important mediators of infectious inflammatory processes. For some viruses, such as influenza virus and rotaviruses, proteases such as trypsin enhance infectivity by a direct proteolytic effect on some virion proteins. By using an in vitro model of herpesvirus infection, the possibility that proteases modulate the viral cycle through signalling delivered to the infected cell was investigated. It is reported that exposure of pseudorabies virus-infected cells to trypsin increased virus production. Moreover, this treatment induced synergistic and sustained activation of the extracellular signal-regulated kinase (ERK) 1/2 signalling pathway, which appeared to be necessary for this increased viral production. These results suggest that herpesviruses could take advantage of the inflammatory context and particularly of the presence of proteases to increase their replication. Thus, these data point to a potentially important role of extracellular proteases in herpesvirus infection.

scholarly journals Functional Analyses of the Diels-Alderase Gene sol5 of Ascochyta rabiei and Alternaria solani Indicate that the Solanapyrone Phytotoxins Are Not Required for Pathogenicity

2015 ◽  
Vol 28 (4) ◽  
pp. 482-496 ◽  
Author(s):  
Wonyong Kim ◽  
Chung-Min Park ◽  
Jeong-Jin Park ◽  
Hajime O. Akamatsu ◽  
Tobin L. Peever ◽  
...  
Keyword(s):  
Ascochyta Blight ◽  
Functional Characterization ◽  
Feedback Regulation ◽  
Alternaria Solani ◽  
Ascochyta Rabiei ◽  
Regulation Mechanism ◽  
Pathogenicity Tests ◽  
Functional Analyses

Ascochyta rabiei and Alternaria solani, the causal agents of Ascochyta blight of chickpea (Cicer arietinum) and early blight of potato (Solanum tuberosum), respectively, produce a set of phytotoxic compounds including solanapyrones A, B, and C. Although both the phytotoxicity of solanapyrones and their universal production among field isolates have been documented, the role of solanapyrones in pathogenicity is not well understood. Here, we report the functional characterization of the sol5 gene, which encodes a Diels-Alderase that catalyzes the final step of solanapyrone biosynthesis. Deletion of sol5 in both Ascochyta rabiei and Alternaria solani completely prevented production of solanapyrones and led to accumulation of the immediate precursor compound, prosolanapyrone II-diol, which is not toxic to plants. Deletion of sol5 did not negatively affect growth rate or spore production in vitro, and led to overexpression of the other solanapyrone biosynthesis genes, suggesting a possible feedback regulation mechanism. Phytotoxicity tests showed that solanapyrone A is highly toxic to several legume species and Arabidopsis thaliana. Despite the apparent phytotoxicity of solanapyrone A, pathogenicity tests showed that solanapyrone-minus mutants of Ascochyta rabiei and Alternaria solani were equally virulent as their corresponding wild-type progenitors, suggesting that solanapyrones are not required for pathogenicity.

scholarly journals Role of Staphylococcus aureus Global Regulators sae and σB in Virulence Gene Expression during Device-Related Infection

2005 ◽  
Vol 73 (6) ◽  
pp. 3415-3421 ◽  
Author(s):  
Christiane Goerke ◽  
Ursula Fluckiger ◽  
Andrea Steinhuber ◽  
Vittoria Bisanzio ◽  
Martina Ulrich ◽  
...  
Keyword(s):  
Gene Expression ◽  
Staphylococcus Aureus ◽  
Virulence Gene ◽  
Northern Analysis ◽  
Virulence Gene Expression ◽  
Global Regulators ◽  
Reverse Transcription Pcr ◽  
Direct Cross

ABSTRACT The ability of Staphylococcus aureus to adapt to different environments is due to a regulatory network comprising several loci. Here we present a detailed study of the interaction between the two global regulators sae and σB of S. aureus and their influence on virulence gene expression in vitro, as well as during device-related infection. The expression of sae, asp23, hla, clfA, coa, and fnbA was determined in strain Newman and its isogenic saeS/R and sigB mutants by Northern analysis and LightCycler reverse transcription-PCR. There was no indication of direct cross talk between the two regulators. sae had a dominant effect on target gene expression during device-related infection. σB seemed to be less active throughout the infection than under induced conditions in vitro.

Investigating the role of arginine 21 in the structure and function of human [alpha]a-crystallin

2018 ◽  
Author(s):  
◽  
Ashutosh Shripad Phadte
Keyword(s):  
Functional Characterization ◽  
Lens Fiber Cell ◽  
University Of Missouri ◽  
Mutant Proteins ◽  
Plausible Mechanism ◽  
And Function ◽  
The University

[ACCESS RESTRICTED TO THE UNIVERSITY OF MISSOURI AT AUTHOR'S REQUEST.] Cataractogenesis in the eye lens occurs as a result of protein aggregation. Of the multiple mutations in [alpha]A-crystallins associated with the development of congenital hereditary cataract, three identified mutations target R21 within the N- terminal domain of the protein. On structural and functional characterization of a recently identified mutant of [alpha]A-crystallin, [alpha]A-R21Q, we revealed the contribution of R21 in dictating the interaction of [alpha]A-crystallin with other proteins. [alpha]A-R21Q showed and enhanced chaperone-like function, and increased binding to lens fiber cell membranes. Transduction of mutant proteins in ARPE-19 cells prevented their apoptosis in the presence of oxidative stress, suggesting a role for R21 in modulating the anti-apoptotic function of [alpha]A-crystallin. In addition, the R21Q point mutation rescued the chaperone-like activity of [alpha]A-G98R crystallin as well as palliated [alpha]A-G98R mediated cytotoxicity otherwise observed in transduction experiments. Although another mutation, R157Q rescued the chaperone-like activity of [alpha]A-G98R, the double mutant exhibited a loss of its cytoprotective function. The results therefore implicate an important role of R21 in regulating the functional aspect of [alpha]A-crystallin. [alpha]A-crystallin derived peptides have been shown to prevent non-specific aggregation of unfolding proteins in vitro. We show that the [alpha]A-crystallin derived mini-chaperone (mini-[alpha]A) mediated stabilization of self-aggregating [alpha]A-G98R crystallin and bovine [subscript]-crystallin occurs via compensation of lost surface charge. The observation therefore suggests a plausible mechanism of action of [alpha]A-crystallin derived peptides of therapeutic interest.

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