scholarly journals Pseudomonas orientalis F9 Pyoverdine, Safracin, and Phenazine Mutants Remain Effective Antagonists against Erwinia amylovora in Apple Flowers

2020 ◽  
Vol 86 (8) ◽  
Author(s):  
Amanda Santos Kron ◽  
Veronika Zengerer ◽  
Marco Bieri ◽  
Vera Dreyfuss ◽  
Tanja Sostizzo ◽  
...  
Keyword(s):  
Fire Blight ◽  
Erwinia Amylovora ◽  
Ex Vivo ◽  
Pythium Ultimum ◽  
Content Type ◽  
Transposon Mutants ◽  
A Site ◽  
Transposon Insertions

ABSTRACT The recently characterized strain Pseudomonas orientalis F9, an isolate from apple flowers in a Swiss orchard, exhibits antagonistic traits against phytopathogens. At high colonization densities, it exhibits phytotoxicity against apple flowers. P. orientalis F9 harbors biosynthesis genes for the siderophore pyoverdine as well as for the antibiotics safracin and phenazine. To elucidate the role of the three compounds in biocontrol, we screened a large random knockout library of P. orientalis F9 strains for lack of pyoverdine production or in vitro antagonism. Transposon mutants that lacked the ability for fluorescence carried transposons in pyoverdine production genes. Mutants unable to antagonize Erwinia amylovora in an in vitro double-layer assay carried transposon insertions in the safracin gene cluster. As no phenazine transposon mutant could be identified using the chosen selection criteria, we constructed a site-directed deletion mutant. Pyoverdine-, safracin-, and phenazine mutants were tested for their abilities to counteract the fire blight pathogen Erwinia amylovora ex vivo on apple flowers or the soilborne pathogen Pythium ultimum in vivo in a soil microcosm. In contrast to some in vitro assays, ex vivo and in vivo assays did not reveal significant differences between parental and mutant strains in their antagonistic activities. This suggests that, ex vivo and in vivo, other factors, such as competition for resources or space, are more important than the tested antibiotics or pyoverdine for successful antagonism of P. orientalis F9 against phytopathogens in the performed assays. IMPORTANCE Pseudomonas orientalis F9 is an antagonist of the economically important phytopathogen Erwinia amylovora, the causal agent of fire blight in pomme fruit. On King’s B medium, P. orientalis F9 produces a pyoverdine siderophore and the antibiotic safracin. P. orientalis F9 transposon mutants lacking these factors fail to antagonize E. amylovora, depending on the in vitro assay. On isolated flowers and in soil microcosms, however, pyoverdine, safracin, and phenazine mutants control phytopathogens as clearly as their parental strains.

scholarly journals Development of Transposon Mutagenesis for Chlamydia muridarum

2019 ◽  
Vol 201 (23) ◽  
Author(s):  
Yibing Wang ◽  
Scott D. LaBrie ◽  
Steven J. Carrell ◽  
Robert J. Suchland ◽  
Zoe E. Dimond ◽  
...  
Keyword(s):  
Genetic Manipulation ◽  
Transposon Mutagenesis ◽  
Content Type ◽  
Chlamydia Muridarum ◽  
Transposon Mutants ◽  
Transposon Insertions ◽  
Glycogen Biosynthesis

ABSTRACT Functional genetic analysis of Chlamydia has been a challenge due to the historical genetic intractability of Chlamydia, although recent advances in chlamydial genetic manipulation have begun to remove these barriers. Here, we report the development of the Himar C9 transposon system for Chlamydia muridarum, a mouse-adapted Chlamydia species that is widely used in Chlamydia infection models. We demonstrate the generation and characterization of an initial library of 33 chloramphenicol (Cam)-resistant, green fluorescent protein (GFP)-expressing C. muridarum transposon mutants. The majority of the mutants contained single transposon insertions spread throughout the C. muridarum chromosome. In all, the library contained 31 transposon insertions in coding open reading frames (ORFs) and 7 insertions in intergenic regions. Whole-genome sequencing analysis of 17 mutant clones confirmed the chromosomal locations of the insertions. Four mutants with transposon insertions in glgB, pmpI, pmpA, and pmpD were investigated further for in vitro and in vivo phenotypes, including growth, inclusion morphology, and attachment to host cells. The glgB mutant was shown to be incapable of complete glycogen biosynthesis and accumulation in the lumen of mutant inclusions. Of the 3 pmp mutants, pmpI was shown to have the most pronounced growth attenuation defect. This initial library demonstrates the utility and efficacy of stable, isogenic transposon mutants for C. muridarum. The generation of a complete library of C. muridarum mutants will ultimately enable comprehensive identification of the functional genetic requirements for Chlamydia infection in vivo. IMPORTANCE Historical issues with genetic manipulation of Chlamydia have prevented rigorous functional genetic characterization of the ∼1,000 genes in chlamydial genomes. Here, we report the development of a transposon mutagenesis system for C. muridarum, a mouse-adapted Chlamydia species that is widely used for in vivo investigations of chlamydial pathogenesis. This advance builds on the pioneering development of this system for C. trachomatis. We demonstrate the generation of an initial library of 33 mutants containing stable single or double transposon insertions. Using these mutant clones, we characterized in vitro phenotypes associated with genetic disruptions in glycogen biosynthesis and three polymorphic outer membrane proteins.

scholarly journals Ex VivoMaturation Assay for Testing Antimalarial Sensitivity of Rodent Malaria Parasites

2016 ◽  
Vol 60 (11) ◽  
pp. 6859-6866 ◽  
Author(s):  
Zi Wei Chang ◽  
Benoit Malleret ◽  
Bruce Russell ◽  
Laurent Rénia ◽  
Carla Claser
Keyword(s):  
Ex Vivo ◽  
Single Step ◽  
Parasite Development ◽  
Ring Stage ◽  
Malaria Parasites ◽  
Rodent Malaria ◽  
Content Type ◽  
Parasite Biology

ABSTRACTEx vivoassay systems provide a powerful approach to studying human malaria parasite biology and to testing antimalarials. For rodent malaria parasites, short-termin vitroculture andex vivoantimalarial susceptibility assays are relatively cumbersome, relying onin vivopassage for synchronization, since ring-stage parasites are an essential starting material. Here, we describe a new approach based on the enrichment of ring-stagePlasmodium berghei,P. yoelii, andP. vinckei vinckeiusing a single-step Percoll gradient. Importantly, we demonstrate that the enriched ring-stage parasites develop synchronously regardless of the parasite strain or species used. Using a flow cytometry assay with Hoechst and ethidium or MitoTracker dye, we show that parasite development is easily and rapidly monitored. Finally, we demonstrate that this approach can be used to screen antimalarial drugs.

scholarly journals Intestinal IgA Regulates Expression of a Fructan Polysaccharide Utilization Locus in Colonizing Gut Commensal Bacteroides thetaiotaomicron

mBio ◽  
2019 ◽  
Vol 10 (6) ◽  
Author(s):  
Payal Joglekar ◽  
Hua Ding ◽  
Pablo Canales-Herrerias ◽  
Pankaj Jay Pasricha ◽  
Justin L. Sonnenburg ◽  
...  
Keyword(s):  
Gut Microbiota ◽  
Ex Vivo ◽  
Microbial Colonization ◽  
Bacteroides Thetaiotaomicron ◽  
Content Type ◽  
Microbial Utilization ◽  
Polysaccharide Utilization Locus ◽  
The Impact

ABSTRACT Gut-derived immunoglobulin A (IgA) is the most abundant antibody secreted in the gut that shapes gut microbiota composition and functionality. However, most of the microbial antigens targeted by gut IgA remain unknown, and the functional effects of IgA targeting these antigens are currently understudied. This study provides a framework for identifying and characterizing gut microbiota antigens targeted by gut IgA. We developed a small intestinal ex vivo culture assay to harvest lamina propria IgA from gnotobiotic mice, with the aim of identifying antigenic targets in a model human gut commensal, Bacteroides thetaiotaomicron VPI-5482. Colonization by B. thetaiotaomicron induced a microbe-specific IgA response that was reactive against diverse antigens, including capsular polysaccharides, lipopolysaccharides, and proteins. IgA against microbial protein antigens targeted membrane and secreted proteins with diverse functionalities, including an IgA specific against proteins of the polysaccharide utilization locus (PUL) that are necessary for utilization of fructan, which is an important dietary polysaccharide. Further analyses demonstrated that the presence of dietary fructan increased the production of fructan PUL-specific IgA, which then downregulated the expression of fructan PUL in B. thetaiotaomicron, both in vivo and in vitro. Since the expression of fructan PUL has been associated with the ability of B. thetaiotaomicron to colonize the gut in the presence of dietary fructans, our work suggests a novel role for gut IgA in regulating microbial colonization by modulating their metabolism. IMPORTANCE Given the significant impact that gut microbes have on our health, it is essential to identify key host and environmental factors that shape this diverse community. While many studies have highlighted the impact of diet on gut microbiota, little is known about how the host regulates this critical diet-microbiota interaction. In our present study, we discovered that gut IgA targeted a protein complex involved in the utilization of an important dietary polysaccharide: fructan. While the presence of dietary fructans was previously thought to allow unrestricted growth of fructan-utilizing bacteria, our work shows that gut IgA, by targeting proteins responsible for fructan utilization, provides the host with tools that can restrict the microbial utilization of such polysaccharides, thereby controlling their growth.

scholarly journals Management of Erwinia amylovora by Potential Bio-Pesticides in vitro and in vivo Conditions

2020 ◽  
Vol 8 (sp1) ◽  
pp. 38-45
Author(s):  
Kubilay Kurtulus Bastas
Keyword(s):  
Fire Blight ◽  
Erwinia Amylovora ◽  
Shoot Length ◽  
Absolute Methanol ◽  
Diffusion Method ◽  
Thymus Vulgaris ◽  
Blight Disease ◽  
Santa Maria

Erwinia amylovora, the causative agent of fire blight disease, threatens a lot of species of the Rosaceae family. Antibiotics and copper compounds in chemical applications are most frequently are applied, but these can be phytotoxic and cause resistant strains of the pathogen. In our experiments, 20 herbal materials were tested for their antimicrobial effectiveness against the fire blight pathogen in vitro and in planta. The air-dried plants ground into fine powder and extraction was performed at room temperature by maceration with 80% (v/v) methanol/distilled water. The minimum inhibitory concentration values were determined by using disc diffusion method and streptomycin was used as control in all experiments. Antimicrobial activity was evaluated by measuring the inhibition zones in reference to the pathogen. Among the tested plants, Szygium aromaticum, Thymus vulgaris and Rhus cararia showed a good antibacterial activity and they inhibited the growth of E. amylovora with inhibition zone diameter ranging from 21 to 27 mm at 20% (w/v) in absolute methanol compared to streptomycin (31 mm) in vitro conditions. In vivo tests were performed by using highly virulent E. amylovora isolate (Eak24b, 91%) grown on TSA medium and inoculation on young shoots of 3-year-old Gala variety of apple and Santa Maria variety of pear seedlings at 107 CFU ml-1 density of the pathogen. Disease severity (%) was assessed by by proportion of blighted shoot length to the whole shoot length and also efficacy of the extracts was determined by using Abbott formula. The highest efficacy was obtained by S. aromaticum and T. vulgaris extracts of reducing shoot blight of cv. Gala and cv. Santa Maria by 67.81% - 64-12% and 51.50% - 51.04% ratios, respectively. Obtaining results showed that some medicinal and aromatic plant extracts might be used against fire blight disease as potential new generation chemicals on pome fruits within integrated and organic control programs.

scholarly journals Listeria monocytogenes MenI Encodes a DHNA-CoA Thioesterase Necessary for Menaquinone Biosynthesis, Cytosolic Survival, and Virulence

2021 ◽  
Vol 89 (5) ◽  
Author(s):  
Hans B. Smith ◽  
Tin Lok Li ◽  
Man Kit Liao ◽  
Grischa Y. Chen ◽  
Zhihong Guo ◽  
...  
Keyword(s):  
Listeria Monocytogenes ◽  
Ex Vivo ◽  
Strong Activity ◽  
Content Type ◽  
Transport Chain ◽  
Intermediate Metabolites ◽  
Bacterial Replication ◽  
Functional Analyses

ABSTRACT Listeria monocytogenes is a Gram-positive, intracellular pathogen that is highly adapted to invade and replicate in the cytosol of eukaryotic cells. Intermediate metabolites in the menaquinone biosynthesis pathway are essential for the cytosolic survival and virulence of L. monocytogenes, independent of the production of menaquinone (MK) and aerobic respiration. Determining which specific intermediate metabolite(s) are essential for cytosolic survival and virulence has been hindered by the lack of an identified 1,4-dihydroxy-2-naphthoyl-coenzyme A (DHNA-CoA) thioesterase essential for converting DHNA-CoA to DHNA in the MK synthesis pathway. Using the recently identified Escherichia coli DHNA-CoA thioesterase as a query, homology sequence analysis revealed a single homolog in L. monocytogenes, LMRG_02730. Genetic deletion of LMRG_02730 resulted in an ablated membrane potential, indicative of a nonfunctional electron transport chain (ETC) and an inability to aerobically respire. Biochemical kinetic analysis of LMRG_02730 revealed strong activity toward DHNA-CoA, similar to its E. coli homolog, further demonstrating that LMRG_02730 is a DHNA-CoA thioesterase. Functional analyses in vitro, ex vivo, and in vivo using mutants directly downstream and upstream of LMRG_02730 revealed that DHNA-CoA is sufficient to facilitate in vitro growth in minimal medium, intracellular replication, and plaque formation in fibroblasts. In contrast, protection against bacteriolysis in the cytosol of macrophages and tissue-specific virulence in vivo requires the production of 1,4-dihydroxy-2-naphthoate (DHNA). Taken together, these data implicate LMRG_02730 (renamed MenI) as a DHNA-CoA thioesterase and suggest that while DHNA, or an unknown downstream product of DHNA, protects the bacteria from killing in the macrophage cytosol, DHNA-CoA is necessary for intracellular bacterial replication.

Applications of monoclonal antibodies in the diagnosis and treatment of primary brain tumors

1985 ◽  
Vol 63 (1) ◽  
pp. 2-16 ◽  
Author(s):  
Dennis E. Bullard ◽  
Darell D. Bigner
Keyword(s):  
Monoclonal Antibodies ◽  
Ex Vivo ◽  
Neuroectodermal Tumor ◽  
Diagnosis And Treatment ◽  
Early Application ◽  
Content Type ◽  
Tumor Associated Antigens ◽  
Neuroectodermal Tumors

✓ The development of monoclonal antibodies has resulted in marked expansion in understanding the central nervous system (CNS). This has been especially true in the study of human neuroectodermal tumors where monoclonal antibodies have been used as physiological probes to define and characterize human neuroectodermal tumor-associated antigens. Utilizing monoclonal antibodies, neuroectodermal tumor-associated antigens have been described in four broad categories; biochemically defined markers, shared nervous systemlymphoid cell markers, shared neuroectodermal-oncofetal markers, and putative restricted tumor markers. Preliminary data have demonstrated the ability to localize animal and human tumors in vitro, ex vivo, and in vivo. Early application of monoclonal antibody technology to neuroimmunology and neuro-oncology has resulted in a new awareness of the complex relationships that exist within the CNS. Their specificity and reproducibility may provide the means to qualitatively and quantitatively define the phenotypic heterogeneity of human neuroectodermal tumors. Potentially, monoclonal antibodies, alone or as carriers of radionuclides, drugs, or toxins, may allow successful diagnosis and treatment of human neuroectodermal tumors.

scholarly journals In Vitro, Ex Vivo, and In Vivo Activities of Diamidines against Trypanosoma congolense and Trypanosoma vivax

2017 ◽  
Vol 61 (5) ◽  
Author(s):  
Kirsten Gillingwater ◽  
Christina Kunz ◽  
Christiane Braghiroli ◽  
David W. Boykin ◽  
Richard R. Tidwell ◽  
...  
Keyword(s):  
Ex Vivo ◽  
Trypanosoma Congolense ◽  
Tsetse Fly ◽  
Sub Saharan Africa ◽  
Content Type ◽  
Lead Compounds ◽  
Single Bolus Dose ◽  
Sub Saharan

ABSTRACT African animal trypanosomosis (AAT) is caused by the tsetse fly-transmitted protozoans Trypanosoma congolense and T. vivax and leads to huge agricultural losses throughout sub-Saharan Africa. Three drugs are available to treat nagana in cattle (diminazene diaceturate, homidium chloride, and isometamidium chloride). With increasing reports of drug-resistant populations, new molecules should be investigated as potential candidates to combat nagana. Dicationic compounds have been demonstrated to have excellent efficacy against different kinetoplastid parasites. This study therefore evaluated the activities of 37 diamidines, using in vitro and ex vivo drug sensitivity assays. The 50% inhibitory concentrations obtained ranged from 0.007 to 0.562 μg/ml for T. congolense and from 0.019 to 0.607 μg/ml for T. vivax. On the basis of these promising results, 33 of these diamidines were further examined using in vivo mouse models of infection. Minimal curative doses of 1.25 mg/kg of body weight for both T. congolense- and T. vivax-infected mice were seen when the diamidines were administered intraperitoneally (i.p.) over 4 consecutive days. From these observations, 15 of these 33 diamidines were then further tested in vivo, using a single bolus dose for administration. The total cure of mice infected with T. congolense and T. vivax was seen with single i.p. doses of 5 and 2.5 mg/kg, respectively. This study identified a selection of diamidines which could be considered lead compounds for the treatment of nagana.

scholarly journals Effect of host-mimicking medium and biofilm growth on the ability of colistin to kill Pseudomonas aeruginosa

Microbiology ◽  
2020 ◽  
Vol 166 (12) ◽  
pp. 1171-1180 ◽  
Author(s):  
Esther Sweeney ◽  
Akshay Sabnis ◽  
Andrew M. Edwards ◽  
Freya Harrison
Keyword(s):  
Pseudomonas Aeruginosa ◽  
Ex Vivo ◽  
Clinical Success ◽  
Biofilm Growth ◽  
Content Type ◽  
The Matrix ◽  
Model Versus ◽  
High Doses

In vivo biofilms cause recalcitrant infections with extensive and unpredictable antibiotic tolerance. Here, we demonstrate increased tolerance of colistin by Pseudomonas aeruginosa when grown in medium that mimics cystic fibrosis (CF) sputum versus standard medium in in vitro biofilm assays, and drastically increased tolerance when grown in an ex vivo CF model versus the in vitro assay. We used colistin conjugated to the fluorescent dye BODIPY to assess the penetration of the antibiotic into ex vivo biofilms and showed that poor penetration partly explains the high doses of drug necessary to kill bacteria in these biofilms. The ability of antibiotics to penetrate the biofilm matrix is key to their clinical success, but hard to measure. Our results demonstrate both the importance of reduced entry into the matrix in in vivo-like biofilm, and the tractability of using a fluorescent tag and benchtop fluorimeter to assess antibiotic entry into biofilms. This method could be a relatively quick, cheap and useful addition to diagnostic and drug development pipelines, allowing the assessment of drug entry into biofilms, in in vivo-like conditions, prior to more detailed tests of biofilm killing.

scholarly journals Infectivity of Plasmodium falciparum in Malaria-Naive Individuals Is Related to Knob Expression and Cytoadherence of the Parasite

2016 ◽  
Vol 84 (9) ◽  
pp. 2689-2696 ◽  
Author(s):  
Danielle I. Stanisic ◽  
John Gerrard ◽  
James Fink ◽  
Paul M. Griffin ◽  
Xue Q. Liu ◽  
...  
Keyword(s):  
Plasmodium Falciparum ◽  
Ex Vivo ◽  
Cell Bank ◽  
Human Environment ◽  
Content Type ◽  
Antibody Recognition ◽  
Cell Banks ◽  
Study Participants

Plasmodium falciparumis the most virulent human malaria parasite because of its ability to cytoadhere in the microvasculature. Nonhuman primate studies demonstrated relationships among knob expression, cytoadherence, and infectivity. This has not been examined in humans. Cultured clinical-gradeP. falciparumparasites (NF54, 7G8, and 3D7B) andex vivo-derived cell banks were characterized. Knob and knob-associated histidine-rich protein expression, CD36 adhesion, and antibody recognition of parasitized erythrocytes (PEs) were evaluated. Parasites from the cell banks were administered to malaria-naive human volunteers to explore infectivity. For the NF54 and 3D7B cell banks, blood was collected from the study participants forin vitrocharacterization. All parasites were infectivein vivo. However, infectivity of NF54 was dramatically reduced.In vitrocharacterization revealed that unlike other cell bank parasites, NF54 PEs lacked knobs and did not cytoadhere. Recognition of NF54 PEs by immune sera was observed, suggestingP. falciparumerythrocyte membrane protein 1 expression. Subsequent recovery of knob expression and CD36-mediated adhesion were observed in PEs derived from participants infected with NF54. Knobless cell bank parasites have a dramatic reduction in infectivity and the ability to adhere to CD36. Subsequent infection of malaria-naive volunteers restored knob expression and CD36-mediated cytoadherence, thereby showing that the human environment can modulate virulence.

scholarly journals Promethazine Exhibits Antiparasitic Properties In Vitro and Reduces Worm Burden, Egg Production, Hepatomegaly, and Splenomegaly in a Schistosomiasis Animal Model

2019 ◽  
Vol 63 (12) ◽  
Author(s):  
Daniel B. Roquini ◽  
Ramon M. Cogo ◽  
Ana C. Mengarda ◽  
Susana F. Mazloum ◽  
Cristiane S. Morais ◽  
...  
Keyword(s):  
Animal Model ◽  
Worm Burden ◽  
Egg Production ◽  
Ex Vivo ◽  
Anticholinergic Drug ◽  
Drug Repurposing ◽  
Content Type ◽  
Parasite Motility

ABSTRACT The treatment and control of schistosomiasis, a neglected disease that affects more than 200 million people worldwide, rely on the use of a single drug, praziquantel. A vaccine has yet to be developed, and since new drug design and development is a lengthy and costly process, drug repurposing is a promising strategy. In this study, the efficacy of promethazine, a first-generation antihistamine, was evaluated against Schistosoma mansoni ex vivo and in a murine model of schistosomiasis. In vitro assays demonstrated that promethazine affected parasite motility and viability, and it induced severe tegumental damage in schistosomes. The 50% lethal concentration (LC50) of the drug was 5.84 μM. Similar to promethazine, schistosomes incubated with atropine, a classical anticholinergic drug, displayed reduced motor activity. In an animal model, promethazine treatment was introduced at an oral dose of 100 mg/kg of body weight for five successive days at different intervals from the time of infection for the evaluation of the stage-specific susceptibility (prepatent and patent infections). Various parasitological criteria indicated the following in vivo antischistosomal effects of promethazine: there were significant reductions in worm burden, egg production, hepatomegaly, and splenomegaly. The highest worm burden reduction was achieved with promethazine in patent infections (>90%). Taken together, considering the importance of the repositioning of drugs in infectious diseases, especially those related to poverty, our data revealed the possibility of promethazine repositioning as an antischistosomal agent.

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