scholarly journals Elizabethkingia anophelis: Molecular Manipulation and Interactions with Mosquito Hosts

2015 ◽  
Vol 81 (6) ◽  
pp. 2233-2243 ◽  
Author(s):  
Shicheng Chen ◽  
Michael Bagdasarian ◽  
Edward D. Walker
Keyword(s):  
Fluorescent Protein ◽  
Developmental Stages ◽  
Genetic Manipulation ◽  
Life Stage ◽  
Expression System ◽  
Aedes Triseriatus ◽  
Content Type ◽  
Mosquito Midgut

ABSTRACTFlavobacteria (members of the familyFlavobacteriaceae) dominate the bacterial community in theAnophelesmosquito midgut. One such commensal,Elizabethkingia anophelis, is closely associated withAnophelesmosquitoes through transstadial persistence (i.e., from one life stage to the next); these and other properties favor its development for paratransgenic applications in control of malaria parasite transmission. However, the physiological requirements ofE. anophelishave not been investigated, nor has its capacity to perpetuate despite digestion pressure in the gut been quantified. To this end, we first developed techniques for genetic manipulation ofE. anophelis, including selectable markers, reporter systems (green fluorescent protein [GFP] and NanoLuc), and transposons that function inE. anophelis. A flavobacterial expression system based on the promoter PompAwas integrated into theE. anophelischromosome and showed strong promoter activity to drive GFP and NanoLuc reporter production. Introduced, GFP-taggedE. anophelisassociated with mosquitoes at successive developmental stages and propagated inAnopheles gambiaeandAnopheles stephensibut not inAedes triseriatusmosquitoes. Feeding NanoLuc-tagged cells toA. gambiaeandA. stephensiin the larval stage led to infection rates of 71% and 82%, respectively. In contrast, a very low infection rate (3%) was detected inAedes triseriatusmosquitoes under the same conditions. Of the initialE. anopheliscells provided to larvae, 23%, 71%, and 85% were digested inA. stephensi,A. gambiae, andAedes triseriatus, respectively, demonstrating thatE. anophelisadapted to various mosquito midgut environments differently. Bacterial cell growth increased up to 3-fold when arginine was supplemented in the defined medium. Furthermore, the number of NanoLuc-tagged cells inA. stephensisignificantly increased when arginine was added to a sugar diet, showing it to be an important amino acid forE. anophelis. Animal erythrocytes promotedE. anophelisgrowthin vivoandin vitro, indicating that this bacterium could obtain nutrients by participating in erythrocyte lysis in the mosquito midgut.

scholarly journals Improving Baculovirus Infectivity by Efficiently Embedding Enhancing Factors into Occlusion Bodies

2017 ◽  
Vol 83 (14) ◽  
Author(s):  
Shili Yang ◽  
Lijuan Zhao ◽  
Ruipeng Ma ◽  
Wei Fang ◽  
Jia Hu ◽  
...  
Keyword(s):  
Protein Expression ◽  
Fluorescent Protein ◽  
Expression System ◽  
Agrotis Segetum ◽  
Foreign Protein ◽  
Content Type ◽  
Occlusion Bodies ◽  
Foreign Proteins ◽  
Novel Strategy

ABSTRACT The relatively low infectivity of baculoviruses to their host larvae limits their use as insecticidal agents on a larger scale. In the present study, a novel strategy was developed to efficiently embed foreign proteins into Autographa californica multiple nucleopolyhedrovirus (AcMNPV) occlusion bodies (OBs) to achieve stable expression of foreign proteins and to improve viral infectivity. A recombinant AcMNPV bacmid was constructed by expressing the 150-amino-acid (aa) N-terminal segment of polyhedrin under the control of the p10 promoter and the remaining C-terminal 95-aa segment under the control of the polyhedrin promoter. The recombinant virus formed OBs in Spodoptera frugiperda 9 cells, in which the occlusion-derived viruses were embedded in a manner similar to that for wild-type AcMNPV. Next, the 95-aa polyhedrin C terminus was fused to enhanced green fluorescent protein, and the recombinant AcMNPV formed fluorescent green OBs and was stably passaged in vitro and in vivo. The AcMNPV recombinants were further modified by fusing truncated Agrotis segetum granulovirus enhancin or truncated Cydia pomonella granulovirus ORF13 (GP37) to the C-terminal 95 aa of polyhedrin, and both recombinants were able to form normal OBs. Bioactivity assays indicated that the median lethal concentrations of these two AcMNPV recombinants were 3- to 5-fold lower than that of the control virus. These results suggest that embedding enhancing factors in baculovirus OBs by use of this novel technique may promote efficient and stable foreign protein expression and significantly improve baculovirus infectivity. IMPORTANCE Baculoviruses have been used as bioinsecticides for over 40 years, but their relatively low infectivity to their host larvae limits their use on a larger scale. It has been reported that it is possible to improve baculovirus infectivity by packaging enhancing factors within baculovirus occlusion bodies (OBs); however, so far, the packaging efficiency has been low. In this article, we describe a novel strategy for efficiently embedding foreign proteins into AcMNPV OBs by expressing N- and C-terminal (dimidiate) polyhedrin fragments (150 and 95 amino acids, respectively) as fusions to foreign proteins under the control of the p10 and polyhedrin promoters, respectively. When this strategy was used to embed an enhancing factor (enhancin or GP37) into the baculovirus OBs, 3- to 5-fold increases in baculoviral infectivity were observed. This novel strategy has the potential to create an efficient protein expression system and a highly efficient virus-based system for insecticide production in the future.

scholarly journals Nonselective Persistence of a Rickettsia conorii Extrachromosomal Plasmid during Mammalian Infection

2016 ◽  
Vol 84 (3) ◽  
pp. 790-797 ◽  
Author(s):  
Sean P. Riley ◽  
Abigail I. Fish ◽  
Daniel A. Garza ◽  
Kaikhushroo H. Banajee ◽  
Emma K. Harris ◽  
...  
Keyword(s):  
Pathogenic Bacteria ◽  
Genetic Manipulation ◽  
Fluorescent Proteins ◽  
Spotted Fever ◽  
Rickettsia Conorii ◽  
Mediterranean Spotted Fever ◽  
Extrachromosomal Dna ◽  
Content Type

Scientific analysis of the genusRickettsiais undergoing a rapid period of change with the emergence of viable genetic tools. The development of these tools for the mutagenesis of pathogenic bacteria will permit forward genetic analysis ofRickettsiapathogenesis. Despite these advances, uncertainty still remains regarding the use of plasmids to study these bacteria inin vivomammalian models of infection, namely, the potential for virulence changes associated with the presence of extrachromosomal DNA and nonselective persistence of plasmids in mammalian models of infection. Here, we describe the transformation ofRickettsia conoriiMalish 7 with the plasmid pRam18dRGA[AmTrCh]. TransformedR. conoriistably maintains this plasmid in infected cell cultures, expresses the encoded fluorescent proteins, and exhibits growth kinetics in cell culture similar to those of nontransformedR. conorii. Using a well-established murine model of fatal Mediterranean spotted fever, we demonstrate thatR. conorii(pRam18dRGA[AmTrCh]) elicits the same fatal outcomes in animals as its untransformed counterpart and, importantly, maintains the plasmid throughout infection in the absence of selective antibiotic pressure. Interestingly, plasmid-transformedR. conoriiwas readily observed both in endothelial cells and within circulating leukocytes. Together, our data demonstrate that the presence of an extrachromosomal DNA element in a pathogenic rickettsial species does not affect eitherin vitroproliferation orin vivoinfectivity in models of disease and that plasmids such as pRam18dRGA[AmTrCh] are valuable tools for the further genetic manipulation of pathogenic rickettsiae.

scholarly journals Development of a Tetracycline-Inducible Gene Expression System for the Study of Helicobacter pylori Pathogenesis

2013 ◽  
Vol 79 (23) ◽  
pp. 7351-7359 ◽  
Author(s):  
Aleksandra W. Debowski ◽  
Phebe Verbrugghe ◽  
Miriam Sehnal ◽  
Barry James Marshall ◽  
Mohammed Benghezal
Keyword(s):  
Helicobacter Pylori ◽  
Animal Models ◽  
Chronic Infection ◽  
Fluorescent Protein ◽  
Expression System ◽  
Content Type ◽  
Conditional Expression ◽  
H Pylori ◽  
Green Fluorescent

ABSTRACTDeletion mutants and animal models have been instrumental in the study ofHelicobacter pyloripathogenesis. Conditional mutants, however, would enable the study of the temporal gene requirement duringH. pyloricolonization and chronic infection. To achieve this goal, we adapted theEscherichia coliTn10-derived tetracycline-inducible expression system for use inH. pylori. TheureApromoter was modified by inserting one or twotetoperators to generate tetracycline-responsive promoters, nameduPtetO, and these promoters were then fused to the reportergfpmut2 and inserted into different loci. The expression of the tetracycline repressor (tetR) was placed under the control of one of three promoters and inserted into the chromosome. Conditional expression of green fluorescent protein (GFP) in strains harboringtetRanduPtetO-GFPwas characterized by measuring GFP activity and by immunoblotting. The twotet-responsiveuPtetOpromoters differ in strength, and induction of these promoters was inducer concentration and time dependent, with maximum expression achieved after induction for 8 to 16 h. Furthermore, the chromosomal location of theuPtetO-GFPconstruct and the nature of the promoter driving expression oftetRinfluenced the strength of theuPtetOpromoters upon induction. Integration ofuPtetO-GFPandtetRconstructs at different genomic loci was stablein vivoand did not affect colonization. Finally, we demonstrate tetracycline-dependent induction of GFP expressionin vivoduring chronic infection. These results open new experimental avenues for dissectingH. pyloripathogenesis using animal models and for testing the roles of specific genes in colonization of, adaptation to, and persistence in the host.

scholarly journals Holotoxin Activity of Botulinum Neurotoxin Subtype A4 Originating from a Nontoxigenic Clostridium botulinum Expression System

2014 ◽  
Vol 80 (23) ◽  
pp. 7415-7422 ◽  
Author(s):  
Marite Bradshaw ◽  
William H. Tepp ◽  
Regina C. M. Whitemarsh ◽  
Sabine Pellett ◽  
Eric A. Johnson
Keyword(s):  
Clostridium Botulinum ◽  
Botulinum Neurotoxin ◽  
Expression System ◽  
Single Amino Acid ◽  
Content Type ◽  
Expression Host ◽  
Toxin Complex ◽  
Human Neuronal Cells

ABSTRACTClostridium botulinumsubtype A4 neurotoxin (BoNT/A4) is naturally expressed in the dual-toxin-producingC. botulinumstrain 657Ba at 100× lower titers than BoNT/B. In this study, we describe purification of recombinant BoNT/A4 (rBoNT/A4) expressed in a nonsporulating and nontoxigenicC. botulinumexpression host strain. The rBoNT/A4 copurified with nontoxic toxin complex components provided intransby the expression host and was proteolytically cleaved to the active dichain form. Activity of the recombinant BoNT/A4 in mice and in human neuronal cells was about 1,000-fold lower than that of BoNT/A1, and the recombinant BoNT/A4 was effectively neutralized by botulism heptavalent antitoxin. A previous report using recombinant truncated BoNT/A4 light chain (LC) expressed inEscherichia colihas indicated reduced stability and activity of BoNT/A4 LC compared to BoNT/A1 LC, which was surmounted by introduction of a single-amino-acid substitution, I264R. In order to determine whether this mutation would also affect the holotoxin activity of BoNT/A4, a recombinant full-length BoNT/A4 carrying this mutation as well as a second mutation predicted to increase solubility (L260F) was produced in the clostridial expression system. Comparative analyses of thein vitro, cellular, andin vivoactivities of rBoNT/A4 and rBoNT/A4-L260F I264R showed 1,000-fold-lower activity than BoNT/A1 in both the mutated and nonmutated BoNT/A4. This indicates that these mutations do not alter the activity of BoNT/A4 holotoxin. In summary, a recombinant BoNT from a dual-toxin-producing strain was expressed and purified in an endogenous clostridial expression system, allowing analysis of this toxin.

scholarly journals Visualization of Periplasmic and Cytoplasmic Proteins with a Self-Labeling Protein Tag

2016 ◽  
Vol 198 (7) ◽  
pp. 1035-1043 ◽  
Author(s):  
Na Ke ◽  
Dirk Landgraf ◽  
Johan Paulsson ◽  
Mehmet Berkmen
Keyword(s):  
Escherichia Coli ◽  
Fluorescent Protein ◽  
Fluorescent Proteins ◽  
Living Cells ◽  
Functional Protein ◽  
Content Type ◽  
Additional Tool

ABSTRACTThe use of fluorescent and luminescent proteins in visualizing proteins has become a powerful tool in understanding molecular and cellular processes within living organisms. This success has resulted in an ever-increasing demand for new and more versatile protein-labeling tools that permit light-based detection of proteins within living cells. In this report, we present data supporting the use of the self-labeling HaloTag protein as a light-emitting reporter for protein fusions within the model prokaryoteEscherichia coli. We show that functional protein fusions of the HaloTag can be detected bothin vivoandin vitrowhen expressed within the cytoplasmic or periplasmic compartments ofE. coli. The capacity to visually detect proteins localized in various prokaryotic compartments expands today's molecular biologist toolbox and paves the path to new applications.IMPORTANCEVisualizing proteins microscopically within living cells is important for understanding both the biology of cells and the role of proteins within living cells. Currently, the most common tool is green fluorescent protein (GFP). However, fluorescent proteins such as GFP have many limitations; therefore, the field of molecular biology is always in need of new tools to visualize proteins. In this paper, we demonstrate, for the first time, the use of HaloTag to visualize proteins in two different compartments within the model prokaryoteEscherichia coli. The use of HaloTag as an additional tool to visualize proteins within prokaryotes increases our capacity to ask about and understand the role of proteins within living cells.

scholarly journals BEM46 Shows Eisosomal Localization and Association with Tryptophan-Derived Auxin Pathway in Neurospora crassa

2014 ◽  
Vol 13 (8) ◽  
pp. 1051-1063 ◽  
Author(s):  
K. Kollath-Leiß ◽  
C. Bönniger ◽  
P. Sardar ◽  
F. Kempken
Keyword(s):  
Neurospora Crassa ◽  
Fluorescent Protein ◽  
Developmental Stages ◽  
Yellow Fluorescent Protein ◽  
Content Type ◽  
Bioinformatic Tools ◽  
Ascospore Germination ◽  
Alternatively Spliced ◽  
Indole Production

ABSTRACTBEM46 proteins are evolutionarily conserved, but their functions remain elusive. We reported previously that the BEM46 protein inNeurospora crassais targeted to the endoplasmic reticulum (ER) and is essential for ascospore germination. In the present study, we established abem46knockout strain ofN. crassa. This Δbem46mutant exhibited a level of ascospore germination lower than that of the wild type but much higher than those of the previously characterizedbem46-overexpressing and RNA interference (RNAi) lines. Reinvestigation of the RNAi transformants revealed two types of alternatively splicedbem46mRNA; expression of either type led to a loss of ascospore germination. Our results indicated that the phenotype was not due tobem46mRNA downregulation or loss but was caused by the alternatively spliced mRNAs and the peptides they encoded. Using theN. crassaortholog of the eisosomal protein PILA fromAspergillus nidulans, we further demonstrated the colocalization of BEM46 with eisosomes. Employing the yeast two-hybrid system, we identified a single interaction partner: anthranilate synthase component II (encoded bytrp-1). This interaction was confirmedin vivoby a split-YFP (yellow fluorescent protein) approach. The Δtrp-1mutant showed reduced ascospore germination and increased indole production, and we used bioinformatic tools to identify a putative auxin biosynthetic pathway. The genes involved exhibited various levels of transcriptional regulation in the differentbem46transformant and mutant strains. We also investigated the indole production of the strains in different developmental stages. Our findings suggested that the regulation of indole biosynthesis genes was influenced bybem46overexpression. Furthermore, we uncovered evidence of colocalization of BEM46 with the neutral amino acid transporter MTR.

scholarly journals Positive and Negative Regulation of a SNARE Protein by Control of Intracellular Localization

2004 ◽  
Vol 15 (4) ◽  
pp. 1802-1815 ◽  
Author(s):  
Hideki Nakanishi ◽  
Pablo de los Santos ◽  
Aaron M. Neiman
Keyword(s):  
Fluorescent Protein ◽  
Genetic Manipulation ◽  
Intracellular Localization ◽  
Negative Regulation ◽  
Snare Protein ◽  
Nuclear Targeting ◽  
Protein Receptor ◽  
Prospore Membrane

In Saccharomyces cerevisiae, the developmentally regulated Soluble N-ethylmaleimide sensitive factor attachment protein receptor (SNARE) protein Spo20p mediates the fusion of vesicles with the prospore membrane, which is required for the formation of spores. Spo20p is subject to both positive and negative regulation by separate sequences in its aminoterminal domain. We report that the positive activity is conferred by a short, amphipathic helix that is sufficient to confer plasma membrane or prospore membrane localization to green fluorescent protein. In vitro, this helix binds to acidic phospholipids, and mutations that reduce or eliminate phospholipid binding in vitro inactivate Spo20p in vivo. Genetic manipulation of phospholipid pools indicates that the likely in vivo ligand of this domain is phosphatidic acid. The inhibitory activity is a nuclear targeting signal, which confers nuclear localization in vegetative cells and in cells entering meiosis. However, as cells initiate spore formation, fusions containing the inhibitory domain exit the nucleus and localize to the nascent prospore membrane. Thus, the SNARE Spo20p is both positively and negatively regulated by control of its intracellular localization.

scholarly journals Superior Pyronaridine Single-Dose Pharmacodynamics Compared to Artesunate, Chloroquine, and Amodiaquine in a Murine Malaria Luciferase Model

2018 ◽  
Vol 62 (9) ◽  
Author(s):  
Winter A. Okoth ◽  
Elijah J. Dukes ◽  
David J. Sullivan
Keyword(s):  
Single Dose ◽  
Fluorescent Protein ◽  
Lag Phase ◽  
Content Type ◽  
Log Reduction ◽  
Drug Inhibition ◽  
Drug Studies ◽  
Dose Dependent

ABSTRACTMany previousin vitroandin vivopreclinical malaria drug studies have relied on low-parasite-number drug inhibition numerically compared to the untreated controls. In contrast, human malaria drug studies measure the high-parasite-density killing near 100 million/ml. Here we compared thein vivosingle-dose pharmacodynamic properties of artesunate and the 4-aminoquinolines pyronaridine, chloroquine, and amodiaquine in aPlasmodium bergheiANKA-green fluorescent protein GFP-luciferase-based murine malaria blood-stage model. Pyronaridine exhibited dose-dependent killing, achieving parasite reductions near 5 to 6 logs at 48 h, with complete cure at 10 mg/kg of body weight compared to artesunate, which exhibited a 48-h dose-dependent killing with a 2-log drop at the noncurative 250-mg/kg dose. Chloroquine, which was noncurative, and amodiaquine, which was partially curative, had nearly the same initial dose-independent killing, with a lag phase of minimal parasite reduction at all doses between 6 and 24 h, followed by a 2.5-log reduction at 48 h. In experiments with drug-treated, washed infected blood transfer to naive mice, chloroquine and amodiaquine showed fewer viable parasites at the 24-h transfer than at the 8-h transfer, measured by a prolonged return to parasitemia, despite a similar parasite log reduction at these time points, in contrast to the correlation of the parasite log reduction to viable parasites with artesunate and pyronaridine. Artesunate in combination with pyronaridine exhibited an initial parasite reduction similar to that achieved with pyronaridine, while with chloroquine or amodiaquine, the reduction was similar to that achieved with artesunate. Single-oral-dose pyronaridine was much more potentin vivothan artesunate, chloroquine, and amodiaquine during the initial decline in parasites and cure.

scholarly journals Plasma Cell Ontogeny Defined by Quantitative Changes in Blimp-1 Expression

2004 ◽  
Vol 200 (8) ◽  
pp. 967-977 ◽  
Author(s):  
Axel Kallies ◽  
Jhagvaral Hasbold ◽  
David M. Tarlinton ◽  
Wendy Dietrich ◽  
Lynn M. Corcoran ◽  
...  
Keyword(s):  
Plasma Cell ◽  
B Lymphocyte ◽  
Plasma Cells ◽  
Fluorescent Protein ◽  
Developmental Stages ◽  
Maturation Protein ◽  
Quantitative Changes ◽  
Green Fluorescent

Plasma cells comprise a population of terminally differentiated B cells that are dependent on the transcriptional regulator B lymphocyte–induced maturation protein 1 (Blimp-1) for their development. We have introduced a gfp reporter into the Blimp-1 locus and shown that heterozygous mice express the green fluorescent protein in all antibody-secreting cells (ASCs) in vivo and in vitro. In vitro, these cells display considerable heterogeneity in surface phenotype, immunoglobulin secretion rate, and Blimp-1 expression levels. Importantly, analysis of in vivo ASCs induced by immunization reveals a developmental pathway in which increasing levels of Blimp-1 expression define developmental stages of plasma cell differentiation that have many phenotypic and molecular correlates. Thus, maturation from transient plasmablast to long-lived ASCs in bone marrow is predicated on quantitative increases in Blimp-1 expression.

scholarly journals Molecular Toolbox for Genetic Manipulation of the Stalked Budding Bacterium Hyphomonas neptunium

2014 ◽  
Vol 81 (2) ◽  
pp. 736-744 ◽  
Author(s):  
Alexandra Jung ◽  
Sabrina Eisheuer ◽  
Emöke Cserti ◽  
Oliver Leicht ◽  
Wolfgang Strobel ◽  
...  
Keyword(s):  
Target Genes ◽  
Fluorescent Protein ◽  
Dynamic Range ◽  
Genetic Manipulation ◽  
Expression System ◽  
Genetic System ◽  
High Dynamic Range ◽  
Content Type ◽  
Depth Analysis ◽  
Inducible Synthesis

ABSTRACTThe alphaproteobacteriumHyphomonas neptuniumproliferates by a unique budding mechanism in which daughter cells emerge from the end of a stalk-like extension emanating from the mother cell body. Studies of this species so far have been hampered by the lack of a genetic system and of molecular tools allowing the regulated expression of target genes. Based on microarray analyses, this work identifies twoH. neptuniumpromoters that are activated specifically by copper and zinc. Functional analyses show that they have low basal activity and a high dynamic range, meeting the requirements for use as a multipurpose expression system. To facilitate their application, the two promoters were incorporated into a set of integrative plasmids, featuring a choice of two different selection markers and various fluorescent protein genes. These constructs enable the straightforward generation and heavy metal-inducible synthesis of fluorescent protein fusions inH. neptunium, thereby opening the door to an in-depth analysis of polar growth and development in this species.

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