scholarly journals Use of Drosophila S2 Cells as a Model for Studying Ehrlichia chaffeensis Infections

2008 ◽  
Vol 74 (6) ◽  
pp. 1886-1891 ◽  
Author(s):  
Alison Luce-Fedrow ◽  
Tonia Von Ohlen ◽  
Daniel Boyle ◽  
Roman R. Ganta ◽  
Stephen K. Chapes
Keyword(s):  
Mammalian Cells ◽  
Genetic Manipulation ◽  
Rrna Gene ◽  
Ehrlichia Chaffeensis ◽  
S2 Cells ◽  
Obligate Intracellular Bacterium ◽  
Obligate Intracellular ◽  
Mammalian Cell Lines ◽  
Drosophila S2 Cells ◽  
Human Monocytic Ehrlichiosis

ABSTRACT Ehrlichia chaffeensis is an obligate intracellular bacterium and the causative agent of human monocytic ehrlichiosis. Although this pathogen grows in several mammalian cell lines, no general model for eukaryotic cellular requirements for bacteria replication has yet been proposed. We found that Drosophila S2 cells are permissive for the growth of E. chaffeensis. We saw morulae (aggregates of bacteria) by microscopy, detected the E. chaffeensis 16S rRNA gene by reverse transcriptase PCR, and used immunocytochemistry to detect E. chaffeensis in S2 and mammalian cells. Bacteria grown in S2 cells reinfected mammalian macrophages. S2 cells were made nonpermissive for E. chaffeensis through incubation with lipopolysaccharide. Our results demonstrate that S2 cells are an appropriate system for studying the pathogenesis of E. chaffeensis. The use of a Drosophila system has the potential to serve as a model system for studying Ehrlichia due to its completed genome, ease of genetic manipulation, and the availability of mutants.

scholarly journals EtpE Binding to DNase X Induces Ehrlichial Entry via CD147 and hnRNP-K Recruitment, Followed by Mobilization of N-WASP and Actin

mBio ◽  
2015 ◽  
Vol 6 (6) ◽  
Author(s):  
Dipu Mohan Kumar ◽  
Mingqun Lin ◽  
Qingming Xiong ◽  
Mathew James Webber ◽  
Comert Kural ◽  
...  
Keyword(s):  
Cell Surface ◽  
Host Cell ◽  
Actin Polymerization ◽  
Ehrlichia Chaffeensis ◽  
Content Type ◽  
Hnrnp K ◽  
Obligate Intracellular ◽  
Human Proteins ◽  
Coated Bead ◽  
Human Monocytic Ehrlichiosis

ABSTRACTObligate intracellular bacteria, such asEhrlichia chaffeensis, perish unless they can enter eukaryotic cells.E. chaffeensisis the etiological agent of human monocytic ehrlichiosis, an emerging infectious disease. To infect cells,Ehrlichiauses theCterminus of the outer membrane invasinentry-triggeringprotein (EtpE) ofEhrlichia(EtpE-C), which directly binds the mammalian cell surface glycosylphosphatidyl inositol-anchored protein, DNase X. How this binding drivesEhrlichiaentry is unknown. Here, using affinity pulldown of host cell lysates with recombinant EtpE-C (rEtpE-C), we identified two new human proteins that interact with EtpE-C: CD147 and heterogeneous nuclear ribonucleoprotein K (hnRNP-K). The interaction of CD147 with rEtpE-C was validated by far-Western blotting and coimmunoprecipitation of native EtpE with endogenous CD147. CD147 was ubiquitous on the cell surface and also present around foci of rEtpE-C-coated-bead entry. Functional neutralization of surface-exposed CD147 with a specific antibody inhibitedEhrlichiainternalization and infection but not binding. Downregulation of CD147 by short hairpin RNA (shRNA) impairedE. chaffeensisinfection. Functional ablation of cytoplasmic hnRNP-K by a nanoscale intracellular antibody markedly attenuated bacterial entry and infection but not binding. EtpE-C also interacted with neuronal Wiskott-Aldrich syndrome protein (N-WASP), which is activated by hnRNP-K. Wiskostatin, which inhibits N-WASP activation, and cytochalasin D, which inhibits actin polymerization, inhibitedEhrlichiaentry. Upon incubation with host cell lysate, EtpE-C but not an EtpE N-terminal fragment stimulatedin vitroactin polymerization in an N-WASP- and DNase X-dependent manner. Time-lapse video images revealed N-WASP recruitment at EtpE-C-coated bead entry foci. Thus, EtpE-C binding to DNase X drivesEhrlichiaentry by engaging CD147 and hnRNP-K and activating N-WASP-dependent actin polymerization.IMPORTANCEEhrlichia chaffeensis, an obligate intracellular bacterium, causes a blood-borne disease called human monocytic ehrlichiosis, one of the most prevalent life-threatening emerging tick-transmitted infectious diseases in the United States. The survival ofEhrlichiabacteria, and hence, their ability to cause disease, depends on their specific mode of entry into eukaryotic host cells. Understanding the mechanism by whichE. chaffeensisenters cells will create new opportunities for developing effective therapies to prevent bacterial entry and disease in humans. Our findings reveal a novel cellular signaling pathway triggered by an ehrlichial surface protein called EtpE to induce its infectious entry. The results are also important from the viewpoint of human cell physiology because three EtpE-interacting human proteins, DNase X, CD147, and hnRNP-K, are hitherto unknown partners that drive the uptake of small particles, including bacteria, into human cells.

scholarly journals Antibody-Mediated Elimination of the Obligate Intracellular Bacterial Pathogen Ehrlichia chaffeensisduring Active Infection

2000 ◽  
Vol 68 (4) ◽  
pp. 2187-2195 ◽  
Author(s):  
Gary M. Winslow ◽  
Eric Yager ◽  
Konstantin Shilo ◽  
Erin Volk ◽  
Andrew Reilly ◽  
...  
Keyword(s):  
Bacterial Pathogen ◽  
Immune Serum ◽  
Intracellular Bacterium ◽  
Bacterial Clearance ◽  
Obligate Intracellular Bacterium ◽  
Active Infection ◽  
Obligate Intracellular ◽  
Humoral Responses ◽  
Human Monocytic Ehrlichiosis ◽  
Dose Dependent

ABSTRACT It is generally accepted that cellular, but not humoral immunity, plays an important role in host defense against intracellular bacteria. However, studies of some of these pathogens have provided evidence that antibodies can provide immunity if present during the initiation of infection. Here, we examined immunity against infection byEhrlichia chaffeensis, an obligate intracellular bacterium that causes human monocytic ehrlichiosis. Studies with mice have demonstrated that immunocompetent strains are resistant to persistent infection but that SCID mice become persistently and fatally infected. Transfer of immune serum or antibodies obtained from immunocompetent C57BL/6 mice to C57BL/6 scid mice provided significant although transient protection from infection. Bacterial clearance was observed when administration occurred at the time of inoculation or well after infection was established. The effect was dose dependent, occurred within 2 days, and persisted for as long as 2 weeks. Weekly serum administration prolonged the survival of susceptible mice. Although cellular immunity is required for complete bacterial clearance, the data show that antibodies can play a significant role in the elimination of this obligate intracellular bacterium during active infection and thus challenge the paradigm that humoral responses are unimportant for immunity to such organisms.

scholarly journals A Store-operated Calcium Channel in Drosophila S2 Cells

2004 ◽  
Vol 123 (2) ◽  
pp. 167-182 ◽  
Author(s):  
Andriy V. Yeromin ◽  
Jack Roos ◽  
Kenneth A. Stauderman ◽  
Michael D. Cahalan
Keyword(s):  
Mammalian Cells ◽  
Divalent Cations ◽  
Noise Analysis ◽  
Reversal Potential ◽  
S2 Cells ◽  
Pipette Solution ◽  
Crac Channels ◽  
Drosophila S2 Cells ◽  
Cell Recording ◽  
Inward Currents

Using whole-cell recording in Drosophila S2 cells, we characterized a Ca2+-selective current that is activated by depletion of intracellular Ca2+ stores. Passive store depletion with a Ca2+-free pipette solution containing 12 mM BAPTA activated an inwardly rectifying Ca2+ current with a reversal potential >60 mV. Inward currents developed with a delay and reached a maximum of 20–50 pA at −110 mV. This current doubled in amplitude upon increasing external Ca2+ from 2 to 20 mM and was not affected by substitution of choline for Na+. A pipette solution containing ∼300 nM free Ca2+ and 10 mM EGTA prevented spontaneous activation, but Ca2+ current activated promptly upon application of ionomycin or thapsigargin, or during dialysis with IP3. Isotonic substitution of 20 mM Ca2+ by test divalent cations revealed a selectivity sequence of Ba2+ > Sr2+ > Ca2+ >> Mg2+. Ba2+ and Sr2+ currents inactivated within seconds of exposure to zero-Ca2+ solution at a holding potential of 10 mV. Inactivation of Ba2+ and Sr2+ currents showed recovery during strong hyperpolarizing pulses. Noise analysis provided an estimate of unitary conductance values in 20 mM Ca2+ and Ba2+ of 36 and 420 fS, respectively. Upon removal of all external divalent ions, a transient monovalent current exhibited strong selectivity for Na+ over Cs+. The Ca2+ current was completely and reversibly blocked by Gd3+, with an IC50 value of ∼50 nM, and was also blocked by 20 μM SKF 96365 and by 20 μM 2-APB. At concentrations between 5 and 14 μM, application of 2-APB increased the magnitude of Ca2+ currents. We conclude that S2 cells express store-operated Ca2+ channels with many of the same biophysical characteristics as CRAC channels in mammalian cells.

scholarly journals Transport of polyamines in Drosophila S2 cells: kinetics, pharmacology and dependence on the plasma membrane proton gradient

2005 ◽  
Vol 393 (2) ◽  
pp. 583-589 ◽  
Author(s):  
Rafael Romero-Calderón ◽  
David E. Krantz
Keyword(s):  
Mammalian Cells ◽  
Organic Anion ◽  
S2 Cells ◽  
Transport Activity ◽  
Molecular Identity ◽  
Polyamine Transport ◽  
Drosophila S2 Cells ◽  
Solute Carrier ◽  
Polyamine Uptake

Polyamine transport activities have been described in diverse multicellular systems, but their bioenergetic mechanisms and molecular identity remain unclear. In the present paper, we describe a high-affinity spermine/spermidine transport activity expressed in Drosophila S2 cells. Ion-replacement experiments indicate that polyamine uptake across the cell membrane is Na+-, K+-, Cl−- and Ca2+-independent, but pH-sensitive. Additional experiments using ionophores suggest that polyamine uptake may be H+-coupled. Pharmacological experiments show that polyamine uptake in S2 cells is selectively blocked by MGBG {methylglyoxal bis(guanylhydrazone) or 1,1′-[(methylethanediylidine)-dinitrilo]diguanidine} and paraquat (N,N-dimethyl-4,4′-bipyridylium), two known inhibitors of polyamine uptake in mammalian cells. In addition, inhibitors known to block the Slc22 (solute carrier 22) family of organic anion/cation transporters inhibit spermine uptake in S2 cells. These data and the genetic tools available in Drosophila will facilitate the molecular identification and further characterization of this activity.

scholarly journals Akt Phosphorylation Influences Persistent Chlamydial Infection and Chlamydia-Induced Golgi Fragmentation Without Involving Rab14

2021 ◽  
Vol 11 ◽  
Author(s):  
Xiaobao Huang ◽  
Jinfeng Tan ◽  
Xiaohong Chen ◽  
Mingna Liu ◽  
Huiling Zhu ◽  
...  
Keyword(s):  
Persistent Infection ◽  
Chlamydial Infection ◽  
Akt Pathway ◽  
Rrna Gene ◽  
Obligate Intracellular Bacterium ◽  
Akt Inhibitor ◽  
Limited Effect ◽  
Akt Phosphorylation ◽  
Obligate Intracellular ◽  
Golgi Fragmentation

Chlamydia trachomatis is an obligate intracellular bacterium that causes multiple diseases involving the eyes, gastrointestinal tract, and genitourinary system. Previous studies have identified that in acute chlamydial infection, C. trachomatis requires Akt pathway phosphorylation and Rab14-positive vesicles to transmit essential lipids from the Golgi apparatus in survival and replication. However, the roles that Akt phosphorylation and Rab14 play in persistent chlamydial infection remain unclear. Here, we discovered that the level of Akt phosphorylation was lower in persistent chlamydial infection, and positively correlated with the effect of activating the development of Chlamydia but did not change the infectivity and 16s rRNA gene expression. Rab14 was found to exert a limited effect on persistent infection. Akt phosphorylation might regulate Chlamydia development and Chlamydia-induced Golgi fragmentation in persistent infection without involving Rab14. Our results provide a new insight regarding the potential of synergistic repressive effects of an Akt inhibitor with antibiotics in the treatment of persistent chlamydial infection induced by penicillin.

scholarly journals Rifalazil Pretreatment of Mammalian Cell Cultures Prevents Subsequent Chlamydia Infection

2006 ◽  
Vol 50 (2) ◽  
pp. 439-444 ◽  
Author(s):  
Robert J. Suchland ◽  
Kara Brown ◽  
David M. Rothstein ◽  
Walter E. Stamm
Keyword(s):  
Mammalian Cell ◽  
Mammalian Cells ◽  
Chlamydia Pneumoniae ◽  
Respiratory Infections ◽  
Host Cells ◽  
Chlamydia Infection ◽  
Intracellular Pathogens ◽  
Obligate Intracellular ◽  
Mammalian Cell Lines ◽  
Mammalian Cell Cultures

ABSTRACT Chlamydia species are widely disseminated obligate intracellular pathogens that primarily cause urogenital, ocular, and respiratory infections. In these studies, we show that exposing mammalian cells to antibacterial agents prior to Chlamydia inoculation protects the host cells against subsequent challenge by chlamydiae (the protective effect [PE]). Rifalazil exhibited a considerably stronger PE than did azithromycin, rifampin, doxycycline, and ofloxacin. Specifically, 0.002 μg/ml rifalazil incubated for 1 day with a monolayer of McCoy cells was sufficient to protect against a challenge 2 days later with Chlamydia trachomatis serovar D (UW-3). The PE was observed with five different mammalian cell lines and with a variety of C. trachomatis and Chlamydia pneumoniae isolates. The duration of the PE was 6 to 12 days for rifalazil (depending on the cell line), a maximum of 3 days for azithromycin, and less than a day for the other drugs tested. For rifalazil, the PE was shown to be mediated by inhibition of the chlamydial RNA polymerase since mutants with altered RNA polymerases had correspondingly altered PEs. These results suggest that rifalazil may be unique in its ability to prevent infection with obligate intracellular pathogens for a considerable time after treatment. This characteristic may be of particular public health value in reducing reinfection with chlamydiae.

scholarly journals Physical and Genetic Map of the Obligate Intracellular Bacterium Coxiella burnetii

1998 ◽  
Vol 180 (15) ◽  
pp. 3816-3822 ◽  
Author(s):  
H. Willems ◽  
Cornelie Jäger ◽  
Georg Baljer
Keyword(s):  
Genetic Map ◽  
Coxiella Burnetii ◽  
Physical Map ◽  
Gene Organization ◽  
Intracellular Bacterium ◽  
Rrna Gene ◽  
Obligate Intracellular Bacterium ◽  
Obligate Intracellular ◽  
Great Heterogeneity ◽  
Physical And Genetic Map

ABSTRACT Pulsed-field gel electrophoresis and PCR techniques have been used to construct a NotI macrorestriction map of the obligate intracellular bacterium Coxiella burnetii Nine Mile. The size of the chromosome has been determined to be 2,103 kb comprising 29NotI restriction fragments. The average resolution is 72.5 kb, or about 3.5% of the genome. Experimental data support the presence of a linear chromosome. Published genes were localized on the physical map by Southern hybridization. One gene, recognized as transposable element, was found to be present in at least nine sites evenly distributed over the whole chromosome. There is only one copy of a 16S rRNA gene. The putative oriC has been located on a 27.5-kb NotI fragment. Gene organization upstream theoriC is almost identical to that of Pseudomonas putida and Bacillus subtilis, whereas gene organization downstream the oriC seems to be unique among bacteria. The physical map will be helpful in investigations of the great heterogeneity in restriction fragment length polymorphism patterns of different isolates and the great variation in genome size. The genetic map will help to determine whether gene order in different isolates is conserved.

scholarly journals Induction of focal adhesions and motility in Drosophila S2 cells

2014 ◽  
Vol 25 (24) ◽  
pp. 3861-3869 ◽  
Author(s):  
Susana A. Ribeiro ◽  
Michael V. D'Ambrosio ◽  
Ronald D. Vale
Keyword(s):  
Focal Adhesion ◽  
Mammalian Cells ◽  
Focal Adhesions ◽  
S2 Cells ◽  
Cell Interior ◽  
Drosophila S2 Cells ◽  
Cytoskeletal Dynamics ◽  
A Cell ◽  
P21 Activated Kinase ◽  
Dynamic Structures

Focal adhesions are dynamic structures that interact with the extracellular matrix on the cell exterior and actin filaments on the cell interior, enabling cells to adhere and crawl along surfaces. We describe a system for inducing the formation of focal adhesions in normally non–ECM-adherent, nonmotile Drosophila S2 cells. These focal adhesions contain the expected molecular markers such as talin, vinculin, and p130Cas, and they require talin for their formation. The S2 cells with induced focal adhesions also display a nonpolarized form of motility on vitronectin-coated substrates. Consistent with findings in mammalian cells, the degree of motility can be tuned by changing the stiffness of the substrate and was increased after the depletion of PAK3, a p21-activated kinase. A subset of nonmotile, nonpolarized cells also exhibited focal adhesions that rapidly assembled and disassembled around the cell perimeter. Such cooperative and dynamic fluctuations of focal adhesions were decreased by RNA interference (RNAi) depletion of myosin II and focal adhesion kinase, suggesting that this behavior requires force and focal adhesion maturation. These results demonstrate that S2 cells, a cell line that is well studied for cytoskeletal dynamics and readily amenable to protein manipulation by RNAi, can be used to study the assembly and dynamics of focal adhesions and mechanosensitive cell motility.

Evaluation of the nucleopolyhedrovirus of Anticarsia gemmatalis as a vector for gene therapy in mammals

2020 ◽  
Vol 20 ◽  
Author(s):  
Cintia N. Parsza ◽  
Diego L. Mengual Gómez ◽  
Jorge Alejandro Simonin ◽  
Mariano Nicolás Belaich ◽  
Pablo Daniel Ghiringhelli
Keyword(s):  
Gene Therapy ◽  
Mammalian Cells ◽  
Insect Cells ◽  
Autographa Californica ◽  
Anticarsia Gemmatalis ◽  
Agricultural Pests ◽  
Mammalian Cell Lines ◽  
Delivery Vector ◽  
Wide Range ◽  
Insect Pathogens

Background: Baculoviruses are insect pathogens with important biotechnological applications that transcend their use as biological controllers of agricultural pests. One species, Autographa californica multiple nucleopolhyedrovirus (AcMNPV) has been extensively exploited as a molecular platform to produce recombinant proteins and as a delivery vector for genes in mammals, because it can transduce a wide range of mammalian cells and tissues without replicating or producing progeny. Objective/Method: To investigate if the budded virions of Anticarsia gemmatalis multiple nucleopolhyedrovirus (AgMNPV) species has the same ability, the viral genome was modified by homologous recombination into susceptible insect cells to integrate reporter genes and then it was evaluated on mammalian cell lines in comparative form with respect to equivalent viruses derived from AcMNPV. Besides, the replicative capacity of AgMNPV´s virions in mammals was determined. Results: The experiments carried out showed that the recombinant variant of AgMNPV transduces and support the expression of delivered genes but not replicates in mammalian cells. Conclusion: Consequently, this insect pathogen is proposed as an alternative of non-infectious viruses in humans to explore new approaches in gene therapy and other applications based on the use of mammalian cells.

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