An inducible expression system for recombinant Sca proteins with an autotransporter domain from Orientia tsutsugamushi in Escherichia coli

2020 ◽  
Vol 27 ◽  
Author(s):  
Hyejin Cho ◽  
Kwang-Sun Kim
Keyword(s):  
Escherichia Coli ◽  
Vaccine Development ◽  
Scrub Typhus ◽  
Expression System ◽  
Protein Yield ◽  
Host Cells ◽  
Dilution Method ◽  
Orientia Tsutsugamushi ◽  
Dna Encoding ◽  
Protein Fragments

Background: Orientia tsutsugamushi (Ot) is an obligate, intracellular, gram-negative bacterium causing scrub typhus. Some of its encoded proteins play key roles in the adhesion and internalization of the Ot strain into host cells and are suitable resources for vaccine development and tools for scrub typhus diagnosis. Surface cell antigen (Sca) proteins, classified as autotransporter (AT) proteins, are one of the largest protein families involved in bacterial pathogenesis and can be promising candidates for vaccine development. These proteins are typically large and contain inhibitory domains; therefore, recombinant proteins without such domains have been evaluated for this purpose. However, the expression for recombinant Sca proteins containing the AT domain, which might largely affect their protective role against scrub typhus, has not been analyzed and optimized. Objective: In this study, we optimized individual genes encoding Sca protein fragments [ScaA (27–1461), ScaC (257–526), ScaD (26–998), and ScaE (35–760)] harboring the AT domain. Methods: To this end we subcloned sequences of codon-optimized DNA encoding Sca protein fragments into the Escherichia coli expression vector. In addition, the expression condition for individual Sca fragments was optimized, and the proteins were purified using one-step histidine-tag column method. The purified proteins were re-folded by serial dilution method, followed by BCA quantification and densitometric analysis to estimate the protein yield and purity. Results: We prepared platforms for expression of recombinant Sca protein fragments [ScaA (27–1461), ScaC (257–526), ScaD (26–998), and ScaE (35–760)] containing an AT domain but no signal peptide and transmembrane (TM) domain. The protein yield per liter of culture with >70% of purity was ScaC (257–576), ScaE (35–760), ScaD (26-998), and ScaA (27- 1461) in order. Conclusion: Our results could be used to develop Sca AT-domain based vaccines and tools for scrub typhus diagnosis with rapid and cost-effective ways.

scholarly journals Orientia tsutsugamushi Nucleomodulin Ank13 Exploits the RaDAR Nuclear Import Pathway To Modulate Host Cell Transcription

mBio ◽  
2021 ◽  
Author(s):  
Haley E. Adcox ◽  
Amanda L. Hatke ◽  
Shelby E. Andersen ◽  
Sarika Gupta ◽  
Nathan B. Otto ◽  
...  
Keyword(s):  
Nuclear Import ◽  
Scrub Typhus ◽  
Bacterial Pathogen ◽  
Host Cells ◽  
Intracellular Bacteria ◽  
Orientia Tsutsugamushi ◽  
Microbial Protein ◽  
Content Type ◽  
Eukaryotic Gene Expression ◽  
Eukaryotic Gene

Nucleomodulins are recently defined effectors used by diverse intracellular bacteria to manipulate eukaryotic gene expression and convert host cells into hospitable niches. How nucleomodulins enter the nucleus, their functional domains, and the genes that they modulate are incompletely characterized. Orientia tsutsugamushi is an intracellular bacterial pathogen that causes scrub typhus, which can be fatal. O. tsutsugamushi Ank13 is the first example of a microbial protein that coopts eukaryotic RaDAR (RanGDP-ankyrin repeats) nuclear import.

Expression of Foreign Proteins in Escherichia coli

2021 ◽  
Author(s):  
Ali Iftikhar
Keyword(s):  
Escherichia Coli ◽  
Protein Expression ◽  
Recombinant Proteins ◽  
Expression System ◽  
Maximum Yield ◽  
Protein Yield ◽  
Foreign Protein ◽  
Low Protein ◽  
Prokaryotic Expression System ◽  
Foreign Proteins

Abstract BackgroundOptimization of conditions for the recombinant production of proteins in a prokaryotic expression system is essential as the recombinant proteins impose a metabolic burden on cell's growth leading to low protein yield and low protein expression resulting from cell death.Main textThe concentration of media components is optimized to accommodate for depleted nutrients due to foreign protein expression. The temperature is optimized to reduce proteolytic degradation and accumulation of protein as inclusion bodies in Escherichia coli. The concentration of inducer and time of induction for high protein yield is also optimized. These optimization conditions depend on the promoter under which the gene of interest is present and the characteristics of the target protein.ConclusionIn the past few years, many optimization conditions for the production of recombinant proteins in Escherichia coli have been studied. These conditions depend mainly upon the promoter used to produce protein and the type of protein produced. Optimizing the expression parameters of protein produced in Escherichia coli ensures maximum yield of the desired protein.

Thermoinducible expression system for producing recombinant proteins in Escherichia coli: advances and insights

2021 ◽  
Author(s):  
Sara Restrepo-Pineda ◽  
Néstor O. Pérez ◽  
Norma A Valdez-Cruz ◽  
Mauricio A Trujillo-Roldán
Keyword(s):  
Escherichia Coli ◽  
Expression System ◽  
Regulatory Elements ◽  
Host Cells ◽  
Chemical Inducers ◽  
E Coli ◽  
Current Scenario ◽  
Industrial Level ◽  
The Relationship ◽  
Potential Use

ABSTRACT Recombinant protein (RP) production from Escherichia coli has been extensively studied to find strategies for increasing product yields. The thermoinducible expression system is commonly employed at the industrial level to produce various RPs, which avoids the addition of chemical inducers, thus minimizing contamination risks. Multiple aspects of the molecular origin and biotechnological uses of its regulatory elements (pL/pR promoters and cI857 thermolabile repressor) derived from bacteriophage λ provide knowledge to improve the bioprocesses using this system. Here, we discuss the main aspects of the potential use of the λpL/pR-cI857 thermoinducible system for RP production in E. coli, focusing on the approaches of investigations that have contributed to the advancement of this expression system. Metabolic and physiological changes that occur in the host cells caused by heat stress and RP overproduction are also described. Therefore, the current scenario and the future applications of systems that use heat to induce RP production are discussed to understand the relationship between the activation of the bacterial heat shock response, RP accumulation and its possible aggregation to form inclusion bodies.

scholarly journals Orientia tsutsugamushi Strain Ikeda Ankyrin Repeat-Containing Proteins Recruit SCF1 Ubiquitin Ligase Machinery via Poxvirus-Like F-Box Motifs

2015 ◽  
Vol 197 (19) ◽  
pp. 3097-3109 ◽  
Author(s):  
Andrea R. Beyer ◽  
Lauren VieBrock ◽  
Kyle G. Rodino ◽  
Daniel P. Miller ◽  
Brittney K. Tegels ◽  
...  
Keyword(s):  
Host Cell ◽  
Ubiquitin Ligase ◽  
Scrub Typhus ◽  
Ankyrin Repeat ◽  
Intracellular Bacterium ◽  
Host Cells ◽  
Orientia Tsutsugamushi ◽  
Ligand Interaction ◽  
Content Type ◽  
Cell Functions

ABSTRACTA rising theme among intracellular microbes is the delivery of ankyrin repeat-containing effectors (Anks) that interact with target proteins to co-opt host cell functions.Orientia tsutsugamushi, an obligate intracellular bacterium and the etiologic agent of scrub typhus, encodes one of the largest Ank repertoires of any sequenced microorganism. They have been previously identified as type 1 secretion system substrates. Here,in silicoand manual sequence analyses revealed that a large proportion ofO. tsutsugamushistrain Ikeda Anks bear a eukaryotic/poxvirus-like F-box motif, which is known to recruit host cell SCF1 ubiquitin ligase machinery. We assessed the Anks for the ability to serve as F-box proteins. Coimmunoprecipitation assays demonstrated that F-box-containing Anks interact with overexpressed and/or endogenous SCF1 components. When coexpressed with FLAG-Ank4_01 or FLAG-Ank9, a glutathioneS-transferase (GST)-tagged version of the SCF1 component SKP1 localized to subcellular sites of FLAG-Ank accumulation. The abilities of recombinant Anks to interact and colocalize with SKP1 were F-box dependent. GST-SKP1 precipitatedO. tsutsugamushi-derived Ank9 from infected host cells, verifying both that the pathogen expresses Ank9 during infection and the protein's capability to bind SKP1. AligningO. tsutsugamushi, poxviral, and eukaryotic F-box sequences delineated three F-box residues that are highly conserved and likely to be functionally important. Substitution of these residues ablated the ability of GFP-Ank9 to interact with GST-SKP1. These results demonstrate thatO. tsutsugamushistrain Ikeda Anks can co-opt host cell polyubiquitination machinery, provide the first evidence that anO. tsutsugamushiAnk does so during infection, and advance overall understanding of microbial F-box proteins.IMPORTANCEAnkyrin repeat-containing proteins (Anks) are important virulence factors of intracellular bacteria that mediate protein-protein interactions with host cell targets.Orientia tsutsugamushi, which causes a debilitating infection called scrub typhus in one of the most densely populated regions of the world, encodes one of the largest Ank armamentariums of any sequenced bacterium. This study demonstrates thatO. tsutsugamushistrain Ikeda Anks also bear F-box motifs that interact with host cell polyubiquitination machinery. By proving that anOrientia-derived Ank interacts with SKP1 in infected cells, this evidences the first bona fideOrientiaeffector and the first example of an endogenous F-box-containing Ank–mammalian-host ligand interaction for any intracellular bacterium. Also, importantly, this work identifies key residues that are essential for microbial F-box function.

scholarly journals Comparison of Gene Expression Between Two Types of Anti-EGFRvIII ScFv Antibodies Having Different Variable Domain Orders in Escherichia coli

2017 ◽  
Vol 21 (1) ◽  
pp. 29
Author(s):  
Kartika Sari Dewi ◽  
Asrul Muhamad Fuad
Keyword(s):  
Escherichia Coli ◽  
Expression System ◽  
Binding Activity ◽  
Extracellular Proteins ◽  
Antigen Binding ◽  
Dna Encoding ◽  
Single Chain ◽  
Colony Pcr ◽  
E Coli ◽  
Variable Domains

Several studies reported that the expression of various kinds of Single-chain variable fragment (scFv) antibodies in Escherichia coli are significantly influenced by the order of their variable domains. To date, the effect of the order of variable domains in the expression of scFv antibodies against epidermal growth factor receptor variant III (EGFRvIII) has not been reported. This study aimed to compare the expression between VH-linker-VL and VL-linker-VH domain orders of the anti-EGFRvIII scFv antibodies in E. coli expression system. Recombinant plasmids inserted with DNA encoding scFv proteins were transformed into E. coli NiCo21(DE3) competent cells and characterized by colony PCR. The expression of scFv proteins was done by using optimum concentration of inducer. Total proteins, soluble periplasmic and cytoplasmic proteins, also extracellular proteins were isolated, subsequently characterized by SDS-PAGE, Slot Blot, and ImageJ software analyses. The antigen-binding activity of both scFvs proteins against EGFRvIII was observed. The results showed that the relative percentage of scFv expression with VH-linker-VL domain order is higher than that of VL-linker-VH in each compartment. Moreover, both of scFvs proteins have antigen-binding activity against EGFRvIII.

scholarly journals The Orientia tsutsugamushi ScaB Autotransporter Protein Is Required for Adhesion and Invasion of Mammalian Cells

2021 ◽  
Vol 12 ◽  
Author(s):  
Yen Thi Hai Nguyen ◽  
Chaewon Kim ◽  
Yuri Kim ◽  
Kyeongseok Jeon ◽  
Hong-il Kim ◽  
...  
Keyword(s):  
Biofilm Formation ◽  
Mammalian Cells ◽  
Expression System ◽  
Bacterial Pathogenesis ◽  
Host Cells ◽  
Gram Negative Bacteria ◽  
Orientia Tsutsugamushi ◽  
Phagocytic Cells ◽  
E Coli ◽  
Adhesion And Invasion

Autotransporter proteins are widely present in Gram-negative bacteria. They play a pivotal role in processes related to bacterial pathogenesis, including adhesion, invasion, colonization, biofilm formation, and cellular toxicity. Bioinformatics analysis revealed that Orientia tsutsugamushi, the causative agent of scrub typhus, encodes six different autotransporter genes (scaA–scaF). Although four of these genes (scaA, scaC, scaD, and scaE) are present in diverse strains, scaB and scaF have been detected in only a limited number of strains. Previous studies have demonstrated that ScaA and ScaC are involved in the adherence of host cells. However, the putative function of other O. tsutsugamushi Sca proteins has not been studied yet. In this study, we show that scaB is transcribed and expressed on the surface of O. tsutsugamushi Boryong strain. Using a heterologous Escherichia coli expression system, we demonstrated that ScaB-expressing E. coli can successfully mediate adherence to and invasion into non-phagocytic cells, including epithelial and endothelial cells. In addition, pretreatment with a recombinant ScaB polypeptide inhibits the entry of O. tsutsugamushi into cultured mammalian cells. Finally, we also identified the scaB gene in the Kuroki and TA686 strains and observed high levels of sequence variation in the passenger domains. Here, we propose that the ScaB protein of O. tsutsugamushi can mediate both adhesion to and invasion into host cells in the absence of other O. tsutsugamushi genes and may play important roles in bacterial pathogenesis.

scholarly journals Orientia tsutsugamushi modulates cellular levels of NF-κB inhibitor p105

2021 ◽  
Vol 15 (4) ◽  
pp. e0009339
Author(s):  
Tanaporn Wangsanut ◽  
Katelynn R. Brann ◽  
Haley E. Adcox ◽  
Jason A. Carlyon
Keyword(s):  
Host Cell ◽  
Nuclear Translocation ◽  
Scrub Typhus ◽  
Case Fatality ◽  
Immune Defense ◽  
Serine Phosphorylation ◽  
Host Cells ◽  
Tissue Culture Cell ◽  
Orientia Tsutsugamushi

Background Scrub typhus is a neglected tropical disease that threatens more than one billion people. If antibiotic therapy is delayed, often due to mis- or late diagnosis, the case fatality rate can increase considerably. Scrub typhus is caused by the obligate intracellular bacterium, Orientia tsutsugamushi, which invades phagocytes and endothelial cells in vivo and diverse tissue culture cell types in vitro. The ability of O. tsutsugamushi to replicate in the cytoplasm indicates that it has evolved to counter eukaryotic host cell immune defense mechanisms. The transcription factor, NF-κB, is a tightly regulated initiator of proinflammatory and antimicrobial responses. Typically, the inhibitory proteins p105 and IκBα sequester the NF-κB p50:p65 heterodimer in the cytoplasm. Canonical activation of NF-κB via TNFα involves IKKβ-mediated serine phosphorylation of IκBα and p105, which leads to their degradation and enables NF-κB nuclear translocation. A portion of p105 is also processed into p50. O. tsutsugamushi impairs NF-κB translocation into the nucleus, but how it does so is incompletely defined. Principal findings Western blot, densitometry, and quantitative RT-PCR analyses of O. tsutsugamushi infected host cells were used to determine if the pathogen’s ability to inhibit NF-κB is linked to modulation of p105. Results demonstrate that p105 levels are elevated several-fold in O. tsutsugamushi infected HeLa and RF/6A cells with only a nominal increase in p50. The O. tsutsugamushi-stimulated increase in p105 is bacterial dose- and protein synthesis-dependent, but does not occur at the level of host cell transcription. While TNFα-induced phosphorylation of p105 serine 932 proceeds unhindered in infected cells, p105 levels remain elevated and NF-κB p65 is retained in the cytoplasm. Conclusions O. tsutsugamushi specifically stabilizes p105 to inhibit the canonical NF-κB pathway, which advances understanding of how it counters host immunity to establish infection.

scholarly journals Establishment of a Rhesus Macaque Model for Scrub Typhus Transmission: Pilot Study to Evaluate the Minimal Orientia tsutsugamushi Transmission Time by Leptotrombidium chiangraiensis Chiggers

Pathogens ◽  
2021 ◽  
Vol 10 (8) ◽  
pp. 1028
Author(s):  
Piyada Linsuwanon ◽  
Sirima Wongwairot ◽  
Nutthanun Auysawasdi ◽  
Taweesak Monkanna ◽  
Allen L. Richards ◽  
...  
Keyword(s):  
Rhesus Macaque ◽  
Vaccine Development ◽  
Scrub Typhus ◽  
Rhesus Macaques ◽  
Disease Onset ◽  
Immunological Response ◽  
Pathogen Transmission ◽  
Type I ◽  
Orientia Tsutsugamushi ◽  
Macaque Model

Recently, an intradermal inoculation of the rhesus macaque model of scrub typhus has been characterized at our institution. The current project was to establish a rhesus macaque model of scrub typhus using the naturally infected chigger challenge method that faithfully mimics the natural route of pathogen transmission to fully understand the host-pathogen-vector interactions influencing pathogen transmission. Unlike the needle-based inoculation route, Orientia tsutsugamushi-infected chiggers introduce both pathogen and chigger saliva into the host epidermis at the bite site. However, information on the interaction or influence of chigger saliva on pathogenesis and immunity of host has been limited, consequently hindering vaccine development and transmission-blocking studies. To characterize chigger inoculated O. tsutsugamushi in rhesus macaques, we determined the minimum chigger attachment time required to efficiently transmit O. tsutsugamushi to the immunocompetent hosts and preliminary assessed clinical parameters, course of bacterial infection, and host’s immunological response to identifying potential factors influencing pathogen infection. Chigger infestation on hosts resulted in: (i) Rapid transmission of O. tsutsugamushi within 1 h and (ii) antigen-specific type I and II T-cell responses were markedly increased during the acute phase of infection, suggesting that both systems play critical roles in response to the pathogen control during the primary infection. In summary, we demonstrate that O. tsutsugamushi infection in rhesus macaques via chigger challenge recapitulates the time of disease onset and bacteremia observed in scrub typhus patients. Levels of proinflammatory cytokines and chemokines were positively correlated with bacteremia.

scholarly journals An Autotransporter Protein fromOrientia tsutsugamushiMediates Adherence to Nonphagocytic Host Cells

2011 ◽  
Vol 79 (4) ◽  
pp. 1718-1727 ◽  
Author(s):  
Na-Young Ha ◽  
Nam-Hyuk Cho ◽  
Yeon-Sook Kim ◽  
Myung-Sik Choi ◽  
Ik-Sang Kim
Keyword(s):  
Mammalian Cells ◽  
Scrub Typhus ◽  
Expression System ◽  
Critical Role ◽  
Cellular Adhesion ◽  
Host Cells ◽  
Mammalian Host ◽  
Functional Conservation ◽  
Pulldown Assay ◽  
Obligate Intracellular Pathogen

ABSTRACTOrientia tsutsugamushi, the causative agent of scrub typhus, is an obligate intracellular pathogen whose mechanism of cellular adhesion and invasion is poorly characterized. Bioinformatic analyses of twoO. tsutsugamushigenomes revealed the presence of a group of genes that encode autotransporter proteins. In this study, we identified 10 autotransporter gene products and categorized them into five groups of orthologs (ScaA to ScaE) based on their sequence similarities. Sequence homology was highest between members of ScaC group, suggesting the functional conservation of bacterium-host interactions. ScaC was actively expressed on the surface ofO. tsutsugamushiand induced antibody responses in scrub typhus patients. Experiments using microbeads conjugated to recombinant ScaC or a surrogateEscherichia coliexpression system showed that ScaC was sufficient to mediate attachment to, but not invasion of, nonphagocytic mammalian cells. In addition, preincubation of host cells with recombinant ScaC significantly inhibited their interaction withO. tsutsugamushi. Finally, fibronectin was identified as a potential receptor for ScaC by using yeast two-hybrid screening, and this was confirmed using a glutathioneS-transferase (GST) pulldown assay. Taken together, these results demonstrate that ScaC is involved in the interaction ofO. tsutsugamushiwith mammalian host cells and suggest that ScaC may play a critical role in bacterial pathogenesis.

Garlic Extract (Allium sativum L.) Effectively Inhibits Staphylococcus aureus and Escherichia coli by Invitro Test

2020 ◽  
Vol 2 (2) ◽  
pp. 61-68
Author(s):  
Agnina Listya Anggraini ◽  
Ratih Dewi Dwiyanti ◽  
Anny Thuraidah
Keyword(s):  
Escherichia Coli ◽  
Staphylococcus Aureus ◽  
Bacterial Infections ◽  
Allium Sativum ◽  
Antibacterial Properties ◽  
Herbal Medicines ◽  
Garlic Extract ◽  
Dilution Method ◽  
Initial Stage

Infection is a disease caused by the presence of pathogenic microbes, including Staphylococcus aureus and Escherichia coli. Garlic (Allium sativum L.) has chemical contents such as allicin, alkaloids, flavonoids, saponins, tannins, and steroids, which can function as an antibacterial against Staphylococcus aureus and Escherichia coli. This study aims to determine the antibacterial properties of garlic extract powder against Staphylococcus aureus and Escherichia coli. This research is the initial stage of the development of herbal medicines to treat Staphylococcus aureus and Escherichia coli infections. The antibacterial activity test was carried out by the liquid dilution method. The concentrations used were 30 mg/mL, 40 mg/mL, 50 mg/mL, 60 mg/mL and 70 mg/mL. The results showed that the Minimum Inhibitory Concentration (MIC) against Staphylococcus aureus and Escherichia coli was 40 mg/mL and 50 mg / mL. Minimum Bactericidal Concentration (MBC) results for Staphylococcus aureus and Escherichia coli are 50 mg/mL and 70 mg/mL. Based on the Simple Linear Regression test, the R2 value of Staphylococcus aureus and Escherichia coli is 0.545 and 0.785, so it can be concluded that there is an effect of garlic extract powder on the growth of Staphylococcus aureus and Escherichia coli by 54.5% and 78.5%. Garlic (Allium sativum L.) extract powder has potential as herbal medicine against bacterial infections but requires further research to determine its effect in vivo.

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