scholarly journals The YadA Protein of Yersinia pseudotuberculosis Mediates High-Efficiency Uptake into Human Cells under Environmental Conditions in Which Invasin Is Repressed

2002 ◽  
Vol 70 (9) ◽  
pp. 4880-4891 ◽  
Author(s):  
Julia Eitel ◽  
Petra Dersch
Keyword(s):  
Extracellular Matrix ◽  
Mammalian Cells ◽  
Yersinia Pseudotuberculosis ◽  
High Efficiency ◽  
Environmental Parameters ◽  
Infection Process ◽  
Nutrient Concentrations ◽  
Integrin Receptors ◽  
Uptake Process ◽  
High Level

ABSTRACT The YadA protein is a major adhesin of Yersinia pseudotuberculosis that promotes tight adhesion to mammalian cells by binding to extracellular matrix proteins. In this study, we first addressed the possibility of competitive interference of YadA and the major invasive factor invasin and found that expression of YadA in the presence of invasin affected neither the export nor the function of invasin in the outer membrane. Furthermore, expression of YadA promoted both bacterial adhesion and high-efficiency invasion entirely independently of invasin. Antibodies against fibronectin and β1 integrins blocked invasion, indicating that invasion occurs via extracellular-matrix-dependent bridging between YadA and the host cell β1 integrin receptors. Inhibitor studies also demonstrated that tyrosine and Ser/Thr kinases, as well as phosphatidylinositol 3-kinase, are involved in the uptake process. Further expression studies revealed that yadA is regulated in response to several environmental parameters, including temperature, ion and nutrient concentrations, and the bacterial growth phase. In complex medium, YadA production was generally repressed but could be induced by addition of Mg2+. Maximal expression of yadA was obtained in exponential-phase cells grown in minimal medium at 37°C, conditions under which the invasin gene is repressed. These results suggest that YadA of Y. pseudotuberculosis constitutes another independent high-level uptake pathway that might complement other cell entry mechanisms (e.g., invasin) at certain sites or stages during the infection process.

scholarly journals Arf6 and Phosphoinositol-4-Phosphate-5-Kinase Activities Permit Bypass of the Rac1 Requirement for β1 Integrin–mediated Bacterial Uptake

2003 ◽  
Vol 198 (4) ◽  
pp. 603-614 ◽  
Author(s):  
Ka-Wing Wong ◽  
Ralph R. Isberg
Keyword(s):  
Target Cell ◽  
Mammalian Cells ◽  
Yersinia Pseudotuberculosis ◽  
Small Gtpase ◽  
Host Cells ◽  
Β1 Integrin ◽  
Integrin Receptors ◽  
Bacterial Binding ◽  
Bacterial Uptake ◽  
Dependent Pathway

Efficient entry of the bacterium Yersinia pseudotuberculosis into mammalian cells requires the binding of the bacterial invasin protein to β1 integrin receptors and the activation of the small GTPase Rac1. We report here that this Rac1-dependent pathway involves recruitment of phosphoinositol-4-phosphate-5-kinase (PIP5K) to form phosphoinositol-4,5-bisphosphate (PIP2) at the phagocytic cup. Reducing the concentration of PIP2 in the target cell by using a membrane-targeted PIP2-specific phosphatase lowered bacterial uptake proportionately. PIP2 formation is regulated by Arf6. An Arf6 derivative defective for nucleotide binding (Arf6N122I) interfered with uptake and decreased the level of PIP2 around extracellular bacteria bound to host cells. This reduction in PIP2 occurred in spite of fact that PIP5K appeared to be recruited efficiently to the site of bacterial binding, indicating a role for Arf6 in activation of the kinase. The elimination of the Rac1-GTP–bound form from the cell by the introduction of the Y. pseudotuberculosis YopE RhoGAP protein could be bypassed by the overproduction of either PIP5K or Arf6, although the degree of bypass was greater for Arf6 transfectants. These results indicate that both Arf6 and PIP5K are involved in integrin-dependent uptake, and that Arf6 participates in both activation of PIP5K as well as in other events associated with bacterial uptake.

scholarly journals Homogenous overexpression of the extracellular matrix protein Netrin-1 in a hollow fiber bioreactor

2021 ◽  
Author(s):  
Aniel Moya-Torres ◽  
Monika Gupta ◽  
Fabian Heide ◽  
Natalie Krahn ◽  
Scott Legare ◽  
...  
Keyword(s):  
Extracellular Matrix ◽  
Hollow Fiber ◽  
Mammalian Cells ◽  
Matrix Protein ◽  
Continuous Production ◽  
Extracellular Matrix Protein ◽  
Close Monitoring ◽  
High Quality ◽  
Biolayer Interferometry ◽  
Hollow Fiber Bioreactor

Abstract The production of recombinant proteins for functional and biophysical studies, especially in the field of structural determination, still represents a challenge as high quality and quantities are needed to adequately perform experiments. This is in part solved by optimizing protein constructs and expression conditions to maximize the yields in regular flask expression systems. Still, work flow and effort can be substantial with no guarantee to obtain improvements. This study presents a combination of workflows that can be used to dramatically increase protein production and improve processing results, specifically for the extracellular matrix protein Netrin-1. This proteoglycan is an axon guidance cue which interacts with various receptors to initiate downstream signaling cascades affecting cell differentiation, proliferation, metabolism, and survival. We were able to produce large glycoprotein quantities in mammalian cells, which were engineered for protein overexpression and secretion into the media using the controlled environment provided by a hollow fiber bioreactor. Close monitoring of the internal bioreactor conditions allowed for stable production over an extended period of time. In addition to this, Netrin-1 concentrations were monitored in expression media through biolayer interferometry which allowed us to increase Netrin-1 media concentrations tenfold over our current flask systems while preserving excellent protein quality and in solution behavior. Our particular combination of genetic engineering, cell culture system, protein purification, and biophysical characterization permitted us to establish an efficient and continuous production of high-quality protein suitable for structural biology studies that can be translated to various biological systems. Key points • Hollow fiber bioreactor produces substantial yields of homogenous Netrin-1 • Biolayer interferometry allows target protein quantitation in expression media • High production yields in the bioreactor do not impair Netrin-1 proteoglycan quality Graphical abstract

scholarly journals Diversity and Dynamics of Seaweed Associated Microbial Communities Inhabiting the Lagoon of Venice

2020 ◽  
Vol 8 (11) ◽  
pp. 1657
Author(s):  
Abdul-Salam Juhmani ◽  
Alessandro Vezzi ◽  
Mohammad Wahsha ◽  
Alessandro Buosi ◽  
Fabio De Pascale ◽  
...  
Keyword(s):  
Microbial Communities ◽  
Bacterial Communities ◽  
Host Response ◽  
Environmental Parameters ◽  
16S Rrna Genes ◽  
Rrna Genes ◽  
Nutrient Concentrations ◽  
Lagoon Of Venice ◽  
Anthropogenic Stressors ◽  
Rich Diversity

Seaweeds are a group of essential photosynthetic organisms that harbor a rich diversity of associated microbial communities with substantial functions related to host health and defense. Environmental and anthropogenic stressors may disrupt the microbial communities and their metabolic activity, leading to host physiological alterations that negatively affect seaweeds’ performance and survival. Here, the bacterial communities associated with one of the most common seaweed, Ulva laetevirens Areshough, were sampled over a year at three sites of the lagoon of Venice affected by different environmental and anthropogenic stressors. Bacterial communities were characterized through Illumina sequencing of the V4 hypervariable region of 16S rRNA genes. The study demonstrated that the seaweed associated bacterial communities at sites impacted by environmental stressors were host-specific and differed significantly from the less affected site. Furthermore, these communities were significantly distinct from those of the surrounding seawater. The bacterial communities’ composition was significantly correlated with environmental parameters (nutrient concentrations, dissolved oxygen saturation, and pH) across sites. This study showed that several more abundant bacteria on U. laetevirens at stressed sites belonged to taxa related to the host response to the stressors. Overall, environmental parameters and anthropogenic stressors were shown to substantially affect seaweed associated bacterial communities, which reflect the host response to environmental variations.

scholarly journals Poly-L-ornithine-mediated transformation of mammalian cells.

1987 ◽  
Vol 7 (6) ◽  
pp. 2286-2293 ◽  
Author(s):  
V C Bond ◽  
B Wold
Keyword(s):  
Cell Lines ◽  
Mammalian Cells ◽  
High Efficiency ◽  
Protein Product ◽  
Marker Genes ◽  
Recipient Mouse ◽  
L Cells ◽  
Dna And Rna ◽  
Selectable Marker Genes

Poly-L-ornithine has been used to introduce DNA and RNA into mammalian cells in culture. Ornithine-mediated DNA transfer has several interesting and potentially useful properties. The procedure is technically straightforward and is easily applied to either small or large numbers of recipient cells. The efficiency of transformation is high. Under optimal conditions, 1 to 2% of recipient mouse L cells take up and continue to express selectable marker genes. DNA content of transformants can be varied reproducibly, yielding cells with just one or two copies of the new gene under one set of conditions, while under a different set of conditions 25 to 50 copies are acquired. Cotransformation and expression of physically unlinked genes occur at high efficiency under conditions favoring multiple-copy transfer. Polyornithine promotes gene transfer into cell lines other than L cells. These include Friend erythroleukemia cells and NIH 3T3 cells. Both are transformed about 1 order of magnitude more efficiently by this procedure than by standard calcium phosphate products. However, the method does not abolish the large transformation efficiency differences between these cell lines that have been observed previously by other techniques. (vi) mRNA synthesized in vitro was also introduced into cells by this method. The RNA was translated resulting in a transient accumulation of the protein product.

High Efficiency Combined Aggregate – Submerged Combustion Melter–Electric Furnace for Vitrification of High-Level Radioactive Wastes

2004 ◽  
Author(s):  
L. S. Pioro ◽  
I. L. Pioro
Keyword(s):  
Electric Furnace ◽  
High Efficiency ◽  
High Capacity ◽  
Main Idea ◽  
Melting Process ◽  
Radioactive Wastes ◽  
Single Stage ◽  
Interface Surface ◽  
High Level ◽  
Submerged Combustion

It is well known that high-level radioactive wastes (HLRAW) are usually vitrified inside electric furnaces. Disadvantages of electric furnaces are their low melting capacity and restrictions on charge preparation. Therefore, a new concept for a high efficiency combined aggregate – submerged combustion melter (SCM)–electric furnace was developed for vitrification of HLRAW. The main idea of this concept is to use the SCM as the primary high-capacity melting unit with direct melt drainage into an electric furnace. The SCM employs a single-stage method for vitrification of HLRAW. The method includes concentration (evaporation), calcination, and vitrification of HLRAW in a single-stage process inside a melting chamber of the SCM. Specific to the melting process is the use of a gas-air or gas-oxygen-air mixture with direct combustion inside a melt. Located inside the melt are high-temperature zones with increased reactivity of the gas phase, the existence of a developed interface surface, and intensive mixing, leading to intensification of the charge melting and vitrification process. The electric furnace clarifies molten glass, thus preparing the high-quality melt for subsequent melt pouring into containers for final storage.

Dissection of homologous translocon operons reveals a distinct role for YopD in type III secretion by Yersinia pseudotuberculosis

Microbiology ◽  
2003 ◽  
Vol 149 (9) ◽  
pp. 2615-2626 ◽  
Author(s):  
Jeanette E. Bröms ◽  
Anna-Lena Forslund ◽  
Åke Forsberg ◽  
Matthew S. Francis
Keyword(s):  
Type Iii Secretion ◽  
Yersinia Pseudotuberculosis ◽  
Infection Process ◽  
Regulatory Control ◽  
Eukaryotic Cells ◽  
Null Mutant ◽  
Type Iii ◽  
In Trans ◽  
Key Residues ◽  
Distinct Role

The homologous pcrGVHpopBD and lcrGVHyopBD translocase operons of Pseudomonas aeruginosa and pathogenic Yersinia spp., respectively, are responsible for the translocation of anti-host effectors into the cytosol of infected eukaryotic cells. In Yersinia, this operon is also required for yop-regulatory control. To probe for key molecular interactions during the infection process, the functional interchangeability of popB/yopB and popD/yopD was investigated. Secretion of PopB produced in trans in a ΔyopB null mutant of Yersinia was only observed when co-produced with its native chaperone PcrH, but this was sufficient to complement the yopB translocation defect. The Yersinia ΔyopD null mutant synthesized and secreted PopD even in the absence of native PcrH, yet this did not restore YopD-dependent yop-regulatory control or effector translocation. Thus, this suggests that key residues in YopD, which are not conserved in PopD, are essential for functional Yersinia type III secretion.

Design of a Retrovirus-Derived Vector for Expression and Transduction of Exogenous Genes in Mammalian Cells

1983 ◽  
Vol 3 (6) ◽  
pp. 1123-1132
Author(s):  
Archibald S. Perkins ◽  
Paul T. Kirschmeier ◽  
Sebastiano Gattoni-Celli ◽  
I. Bernard Weinstein
Keyword(s):  
Mammalian Cells ◽  
High Efficiency ◽  
Leukemia Virus ◽  
Restriction Enzymes ◽  
Virus Genome ◽  
Opposite Polarity ◽  
High Yield ◽  
Animal Cells ◽  
Murine Sarcoma Virus ◽  
Sarcoma Virus

We have developed a transfection vector for animal cells that contains long terminal repeat (LTR) sequences to promote expression. Plasmid p101/101, a derivative of plasmid pBR322 containing the complete Moloney murine sarcoma virus genome, was cut with restriction enzymes and religated so that both the 5′ and 3′ LTRs were retained and all but about 700 base pairs of the intervening viral sequences were removed. To test this vector, the Escherichia coli gene gpt was cloned into a unique Pst I site, between the two LTRs, with guanine and cytosine tailing, a method that can be generalized for insertion of any DNA segment into this vector. When DNA from recombinant plasmids in which the gpt gene was inserted in the same transcriptional polarity as the LTR sequences was transfected onto murine or rat fibroblast cultures, we obtained a high yield of Gpt + colonies. However, plasmid constructs with the gpt gene in the opposite polarity were virtually devoid of activity. With gpt in the proper orientation, restriction enzyme cuts within the LTRs or between the 5′ LTR and the gpt gene reduced transfection by more than 98%, whereas a cut between the gpt gene and the 3′ LTR gave an 80% reduction in activity. Thus, both 5′ and 3′ LTR sequences are essential for optimal gpt expression, although the 5′ LTR appears to play a more important role. When the LTR- gpt plasmid was transfected onto murine leukemia virus-infected mouse fibroblasts, we obtained evidence that RNA copies became pseudotyped into viral particles which could transfer the Gpt + phenotype into rodent cells with extremely high efficiency. This vector should prove useful for high-efficiency transduction of a variety of genes in mammalian cells.

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