Luisa F. Jimenez-SotoWalther Straub Institute of Pharmacology and Toxicology - LMU
Luisa F. Jimenez-Soto
PhD (Dr.rer.biol.hum)
Establishing my lab under Open Science principles: https://www.youtube.com/channel/UCscynZPlTowZ_IwyLyLjL0g
About
69
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Introduction
Exotoxins are known because of their role in immune evasion and its association with disease. New ideas and experiments are showing a novel exotoxin role in survival from natural predation by helping to escape ciliates and amoeba. By learning how natural predators avoid cellular damage caused by exotoxins, we can help the human immune system cells to recover from them. We will approach this challenge by combining recent advances in Natural Language Processing with toxicological assays.
Education
April 2018 - April 2021
January 1995 - March 1999
Publications
Publications (69)
http://www.internationalinnovation.com/misunderstood-microbe-new-approaches-helicobacter-pylori-co-infection/
Many pathogenic Gram-negative bacteria possess Type-IV Secretion Systems (T4SS) to inject effector proteins directly into host cells to modulate cellular processes to their benefit. The human bacterial pathogen Helicobacter pylori, a major etiological agent in the development of chronic gastritis, duodenal ulcer and gastric carcinoma, harbours the...
CagA is one of the most studied pathogenicity factors of the bacterial pathogen Helicobacter pylori. It is injected into host cells via the H. pylori cag-Type IV secretion system. Due to its association with gastric cancer, CagA is classified as oncogenic protein. At the same time CagA represents the 4th most abundant protein produced by H. pylori,...
Infection with the gastric pathogen Helicobacter pylori is a risk factor for the development of gastric cancer. Pathogenic strains of H. pylori carry a type IV secretion system (T4SS) responsible for the injection of the oncoprotein CagA into host cells. H. pylori and its cag-T4SS exploit α5β1 integrin as a receptor for CagA translocation. Injected...
Translocation of the Helicobacter pylori (Hp) cytotoxin-associated gene A (CagA) effector protein via the cag-Type IV Secretion System (T4SS) into host cells is a major risk factor for severe gastric diseases, including gastric cancer. However, the mechanism of translocation and the requirements from the host cell for that event are not well unders...
For axenic growth of Tetrahymena thermophila in the lab, NEFF media requires a final concentration of 33 µM (see Tetrahymena Stock Center (RRID:SCR_008362)). To guarantee that the iron in the media is as fresh as possible, we complement NEF media with iron (resulting in NEFF) shortly before use, and store the complete medium for no more than a week...
Some liquids are not suitable for autoclaving but need to be sterile for use. Here we describe the process of filtering any solution to achieve sterility (at least removal of organisms bigger than 0.2 µm). In this protocol we show how we filter solutions in our lab that cannot be autoclaved, while we keep sterility, and reducing the amount of garba...
In this protocol we specify the steps needed to create a 250 ml stock solution of 1M Tris-HCl solution used for the preparation of Tetrahymena thermophila cells for mating. The final concentration for inducing mating behavior is 10 mM (see Bruns PJ and Brussard TB, 1974 and Cassidy-Hanley, 2012). For general information about Tris based buffers, ch...
Potassium phosphate buffers, sometimes referred to as "Gomori buffers" are a combination of monobasic dihydrogen phosphate and dibasic monohydrogen phosphate in an aquaeous solution1 . It has a high buffering range, which can be shifted by adjusting the amount of either one of the phosphate salts.1 This buffer is described by Bio-Rad as component r...
This protocol is performed as described by the ASM (American Society of Microbiologists) in their document Gram Staining Protocol, 1981. Our aim is to include all instructions that could be useful for the absolute beginner. Procedure: The first step of the procedure is the primary stain uses crystal violet, which is then fixed with iodine (the mord...
Terrific Broth (TB) as described by Tartoff, K.D. and Hobbs, C.A. (1987) is a nutritionally rich medium, with higher concentrations of peptone, yeast extract, and glycerol as a carbon source (Thermo Scientific | Terrific Broth). It supports high cell titers (Wood et al. 2017), and subsequent yields for plasmid DNA (Wood et al. 2017). While it has b...
Objective
Existing toxins data sets include a mixture of proteins and toxin peptides. In this study we present two curated data sets of toxic proteins free of associated proteins: bacterial exotoxins and animal toxins. Our stringent selection criteria resulted in two data sets with only toxins that directly target or disrupt vital molecular mechani...
The ciliate Tetrahymena thermophila has been a research organism used by a great community of scientists including biologist, geneticists and toxicologists. T. thermophila is a bacterial predator; however, we can grow it in the lab without bacteria, in axenic media, if needed. The media presented here is a modification of the axenic NEF media publi...
Since the rise of cellular organisms, transmembrane proteins (TMPs) are crucial to a variety of cellular processes due to their central role as gates and gatekeepers. Despite their importance, experimental high-resolution structures for TMPs remain underrepresented due to technical limitations. With structure prediction methods coming of age, predi...
Helicobacter pylori colonizes half of the global population and causes gastritis, peptic ulcer disease or gastric cancer. In this study, we were interested in human annexin (ANX), which comprises a protein family with diverse and partly unknown physiological functions, but with a potential role in microbial infections and possible involvement in ga...
The MinION sequencer by Oxford Nanopore Technologies turns DNA and RNA sequencing into a routine task in biology laboratories or in field research. For downstream analysis it is required to have a sufficien[t amount of target reads. Especially prokaryotic or bacteriophagic sequencing samples can contain a significant amount of off-target sequences...
Introduction:
The presence of H. pylori in the stomach is associated with gastric pathologies. However, its diagnosis through culture methods is challenging because of its complex nutritional requirements and microaerophilic conditions for optimal growth. The preferred method for rapid diagnosis of H. pylori is the Rapid Urease Test (RUT) from hum...
Because of its association with severe gastric pathologies, including gastric cancer, Helicobacter pylori has been subject of research for more than 30 years. Its capacity to adapt and survive in the human stomach can be attributed to its genetic flexibility. Its natural competence and its capacity to turn genes on and off allows H. pylori to adapt...
Translocation of the Helicobacter pylori (Hp) cytotoxin-associated gene A (CagA) effector protein via the cag-Type IV Secretion System (cag-T4SS) into host cells is a hallmark of infection with Hp and a major risk factor for severe gastric diseases, including gastric cancer. To mediate the injection of CagA, Hp uses a membrane-embedded syringe-like...
Verification of targeted deletions within integrin genes of AGS and KatoIII cells by gene amplification and DNA sequencing.
The top line shows the corresponding sequence of human integrin β1 A), the integrin αv B) or the β4 gene C) showing the Guide A and Guide B sequences (blue, underlined), the PAM sequence and putative cleavage sites of Cas9 nic...
Characterization of AGS wild type and integrin knockout cell lines for their ability to induce the hummingbird phenotype.
(A) AGS wild type, AGS αvβ4 or AGS β1β4 cells were infected with P12 wt, P12ΔhopQ, or a complemented P12ΔhopQ/hopQ Hp strain re-expressing wt hopQ gene for 4 h. As compared to non-infected controls, AGS wild type and AGS knockou...
Integrin profiling in different integrin-depletion cell lines.
Wild type cell lines and integrin-depletion cell lines were stained with antibodies specific to ITGAv, ITGB1, ITGB2, ITGB3, ITGB4, ITGB5, ITGB6, ITGB7 and ITGB8, and were subsequently monitored by flow cytometry in the FITC-A channel. FITC median were obtained and analyzed with the Flow...
Strategy for a targeted deletion within exon 2 of the CEACAM1 gene in KatoIII cells.
Streptococcus pyogenes Cas9 nickase binding sites (20 bp, highlighted in blue) are immediately followed by the 5’-NGG PAM (protospacer adjacent motif). The short guide RNA (sgRNA) pairs are located on both strands of the target DNA with a 25 bp gap. Cloning scheme...
Verification of the loss of integrin and CEACAM protein production by immunoblotting.
Lysates of AGS wild type and integrin knockout cell lines (A) and KatoIII wild type and integrin- or CEACAM1/5/6 knockout cell lines (B) were analyzed by immunoblotting using specific antibodies against human integrins as indicated. Loading controls are presented...
Strategy for targeted deletion of integrin αv gene in exon 4.
Streptococcus pyogenes Cas9 nickase binding sites (20 bp, highlighted in blue) are immediately followed by the 5’-NGG PAM (protospacer adjacent motif). The short guide RNA (sgRNA) pairs are located on both strands of the target DNA with a 25 bp gap. Cloning scheme of the CRISPR plasmids...
Determination of IL-8 induction in AGS integrin-depletion cell lines.
The induction of IL-8 was determined after infection of AGS wild type or integrin knockout cell lines for 4 h with P12 wt, P12ΔhopQ, P12ΔhopQ/hopQ or other Hp lab strains. Statistics: n = 4, one way Anova, ***, p<0.001. Values are means +/- SEM.
(TIF)
Verification of targeted deletions within the CEACAM1, CEACAM5 and CEACAM6 genes of KatoIII cells by gene amplification and DNA sequencing.
The top line shows the corresponding sequence of human CEACAM1 (A), CEACAM5 (B) and CEACAM6 gene (C) with the Guide A and Guide B sequences (blue, underlined), the PAM sequence and putative cleavage sites of Ca...
Strategy for targeted deletion of integrin β4 gene in exon 6.
Streptococcus pyogenes Cas9 nickase binding sites (20 bp, highlighted in blue) are immediately followed by the 5’-NGG PAM (protospacer adjacent motif). The short guide RNA (sgRNA) pairs are located on both strands of the target DNA with a 25 bp gap. Cloning scheme of the CRISPR plasmids...
Integrin profiling in KatoIII wild type and KatoIII cells lacking CEACAM1, CEACAM5 and CEACAM6 (CEACAM1/5/6 KO) cells.
KatoIII cells and integrin-depletion cell lines were stained with antibodies specific to ITGB1, ITGB2, ITGB3, ITGB4, ITGB5, ITGB6, ITGB7 and ITGB8, and ITGAv and were subsequently monitored by flow cytometry in the FITC-A channel....
Quantitative evaluation of CagA translocation into wild type integrin-knockout AGS or KatoIII cell lines by the TEM-1 β-lactamase reporter assay.
A) Raw data of KatoIII cells and derivatives thereof measured by flow cytometry, as shown in Fig 3A. B) Raw data of KatoIII cells and derivatives thereof measured by flow cytometry, as shown in Fig 3B.
(T...
Sequences of paired sgRNAs designed for targeting ITGB1, ITGAv and ITGB4 genes.
(PDF)
CRISPR constructs and targeted cell lines for the generation of integrin-depletion AGS and KatoIII cell lines.
(PDF)
Bacterial strains used in this study.
(PDF)
Objetivo:
este estudio caracteriza la diversidad de los genes de virulencia cagA (gen asociado con la citotoxina A) y vacA (citotoxina vacuolizante) en pacientes colombianos para determinar posibles asociaciones entre estos 2 genes y la severidad de los hallazgos endoscópicos teniendo en cuenta todos los genotipos reportados para el gen vacA (s, m...
Whole article under https://sciencematters.io/articles/201706000006.
Since its discovery in the human stomach, the bacterium Helicobacter pylori has been branded as the cause of gastric diseases. This association is linked to the oncogenic toxin CagA produced by certain H. pylori strains, which causes severe damages but needs to be injected into th...
The more severe strains of the bacterial human pathogen Helicobacter pylori produce a type IV secretion system (cagT4SS) to inject the oncoprotein CagA into gastric cells. This syringe-like molecular apparatus is prolonged by an external pilus that exploits integrins as receptors to mediate the injection of CagA. The molecular determinants of the i...
Protocol established for shipment of H. pylori samples from Colombia to Germany, which has a time of 5 to 6 days. It was necessary after shipments using Portagerm Pylori (BioMerieux) did not survive this long shipping time.
Protocol established by Anna F. Zetler and published by Luisa F. Jiménez-Soto This protocol is the final adaptation of protocols used in the laboratory of Prof. Rainer haas. The following people contributed with their ideas and experience: Benjamin Busch, Bettina Vogl-Gebert and Luisa F. Jiménez-Soto
The protocol was creating adapting components from the manual from Bio-Rad for the analysis software ImageLab. Data analyzed using the protocol here described have appear in the following publications: Zeitler AF, Gerrer KH, Haas R, Jiménez-Soto LF. Optimized semi-quantitative blot analysis in infection assays using the Stain-Free technology. J Mic...
This is a variation of the CagA translocation assay performed in our lab. This protocol has been used in the publication DOI: 10.1111/cmi.12166
This is a variation of the CagA translocation assay performed in the lab. The protocol was used in the publication DOI: 10.1111/cmi.12166
The IL-8 ELISA protocol here described was established by Stephan Odenbreit and modified in this form by Luisa F. Jiménez-Soto. Here are the publications using this protocol: DOI:10.1371/journal.pone.0035341 DOI:10.1128/IAI.00364-09 DOI:10.1078/1438-4221-00205 and PMID:11886563
This protocol was standarized in our lab based on the publication of Ahn T et al (2001): Polyacrylamide Gel Electrophoresis without a stacking Gel: Use of Amino Acids as Electrolytes. DOI:10.1006/abio.2001.5038 These gels run similar to a gradient gel: In a 6% gel you will be able to separate proteins ranging from 270 kDa to 25 kD an dhave them in...
This is our transfer protocol for the transfer of proteins using the Trans-Blot Turbo Transfer system from Bio-Rad. We use it with our single gel we have made based the One-Gel system by Ahn T et al (2001) (DOI:10.1006/abio.2001.5038). It should work the same for Laemmli-based gel systems. We have achieved transfer from proteins ranging from 270 kD...
Western blots are a commonly used method for protein detection and quantification in biological samples. Compensation of loading variations is achieved by housekeeping protein (HKP) normalization and/or total protein normalization (TPN). However, under infection conditions, HKP normalization, traditionally used in cell biology for quantification of...
Use of the Colony Lift Immunoassay has been described for several Gram negative bacteria of medical interest. In all cases detection was limited to the use of antibodies against outer membrane proteins. Here we describe the adaptation of this method for detection of the cytoplasmic CagA toxin from Helicobacter pylori.
Use of the Colony Lift Immunoassay has been described for several Gram negative bacteria of medical interest. In all cases detection was limited to the use of antibodies against outer membrane proteins. Here we describe the adaptation of this method for detection of the cytoplasmic CagA toxin from Helicobacter pylori.
The human pathogen Helicobacter pylori colonises half of the global population. Residing at the stomach epithelium, it contributes to the development of diseases like gastritis, duodenal and gastric ulcers, and gastric cancer. A major factor is the secreted vacuolating toxin VacA, which forms anion-selective channels in the endosome membrane that c...
The current paradigm suggests that Yersinia entercolitica (Ye) adheres to host cells via the outer membrane proteins YadA or invasin (Inv) to facilitate injection of Yops by the type III secretion system; in this process Inv binds directly to β1 integrins of host cells while YadA may bind indirectly via extracellular matrix proteins to β1 integrins...
Helicobacter pylori, the causative agent of type B gastritis, peptic ulcers, gastric adenocarcinoma and MALT lymphoma, uses the Cag type IV secretion system to induce a strong proinflammatory response in the gastric mucosa and to inject its effector protein CagA into gastric cells. CagA translocation results in altered host cell gene expression pro...
Colonization of the gastric mucosa by Helicobacter pylori is often associated with chronic gastric pathologies in humans. Development of disease correlates with the presence of distinct bacterial pathogenicity factors, such as the cag type IV secretion system (cag-T4SS), the vacuolating cytotoxin (VacA), or the ability of the bacteria to acquire an...
Meningococci are facultative-pathogenic bacteria endowed with a set of adhesins allowing colonization of the human upper respiratory
tract, leading to fulminant meningitis and septicemia. The Neisseria adhesin NadA was identified in about 50% of N. meningitidis isolates and is closely related to the Yersinia adhesin YadA, the prototype of the oligo...
Meningococci are facultative-pathogenic bacteria endowed with a set of adhesins allowing colonization of the human upper respiratory
tract, leading to fulminant meningitis and septicemia. The Neisseria adhesin NadA was identified in about 50% of N. meningitidis isolates and is closely related to the Yersinia adhesin YadA, the prototype of the oligo...
Gastric infection by Helicobacter pylori (Hp) is associated with development of gastritis, ulcerations and gastric adenocarcinoma. Production and secretion of the vacuolating cytotoxin (VacA) is an essential Hp virulence factor. VacA is a multifunctional toxin, which exerts immunosuppressive effects on human T lymphocytes via inhibition of cell pro...
Oligonucleotides used for the generation of GST fusion proteins
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Cell lines used in this study and their growth conditions
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High resolution FESEM micro-graphs of AGS cells infected with H. pylori. (A–F) CagA labelling is repeatedly found on the tip region of the secretion pilus using immunogold labelled antibody AK273 (arrows) (G–I) P12ΔPAI strain showing only few gold-particles on the AGS cell surface (arrows in G and H); in control experiments with H. pylori-infected...
Pharmacological Iinhibitors interfering with CagA translocation in AGS epithelial cells. CagA translocation into AGS cells is inhibited by calpeptin (CP), an inhibitor of the Ca2+-dependent protease calpain. CP, calpain. Filled arrowheads mark CagAP-tyr, open arrowheads mark CagA bands.
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Supporting methods file
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Protein-protein interactions among H. pylori cag-PAI proteins and β1 integrin. Yeast cells co-transformed with plasmids expressing β1 integrin and either HP0527a (CagYa), HP0527b (CagYb), HP0527c (CagYc), HP0540 (CagI), HP0547a (CagAa) or HP0547b (CagAb), growing on minimal SD medium lacking tryptophan and leucine (SD-Trp-Leu) or on selective minim...
Integrin binding assays. (A) General procedure to generate Hp extract for pulldown assay with β1 integrin beads. Total lysate depicts the presence of CagA and CagY in P12 wt strain. (B) Quantification of binding of purified GST-Cagβ, GST-CagG, GST-CagZ (negative controls for integrin binding) or GST-Inv397 (positive control) (30mg/1×106 cells) to i...
The spread of retroviruses between cells is estimated to be 2-3 orders of magnitude more efficient when cells can physically interact with each other. The underlying mechanism is largely unknown, but transfer is believed to occur through large-surface interfaces, called virological or infectious synapses. Here, we report the direct visualization of...
Retroviral assembly and budding is driven by the Gag polyprotein and requires the host-derived vacuolar protein sorting (vps) machinery. With the exception of human immunodeficiency virus (HIV)-infected macrophages, current models predict that the vps machinery is recruited by Gag to viral budding sites at the cell surface. However, here we demonst...
Questions
Questions (18)
Dear all. I was planning on using the Gateway system with my gene in a pDONR vector to reombine it with the pEXT1 - DEST and pEXT2 -DEST vectors. We ordered EVERYTHING on Thursday with Life Technologies - Thermo Scientific and we got an e-mail that they do not sell the pEXT - DEST vectors. I was not planing with it. We found another kit using bacteria ribosomal system with NEB but I cannot find a vector with his-Tag and T7 promoter with RBS that does not have the lac promoter in between. Any idea what we could use? All the pET I found are inducible.
Thank you for your time,
Luisa
Dear All,
We performed some experiments and used the values of absorbance at 570 nm and 600 nm to calculate what the company calls "percentage of reduction". However, with the long time incubations (over 22 hours) we got values with over 100% (111%- 134%). Since these seems to be off, we have checked manually our code and verified that every value was used correctly. As we looked at the formula we noticed that it is measuring a ratio value, but not against a absolute reduced value. See snapshot attached. Are we interpreting it right and the "percentage" given by the companies is wrong?
Any help will be greatly appreciated.
P.S. Because of copyrights I cannot give you a full snapshot of the values.For the full equation please look under "AlamarBlue percentage reduction"
Dear All,
I was just informed that the company Smolecule is promoting their product 2-Chloroethanol with my research and other publications using trichloroethanol for visualization of proteins in gels (https://www.smolecule.com/products/s566426). PLEASE, I am not aware of 2-Chloroethanol working for the visualization of proteins in gels. Do not get confused. We have used ONLY the trichloroethanol and all my research on the subject, as well as the original paper, used this form. I have asked the Smolecule company to bring the evidence that their molecule works identically to the published one or remove my reference. I will keep you updated if I get any answer.
