This work was funded by NIH grant R01 AI073783 to K. Teter. We thank Dr. Shane Massey for assistance in the development of the subcellular fractionation protocol and Helen Burress for critical reading of the manuscript.

1. Preparation of digitonin
<ol>
	<li>Add 500 &mu;L of 100% ethanol to a microcentrifuge tube and place it in a heat block set at 80 &deg;C for 10 min.</li>
	<li>Dissolve 2.5 mg of digitonin in 250 &mu;L of the heated ethanol to produce a 1% stock solution of digitonin.</li>
	<li>To generate a working solution of 0.04% digitonin, add 40 &mu;L of the digitonin stock solution to 960 &mu;L of HCN buffer (50 mM Hepes pH 7.5, 150 mM NaCl, 2 mM CaCl2, 10 mM N-ethylmaleimide, and a protease inhibitor cocktail).</li>
</ol>
2. Cell intoxication and permeabilization
Our translocation assay can be applied to a range of toxins and cell lines. Below, we provide a detailed protocol for the detection of cholera toxin (CT). An overview of the procedure is provided in Figure 1.
<ol>
	<li>HeLa cells are seeded in 6-well plates containing 1 mL DMEM supplemented with 10% fetal bovine serum and antibiotic-antimycotic to achieve 1x106 cells/well after an overnight incubation at 37 &deg;C. To generate enough cytosolic toxin for reproducible detection, triplicate wells are required for each condition.</li>
	<li>Following an overnight incubation, replace the culture medium with 1 mL DMEM containing 100 ng/mL of ganglioside GM1. This increases the number of binding sites for CT, as the GM1 receptor of CT will intercalate into the plasma membrane of cells exposed to a solution of GM1.</li>
	<li>After a 1 hr incubation at 37 &deg;C, remove the GM1-containing medium and wash the cells twice with DMEM. Then, place the cells at 4 &deg;C for 30 min in 1 mL DMEM containing 1 &mu;g/mL of CT.</li>
	<li>Remove the toxin-containing medium, wash the cells twice with DMEM, and incubate the cells in 1 mL DMEM at 37 &deg;C for the desired time interval(s).</li>
	<li>At the end of each chase period, wash the cells with PBS and incubate each well with 250 &mu;L of 0.5 mM EDTA in PBS. Let the cells sit for 10 min at room temperature and then remove them from the 6-well plate by vigorous trituration with a p1000 pipetman. After one well of cells has been collected, combine it with the second and then third well from the same condition. Cell scrapers can also be used to collect the cells.</li>
	<li>Place the combined cell suspension from the three replicate wells (750 &mu;L total volume) in a single microcentrifuge tube, and spin it in a tabletop microcentrifuge for 5 min at 5,000 x g. Cell pellets of roughly equivalent size should be obtained for all conditions.</li>
	<li>Discard the supernatant and resuspend the cell pellet in 100 &mu;L of the 0.04% working solution of digitonin. Place the cell suspension on ice for 10 min.</li>
	<li>Spin the digitonin-permeabilized cells at 16,000 x g for 10 min in a tabletop microcentrifuge. Transfer the cytosol-containing supernatant fraction to a fresh microcentrifuge tube, and retain the organelle-containing pellet fraction.</li>
</ol>
3. Sample preparation
<ol>
	<li>Cytosol-containing supernatant fractions are diluted in HCN buffer to a final volume of 1 mL. Sample volumes of at least 1 mL are necessary to ensure air is purged from the 500 &mu;L sample loop before injection.</li>
	<li>Organelle-containing pellet fractions are resuspended in 1 mL of HCN buffer containing 1% Triton X-100. Addition of detergent is necessary to release the membrane-encased pool of toxin.</li>
	<li>Toxin standards at concentrations of 100, 10, 1, and 0.1 ng/mL are prepared in HCN buffer.</li>
	<li>Parallel sets of cytosolic and organelle fractions are prepared for a control experiment to confirm the fidelity of the fractionation procedure. Fractions generated as described in Section 2 are resuspended in 20 &mu;L of 4x sample buffer (cytosolic fraction) or 120 &mu;L of 1x sample buffer (organelle fraction). Equivalent volumes of each fraction are resolved by sodium dodecyl sulfate polyacrylamide gel electrophoresis and probed by Western blot analysis to demonstrate the partitioning of a cytosolic protein in the supernatant fraction and a soluble, resident ER protein in the pellet fraction16-19.</li>
</ol>
4. SPR slide preparation
<ol>
	<li>The Reichert SR7000 SPR Refractometer is used for SPR experiments.</li>
	<li>Set a gold-plated glass slide with a self-assembled monolayer in the SPR instrument. To activate the sensor slide, perfuse a 1:1 (v:v) solution of 0.4 M EDC and 0.1 M NHS over the slide for 10 min at a flow rate of 41 &mu;L/min. All subsequent perfusions will use the same flow rate.</li>
	<li>To remove the EDC:NHS activation buffer, wash the plate for 5 min with PBS containing 0.05% Tween 20 (PBST). The plate now contains reactive amide tethers due to uncapping by the EDC:NHS solution.</li>
	<li>Perfuse an anti-CTA antibody over the activated sensor slide at a dilution of 1:20,000 in 20 mM sodium acetate (pH 5.5) for 15 min. An initial drop in the refractive index unit (RIU) will be seen due to the pH change. This will be followed by an increase in the RIU as the antibody is captured by the amide-reactive tethers on the sensor slide.</li>
	<li>Remove unbound antibody from the sensor slide with a 5 minute PBST wash. The RIU signal produced by the captured antibody will plateau and stabilize, providing a new baseline signal.</li>
	<li>Perfuse 1 M ethanolamine (pH 8.5) over the sensor slide for 5 min. This caps and inactivates any unbound tethers left on the sensor slide.</li>
</ol>
5. SPR analysis of samples
<ol>
	<li>To establish a baseline reading, perfuse PBST over the antibody-coated sensor for 5 min.</li>
	<li>Perfuse an experimental sample or toxin standard over the sensor for 300 sec. Remove the ligand from the buffer and perfuse PBST over the sensor for another 200 sec.</li>
	<li>Following each perfusion, bound toxin is removed from the sensor by a 100 sec wash with PBST at pH 5.0. This will return the signal to its initial baseline reading, thus allowing another sample to be processed on the same sensor.</li>
	<li>Before loading a new sample, push a small amount of air through the sample loop to expel any residual fluid from the prior sample. Using the procedure outlined in 5.2-5.4, we have run up to 12 samples on one slide without substantial loss of the baseline signal.</li>
	<li>Data analysis is performed with the Scrubber 2 software, and the Igor software is used for figure preparation.</li>
</ol>
6. Representative Results
Pertussis toxin (PT) is an AB toxin that moves from the cell surface to the ER before its A chain (PTS1) enters the cytosol3, 12. As shown in Figure 2, our SPR-based translocation assay could detect PTS1 in the cytosol of intoxicated CHO cells. No signal was generated from the cytosol of unintoxicated cells, which confirmed the anti-PTS1 antibody did not cross-react with a component of the host cytosol. The cytosolic fraction from cells intoxicated in the presence of brefeldin A (BfA) also failed to produce a positive signal. BfA prevents toxin transport to the ER translocation site6-8, 12, 20-25 and, thus, A chain delivery to the cytosol. At the end of each run, bound toxin is stripped from the sensor slide. This allowed multiple samples to be screened on a single sensor slide and thereby provided a direct comparison between results obtained with different experimental conditions.
CT is another AB-type, ER-translocating toxin4. In Figure 3A, CTA1 was detected in the cytosolic fraction from CT-treated HeLa cells. This emphasized that our methodology works with multiple cell types and can be applied to any toxin for which an anti-A chain antibody is available. No signal was detected when the cytosolic fraction from CT-treated cells was perfused over an SPR sensor coated with an anti-CTB antibody (Fig. 3B), thus demonstrating that the CTA1 subunit but not the cell-binding CTB pentamer enters the cytosol. Figure 3C shows the signal from the organelle fraction is off-scale in comparison to the weaker signal from the cytosolic fraction. This was consistent with the known inefficiency of CT transport to the ER translocation site6, 7, which in turn limits the amount of toxin that can reach the cytosol. Furthermore, the organelle fraction contains CT holotoxin as well as ER-localized CTA1, so the resulting SPR signal for the organelle fraction is inflated by the additional mass of the holotoxin-associated CTB pentamer. Thus, it is not practical to plot data from the organelle and cytosolic fractions on the same sensorgram.
Our assay can monitor the time-dependent accumulation of translocated, cytosolic toxin (Fig 4). Cells were exposed to CT at 4 &deg;C, a temperature that allows toxin binding to the plasma membrane but prevents internalization of the cell-associated toxin. After the removal of unbound toxin, the cells were warmed to 37 &deg;C. Both toxin transport to the ER and A chain translocation to the cytosol can occur at this temperature. No toxin was detected in the cytosol 15 minutes after warming to 37 &deg;C. This reflected the lag time required for (i) holotoxin trafficking to the ER; (ii) A/B subunit dissociation in the ER; and (iii) A chain export to the cytosol. A minor pool of cytosolic toxin was detected after 30 minutes at 37 &deg;C, and progressively larger quantities of cytosolic toxin were detected after the 45 and 60 minute chase intervals. Even greater levels of cytosolic toxin were detected after a 5 hour chase interval17, thus demonstrating a continual, long-term delivery of cell-associated toxin to the host cytosol.
Our assay can also detect the inhibition of toxin translocation to the cytosol (Fig. 5). Cells treated with 10% dimethyl sulfoxide (DMSO), a chemical chaperone that prevents the thermal disordering of the isolated CTA1 subunit (T. Banerjee and K. Teter, unpublished observations), exhibited low levels of cytosolic CTA1 in comparison to the untreated control cells. Unfolding of the toxin A chain is a prerequisite for translocation to the cytosol16-18, so the DMSO-induced stabilization of CTA1 accordingly prevented its movement from the ER to the cytosol.
The association rate constant (ka) calculated from SPR experiments is directly proportional to the concentration of ligand in the perfusion buffer14, 15, 26. Thus, it is possible to determine the concentration of cytosolic toxin from a graph that plots the ka values for toxin standards as a function of toxin concentration. This procedure was used to quantify the DMSO-induced block of toxin translocation presented in Figure 5: the standard curve generated from known concentrations of toxin was used to calculate a cytosolic CTA1 concentration of 0.3 ng/mL for untreated cells and 0.1 ng/mL for DMSO-treated cells (Fig. 6). The inhibition of CTA1 unfolding by DMSO thus generated a 3-fold reduction in the ER-to-cytosol translocation of CTA1.
<img alt="Figure 1" src="/files/ftp_upload/3686/3686fig1.jpg" /> 
Figure 1. Protocol overview. (A) Cells are incubated with the AB toxin at 4 &deg;C, a temperature that allows toxin binding to the cell surface but prevents toxin endocytosis. The A and B subunits of the toxin are represented by red and blue circles, respectively. (B) Unbound toxin is removed from the medium, and cells are warmed to 37 &deg;C in order to promote endocytosis and retrograde transport of the holotoxin to the ER. Holotoxin dissociation occurs in the ER, which allows the isolated A chain to enter the cytosol by passing through a protein-conducting channel(s) in the ER membrane. (C) Cells are treated with digitonin in order to selectively permeabilize the plasma membrane. (D) Centrifugation is used to partition the cells into separate cytosolic and organelle fractions. The cytosol is squeezed out of the cell through the digitonin-generated pores and is located in the supernatant. The intact, membrane-bound organelles are found in the pellet fraction. (E) To detect the translocated pool of toxin A chain in the host cytosol, the supernatant fraction is perfused over an SPR sensor coated with an anti-A chain antibody.
<img alt="Figure 2" src="/files/ftp_upload/3686/3686fig2.jpg" /> 
Figure 2. Detection of PTS1 translocation into the host cytosol. CHO cells were pulse-labeled at 4 &deg;C for 30 min with 1 &mu;g/mL of PT. The cells were then chased for 3 hr at 37 &deg;C in toxin-free medium containing no additions (intoxicated) or 5 &mu;g BfA/mL (+BfA intoxicated). Permeabilization of the plasma membrane with digitonin was used to partition cell extracts into separate organelle and cytosolic fractions. An SPR sensor coated with an anti-PTS1 antibody was used to detect the cytosolic pool of PTS1 from untreated or BfA-treated cells. PTS1 standards were perfused over the sensor as positive controls, while the cytosolic fraction from unintoxicated cells was perfused over the sensor slide as a negative control. At the end of each run, bound sample was stripped from the sensor slide.
<img alt="Figure 3" src="/files/ftp_upload/3686/3686fig3.jpg" /> 
Figure 3. Detection of CTA1 translocation into the host cytosol. HeLa cells pulse-labeled at 4 &deg;C with 1 &mu;g/mL of CT were chased for 2 hr at 37 &deg;C in toxin-free medium containing no additions (intoxicated) or 5 &mu;g BfA/mL (+BfA intoxicated). Permeabilization of the plasma membrane with digitonin was used to partition cell extracts into separate organelle and cytosolic fractions. (A) The cytosolic fractions were perfused over an SPR sensor coated with an anti-CTA antibody. Known quantities of CTA were used as positive controls, while the cytosol from unintoxicated cells was used as a negative control. (B) The cytosolic fraction from cells intoxicated in the absence of BfA was perfused over an SPR sensor coated with an anti-CTB antibody. A purified CTB pentamer was perfused over the slide as a positive control. (C) The organelle fraction was solubilized with 1% Triton X-100 before perfusion over a SPR sensor coated with an anti-CTA antibody. For comparative purposes, the cytosolic fraction (1 mL final volume) from the same cell extract and a CTA standard were also perfused over the sensor. For all panels, bound sample was stripped from the sensor at the end of each run.
<img alt="Figure 4" src="/files/ftp_upload/3686/3686fig4.jpg" /> 
Figure 4. Kinetics of CTA1 entry into the cytosol. HeLa cells pulse-labeled at 4 &deg;C with 1 &mu;g/mL of CT were chased for 15, 30, 45, or 60 min at 37 &deg;C in toxin-free medium. To detect the translocated pool of toxin, cytosolic fractions from digitonin-permeabilized cells were perfused over an SPR sensor coated with an anti-CTA antibody. CTA standards were perfused over the sensor as well. At the end of each run, bound sample was stripped from the sensor slide.
<img alt="Figure 5" src="/files/ftp_upload/3686/3686fig5.jpg" /> 
Figure 5. Inhibition of CTA1 translocation by DMSO. HeLa cells pulse-labeled at 4 &deg;C with 1 &mu;g/mL of CT were chased for 2 hr at 37 &deg;C in toxin-free medium containing no additions (intoxicated) or 10% DMSO (+DMSO intoxicated). To detect the translocated pool of toxin, cytosolic fractions from digitonin-permeabilized cells were perfused over an SPR sensor coated with an anti-CTA antibody. CTA standards (100, 10, 1, and 0.1 ng/mL) were perfused over the sensor as positive controls; only the 1 and 0.1 ng/mL standards are shown for scaling purposes. The cytosol from unintoxicated cells was used as a negative control. At the end of each run, bound sample was stripped from the sensor slide.
<img alt="Figure 6" src="/files/ftp_upload/3686/3686fig6.jpg" /> 
Figure 6. Calculation of cytosolic CTA1. ka values for the CTA standards from Figure 5 were plotted as a function of toxin concentration. The resulting standard curve was used to determine, based on the ka values of the experimental samples from Figure 5, the concentration of cytosolic CTA1 in untreated and DMSO-treated cells. Toxin standards are presented as filled circles; the untreated cytosol is presented as an open square; and the DMSO-treated cytosol is presented as an open circle. The averages &plusmn; ranges of two independent experiments are shown.

No conflicts of interest declared.

Comparison to existing methodology
Our SPR-based translocation assay represents a rapid, sensitive, and quantitative method to detect toxin delivery into the host cytosol. The technique does not require radiolabeling or other modifications to the toxin, and it can be applied to any toxin for which an anti-toxin A chain antibody is available. Existing methods to monitor toxin passage into the cytosol also rely upon a subcellular fractionation protocol to partition cell extracts into separate cyt...

<table border="1">
 <tbody>
 <tr>
 <td>Name of the reagent</td>
 <td>Company</td>
 <td>Catalogue number</td>
 </tr>
 <tr>
 <td>Digitonin</td>
 <td>Sigma</td>
 <td>D141</td>
 </tr>
 <tr>
 <td>Ethanol</td>
 <td>Acros</td>
 <td>61509-0010</td>
 </tr>
 <tr>
 <td>DMEM</td>
 <td>Invitrogen</td>
 <td>11995065</td>
 </tr>
 <tr>
 <td>Fetal Bovine Serum</td>
 <td>Atlanta Biologicals</td>
 <td>S11550</td>
 </tr>
 <tr>
 <td>Ganglioside GM1</td>
 <td>Sigma</td>
 <td>G7641</td>
 </tr>
 <tr>
 <td>CTA</td>
 <td>Sigma</td>
 <td>C2398</td>
 </tr>
 <tr>
 <td>PTS1</td>
 <td>List</td>
 <td>182</td>
 </tr>
 <tr>
 <td>NHS (N-Hydroxysuccinimide)</td>
 <td>Pierce</td>
 <td>24500</td>
 </tr>
 <tr>
 <td>EDC (1-Ethyl-3-(3-dimethylaminopropyl)carbodiimide)</td>
 <td>Thermo Scientific</td>
 <td>22981</td>
 </tr>
 <tr>
 <td>Ethanolamine </td>
 <td>Sigma</td>
 <td>E0135</td>
 </tr>
 <tr>
 <td>PBST</td>
 <td>Medicago</td>
 <td>09-8903-100</td>
 </tr>
 <tr>
 <td>Anti-CTA antibody</td>
 <td>Santa Cruz Biotech</td>
 <td>sc-80747</td>
 </tr>
 <tr>
 <td>Anti-CTB antibody</td>
 <td>Calbiochem</td>
 <td>227040</td>
 </tr>
 <tr>
 <td>Anti-PTS1 antibody</td>
 <td>Santa Cruz Biotech</td>
 <td>sc-57639 </td>
 </tr>
 <tr>
 <td>Refractometer</td>
 <td>Reichert</td>
 <td>SR7000, SR7000DC</td>
 </tr>
 <tr>
 <td>SPR sensor slides</td>
 <td>Reichert</td>
 <td>13206060</td>
 </tr>
 <tr>
 <td>Syringe pump</td>
 <td>Cole Palmer</td>
 <td>780200C</td>
 </tr>
 </tbody>
</table>

detection of toxin translocation into the host cytosol by surface plasmon resonance

AB toxins consist of an enzymatic A subunit and a cell-binding B subunit1. These toxins are secreted into the extracellular milieu, but they act upon targets within the eukaryotic cytosol. Some AB toxins travel by vesicle carriers from the cell surface to the endoplasmic reticulum (ER) before entering the cytosol2-4. In the ER, the catalytic A chain dissociates from the rest of the toxin and moves through a protein-conducting channel to reach its cytosolic target5. The translocated, cytosolic A chain is difficult to detect because toxin trafficking to the ER is an extremely inefficient process: most internalized toxin is routed to the lysosomes for degradation, so only a small fraction of surface-bound toxin reaches the Golgi apparatus and ER6-12.

To monitor toxin translocation from the ER to the cytosol in cultured cells, we combined a subcellular fractionation protocol with the highly sensitive detection method of surface plasmon resonance (SPR)13-15. The plasma membrane of toxin-treated cells is selectively permeabilized with digitonin, allowing collection of a cytosolic fraction which is subsequently perfused over an SPR sensor coated with an anti-toxin A chain antibody. The antibody-coated sensor can capture and detect pg/mL quantities of cytosolic toxin. With this protocol, it is possible to follow the kinetics of toxin entry into the cytosol and to characterize inhibitory effects on the translocation event. The concentration of cytosolic toxin can also be calculated from a standard curve generated with known quantities of A chain standards that have been perfused over the sensor. Our method represents a rapid, sensitive, and quantitative detection system that does not require radiolabeling or other modifications to the target toxin.

Watch this Scientific Journal Video about Detection of Toxin Translocation into the Host Cytosol by Surface Plasmon Resonance at JoVE.com

Detection of Toxin Translocation into the Host Cytosol by Surface Plasmon Resonance

In this report, we describe how surface plasmon resonance is used to detect toxin entry into the host cytosol. This highly sensitive method can provide quantitative data on the amount of cytosolic toxin, and it can be applied to a range of toxins.

AB toxins consist of an enzymatic A subunit and a cell-binding B subunit1.  These toxins are secreted into the extracellular milieu, but they act upon ...

detection-toxin-translocation-into-host-cytosol-surface-plasmon

Universidad de Cartagena UDEC

Research

JoVE Journal

Immunology and Infection

13.2K Views.  University of Central Florida. In this report, we describe how surface plasmon resonance is used to detect toxin entry into the host cytosol. This highly sensitive method can provide quantitative data on the amount of cytosolic toxin, and it can be applied to a range of toxins.

Video: Detection of Toxin Translocation into the Host Cytosol by Surface Plasmon Resonance

Detection of Infectious Virus from Field-collected Mosquitoes by Vero Cell Culture Assay

Mosquitoes transmit a number of distinct viruses including important human pathogens such as West Nile virus, dengue virus, and chickungunya virus. Many of these viruses have intensified in their endemic ranges and expanded to new territories, necessitating effective surveillance and control programs to respond to these threats. One strategy to monitor virus activity involves collecting large numbers of mosquitoes from endemic sites and testing them for viral infection. In this article, we describe how to handle, process, and screen field-collected mosquitoes for infectious virus by Vero cell culture assay. Mosquitoes are sorted by trap location and species, and grouped into pools containing &le;50 individuals. Pooled specimens are homogenized in buffered saline using a mixer-mill and the aqueous phase is inoculated onto confluent Vero cell cultures (Clone E6). Cell cultures are monitored for cytopathic effect from days 3-7 post-inoculation and any viruses grown in cell culture are identified by the appropriate diagnostic assays. By utilizing this approach, we have isolated 9 different viruses from mosquitoes collected in Connecticut, USA, and among these, 5 are known to cause human disease. Three of these viruses (West Nile virus, Potosi virus, and La Crosse virus) represent new records for North America or the New England region since 1999. The ability to detect a wide diversity of viruses is critical to monitoring both established and newly emerging viruses in the mosquito population.

We describe a method to process and screen field-collected mosquitoes for a diversity of viruses by Vero cell culture assay. By employing this technique, we have detected 9 different viruses from 4 taxonomic families in mosquitoes collected in Connecticut.

Mosquitoes transmit a number of distinct viruses including important human pathogens such as West Nile virus, dengue virus, and chickungunya virus. Many ...

Amplifying and Quantifying HIV-1 RNA in HIV Infected Individuals with Viral Loads Below the Limit of Detection by Standard Clinical Assays

Amplifying viral genes and quantifying HIV-1 RNA in HIV-1 infected individuals with viral loads below the limit of detection by standard assays (below 50-75 copies/ml) is necessary to gain insight to viral dynamics and virus host interactions in patients who naturally control the infection and those who are on combination antiretroviral therapy (cART). 
Here we describe how to amplify viral genomes by single genome sequencing (the SGS protocol) 13, 19 and how to accurately quantify HIV-1 RNA in patients with low viral loads (the single-copy assay (SCA) protocol) 12, 20. 
The single-copy assay is a real-time PCR assay with sensitivity depending on the volume of plasma being assayed. If a single virus genome is detected in 7 ml of plasma, then the RNA copy number is reported to be 0.3 copies/ml. The assay has an internal control testing for the efficiency of RNA extraction, and controls for possible amplification from DNA or contamination. Patient samples are measured in triplicate.
The single-genome sequencing assay (SGS), now widely used and considered to be non-labor intensive 3, 7, 12, 14, 15,is a limiting dilution assay, in which endpoint diluted cDNA product is spread over a 96-well plate. According to a Poisson distribution, when less than 1/3 of the wells give product, there is an 80% chance of the PCR product being resultant of amplification from a single cDNA molecule. SGS has the advantage over cloning of not being subjected to resampling and not being biased by PCR-introduced recombination 19. However, the amplification success of SCA and SGS depend on primer design. Both assays were developed for HIV-1 subtype B, but can be adapted for other subtypes and other regions of the genome by changing primers, probes, and standards.

Quantifying levels of HIV-1 RNA in plasma and sequencing single HIV-1 genomes from individuals with viral loads below the limit of detection (50-75 copies/ml) is difficult. Here we describe how to extract and quantify plasma viral RNA using a real time PCR assay that reliably measures HIV-1 RNA down to 0.3 copies/ml and how to amplify viral genomes by single genome sequencing, from samples with very low viral loads.

Amplifying viral genes and quantifying HIV-1 RNA in HIV-1 infected individuals with viral loads below the limit of detection by standard assays (below ...

Detection of Invasive Pulmonary Aspergillosis in Haematological Malignancy Patients by using Lateral-flow Technology

Invasive pulmonary aspergillosis (IPA) is a leading cause of morbidity and mortality in haematological malignancy patients and hematopoietic stem cell transplant recipients1. Detection of IPA represents a formidable diagnostic challenge and, in the absence of a 'gold standard', relies on a combination of clinical data and microbiology and histopathology where feasible. Diagnosis of IPA must conform to the European Organization for Research and Treatment of Cancer and the National Institute of Allergy and Infectious Diseases Mycology Study Group (EORTC/MSG) consensus defining "proven", "probable", and "possible" invasive fungal diseases2. Currently, no nucleic acid-based tests have been externally validated for IPA detection and so polymerase chain reaction (PCR) is not included in current EORTC/MSG diagnostic criteria.
Identification of Aspergillus in histological sections is problematic because of similarities in hyphal morphologies with other invasive fungal pathogens3, and proven identification requires isolation of the etiologic agent in pure culture. Culture-based approaches rely on the availability of biopsy samples, but these are not always accessible in sick patients, and do not always yield viable propagules for culture when obtained.
An important feature in the pathogenesis of Aspergillus is angio-invasion, a trait that provides opportunities to track the fungus immunologically using tests that detect characteristic antigenic signatures molecules in serum and bronchoalveolar lavage (BAL) fluids. This has led to the development of the Platelia enzyme immunoassay (GM-EIA) that detects Aspergillus galactomannan and a 'pan-fungal' assay (Fungitell test) that detects the conserved fungal cell wall component (1 &rarr;3)-&beta;-D-glucan, but not in the mucorales that lack this component in their cell walls1,4. Issues surrounding the accuracy of these tests1,4-6 has led to the recent development of next-generation monoclonal antibody (MAb)-based assays that detect surrogate markers of infection1,5.
Thornton5 recently described the generation of an Aspergillus-specific MAb (JF5) using hybridoma technology and its use to develop an immuno-chromatographic lateral-flow device (LFD) for the point-of-care (POC) diagnosis of IPA. A major advantage of the LFD is its ability to detect activity since MAb JF5 binds to an extracellular glycoprotein antigen that is secreted during active growth of the fungus only5. This is an important consideration when using fluids such as lung BAL for diagnosing IPA since Aspergillus spores are a common component of inhaled air. The utility of the device in diagnosing IPA has been demonstrated using an animal model of infection, where the LFD displayed improved sensitivity and specificity compared to the Platelia GM and Fungitell (1 &rarr; 3)-&beta;-D-glucan assays7.
Here, we present a simple LFD procedure to detect Aspergillus antigen in human serum and BAL fluids. Its speed and accuracy provides a novel adjunct point-of-care test for diagnosis of IPA in haematological malignancy patients.

A rapid and accurate point-of-care test for invasive pulmonary aspergillosis is presented. It takes advantage of lateral-flow technology using a specific monoclonal antibody that binds to an Aspergillus antigen secreted during pulmonary infections. The assay is compatible with serum and brochoalveolar lavage and represents a novel adjunct test for disease diagnosis.

Invasive pulmonary aspergillosis (IPA) is a leading cause of morbidity and mortality in haematological malignancy patients and hematopoietic stem cell ...

Analysis of Yersinia enterocolitica Effector Translocation into Host Cells Using Beta-lactamase Effector Fusions

Many gram-negative bacteria including pathogenic Yersinia spp. employ type III secretion systems to translocate effector proteins into eukaryotic target cells. Inside the host cell the effector proteins manipulate cellular functions to the benefit of the bacteria. To better understand the control of type III secretion during host cell interaction, sensitive and accurate assays to measure translocation are required. We here describe the application of an assay based on the fusion of a Yersinia enterocolitica effector protein fragment (Yersinia outer protein; YopE) with TEM-1 beta-lactamase for quantitative analysis of translocation. The assay relies on cleavage of a cell permeant FRET dye (CCF4/AM) by translocated beta-lactamase fusion. After cleavage of the cephalosporin core of CCF4 by the beta-lactamase, FRET from coumarin to fluorescein is disrupted and excitation of the coumarin moiety leads to blue fluorescence emission. Different applications of this method have been described in the literature highlighting its versatility. The method allows for analysis of translocation in vitro and also in in vivo, e.g., in a mouse model. Detection of the fluorescence signals can be performed using plate readers, FACS analysis or fluorescence microscopy. In the setup described here, in vitro translocation of effector fusions into HeLa cells by different Yersinia mutants is monitored by laser scanning microscopy. Recording intracellular conversion of the FRET reporter by the beta-lactamase effector fusion in real-time provides robust quantitative results. We here show exemplary data, demonstrating increased translocation by a Y. enterocolitica YopE mutant compared to the wild type strain.

Analysis of Yersinia enterocolitica Effector Translocation into Host Cells Using Beta-lactamase Effector Fusions

Effector translocation into host cells via a type III secretion system is a common virulence strategy among gram-negative bacteria. A beta-lactamase effector fusion based assay for quantitative analysis of translocation was applied. In Yersinia infected cells, conversion of a FRET reporter by the beta-lactamase is monitored using laser scanning microscopy.

Many gram-negative bacteria including pathogenic Yersinia spp. employ type III secretion systems to translocate effector proteins into eukaryotic target ...

Applying Fluorescence Resonance Energy Transfer (FRET) to Examine Effector Translocation Efficiency by Coxiella burnetii during siRNA Silencing

Coxiella burnetii, the causative agent of Q fever, is an intracellular pathogen that relies on a Type IV Dot/Icm Secretion System to establish a replicative niche. A cohort of effectors are translocated through this system into the host cell to manipulate host processes and allow the establishment of a unique lysosome-derived vacuole for replication. The method presented here involves the combination of two well-established techniques: specific gene silencing using siRNA and measurement of effector translocation using a FRET-based substrate that relies on &#946;-lactamase activity. Applying these two approaches, we can begin to understand the role of host factors in bacterial secretion system function and effector translocation. In this study we examined the role of Rab5A and Rab7A, both important regulators of the endocytic trafficking pathway. We demonstrate that silencing the expression of either protein results in a decrease in effector translocation efficiency. These methods can be easily modified to examine other intracellular and extracellular pathogens that also utilize secretion systems. In this way, a global picture of host factors involved in bacterial effector translocation may be revealed.

Applying Fluorescence Resonance Energy Transfer FRET to Examine Effector Translocation Efficiency by Coxiella burnetii during siRNA Silencing

Investigating the interactions between bacterial pathogens and their hosts is an important area of biological research. Here, we describe the necessary techniques to measure effector translocation by Coxiella burnetii during siRNA gene silencing using BlaM substrate.

Coxiella burnetii, the causative agent of Q fever, is an intracellular pathogen that relies on a Type IV Dot/Icm Secretion System to establish a ...

Detection of Trypanosoma brucei Variant Surface Glycoprotein Switching by Magnetic Activated Cell Sorting and Flow Cytometry

Trypanosoma brucei, a protozoan parasite that causes both Human and Animal African Trypanosomiasis (known as sleeping sickness and nagana, respectively) cycles between a tsetse vector and a mammalian host. It evades the mammalian host immune system by periodically switching the dense, variant surface glycoprotein (VSG) that covers its surface. The detection of antigenic variation in Trypanosoma brucei can be both cumbersome and labor intensive. Here, we present a method for quantifying the number of parasites that have 'switched' to express a new VSG in a given population. The parasites are first stained with an antibody against the starting VSG, and then stained with a secondary antibody attached to a magnetic bead. Parasites expressing the starting VSG are then separated from the rest of the population by running the parasites over a column attached to a magnet. Parasites expressing the dominant, starting VSG are retained on the column, while the flow-through contains parasites that express a new VSG as well as some contaminants expressing the starting VSG. This flow-through population is stained again with a fluorescently labeled antibody against the starting VSG to label contaminants, and propidium iodide (PI), which labels dead cells. A known number of absolute counting beads that are visible by flow cytometry are added to the flow-through population. The ratio of beads to number of cells collected can then be used to extrapolate the number of cells in the entire sample. Flow cytometry is used to quantify the population of switchers by counting the number of PI negative cells that do not stain positively for the starting, dominant VSG. The proportion of switchers in the population can then be calculated using the flow cytometry data.

Detection of Trypanosoma brucei Variant Surface Glycoprotein Switching by Magnetic Activated Cell Sorting and Flow Cytometry

African trypanosomes grown in vitro undergo antigenic variation at a low rate, such that populations are made up of parasites expressing a dominant variant surface glycoprotein (VSG) type and a small population of "switched" variants. This protocol describes a fast method for detecting and quantifying these populations.

Trypanosoma brucei, a protozoan parasite that causes both Human and Animal African Trypanosomiasis (known as sleeping sickness and nagana, respectively) ...

Protocols for Investigating the Host-tissue Distribution, Transmission-mode, and Effect on the Host Fitness of a Densovirus in the Cotton Bollworm

Many novel viruses have been discovered in animal hosts using next-generation sequencing technologies. Previously, we reported a mutualistic virus, Helicoverpa armigera densovirus (HaDV2), in a lepidopteran species, the cotton bollworm, Helicoverpa armigera (Hubner). Here, we describe the protocols that are currently used to study the effect of HaDV2 on its host. First, we establish a HaDV2-free cotton bollworm colony from a single breeding pair. Then, we orally inoculate some neonate larval offspring with HaDV2-containing filtered liquid to produce two colonies with the same genetic background: one HaDV2-infected, the other uninfected. A protocol to compare life table parameters (e.g., larval, pupal, and adult periods and fecundity) between the HaDV2-infected and -uninfected individuals is also presented, as are the protocols for determining the host-tissue distribution and transmission efficiency of HaDV2. These protocols would also be suitable for investigating the effects of other orally transmitted viruses on their insect hosts, lepidopteran hosts in particular.

Here, we present a protocol to investigate the host-tissue distribution, transmission mode, and effect on the host fitness of a densovirus within a lepidopteran species, the cotton bollworm. This protocol can also be used for studying the interaction between other orally-transmitted viruses and their insect hosts.

Many novel viruses have been discovered in animal hosts using next-generation sequencing technologies. Previously, we reported a mutualistic virus, ...

Detection of Enterohemorrhagic Escherichia Coli Colonization in Murine Host by Non-invasive In Vivo Bioluminescence System

Enterohemorrhagic E. coli (EHEC) O157:H7, which is a foodborne pathogen that causesdiarrhea, hemorrhagic colitis (HS), and hemolytic uremic syndrome (HUS), colonize to the intestinal tract of humans. To study the detailed mechanism of EHEC colonization in vivo, it is essential to have animal models to monitor and quantify EHEC colonization. We demonstrate here a mouse-EHEC colonization model by transforming the bioluminescent expressing plasmid to EHEC to monitor and quantify EHEC colonization in living hosts. Animals inoculated with bioluminescence-labeled EHEC show intense bioluminescent signals in mice by detection with a non-invasive in vivo imaging system. After 1 and 2 days post infection, bioluminescent signals could still be detected in infected animals, which suggests that EHEC colonize in hosts for at least 2 days. We also demonstrate that these bioluminescent EHEC locate to mouse intestine, specifically in the cecum and colon, from ex vivo images. This mouse-EHEC colonization model may serve as a tool to advance the current knowledge of the EHEC colonization mechanism.

Detection of Enterohemorrhagic Escherichia Coli Colonization in Murine Host by Non-invasive In Vivo Bioluminescence System

A detailed protocol of a mouse model for enterohemorrhagic E. coli (EHEC) colonization by using bioluminescence-labeled bacteria is presented. The detection of these bioluminescent bacteria by a non-invasive in vivo imaging system in live animals can advance our current understanding of EHEC colonization.

Enterohemorrhagic E. coli (EHEC) O157:H7, which is a foodborne pathogen that causesdiarrhea, hemorrhagic colitis (HS), and hemolytic uremic syndrome ...

A High-throughput Platform for the Screening of Salmonella spp./Shigella spp.

Fecal-oral transmission of acute gastroenteritis occurs from time to time, especially when people who handled food and water are infected by Salmonella spp./Shigella spp. The gold standard method for the detection of Salmonella spp./Shigella spp. is direct culture but this is labor-intensive and time-consuming. Here, we describe a high-throughput platform for Salmonella spp./Shigella spp. screening, using real-time polymerase chain reaction (PCR) combined with guided culture. There are two major stages: real-time PCR and the guided culture. For the first stage (real-time PCR), we explain each step of the method: sample collection, pre-enrichment, DNA extraction and real-time PCR. If the real-time PCR result is positive, then the second stage (guided culture) is performed: selective culture, biochemical identification and serological characterization. We also illustrate representative results generated from it. The protocol described here would be a valuable platform for the rapid, specific, sensitive and high-throughput screening of Salmonella spp./Shigella spp.

A High-throughput Platform for the Screening of Salmonella spp./Shigella spp.

Salmonella spp./Shigella spp. are common pathogens attributed to diarrhea. Here, we describe a high-throughput platform for the screening of Salmonella spp./Shigella spp. using real-time PCR combined with guided culture.

Fecal-oral transmission of acute gastroenteritis occurs from time to time, especially when people who handled food and water are infected by Salmonella ...

Temporal Analysis of the Nuclear-to-cytoplasmic Translocation of a Herpes Simplex Virus 1 Protein by Immunofluorescent Confocal Microscopy

Infected cell protein 0 (ICP0) of herpes simplex virus 1 (HSV-1) is an immediate early protein containing a RING-type E3 ubiquitin ligase. It is responsible for the proteasomal degradation of host restrictive factors and the subsequent viral gene activation. ICP0 contains a canonical nuclear localization sequence (NLS). It enters the nucleus immediately after de novo synthesis and executes its anti-host defense functions mainly in the nucleus. However, later in infection, ICP0 is found solely in the cytoplasm, suggesting the occurrence of a nuclear-to-cytoplasmic translocation during HSV-1 infection. Presumably ICP0 translocation enables ICP0 to modulate its functions according to its subcellular locations at different infection phases. In order to delineate the biological function and regulatory mechanism of ICP0 nuclear-to-cytoplasmic translocation, we modified an immunofluorescent microscopy method to monitor ICP0 trafficking during HSV-1 infection. This protocol involves immunofluorescent staining, confocal microscope imaging, and nuclear vs. cytoplasmic distribution analysis. The goal of this protocol is to adapt the steady state confocal images taken in a time course into a quantitative documentation of ICP0 movement throughout the lytic infection. We propose that this method can be generalized to quantitatively analyze nuclear vs. cytoplasmic localization of other viral or cellular proteins without involving live imaging technology.

ICP0 undergoes nuclear-to-cytoplasmic translocation during HSV-1 infection. The molecular mechanism of this event is not known. Here we describe the use of confocal microscope as a tool to quantify ICP0 movement in HSV-1 infection, which lays the groundwork for quantitatively analyzing ICP0 translocation in future mechanistic studies.

Detection of Toxin Translocation into the Host Cytosol by Surface Plasmon Resonance

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Detection of Trypanosoma brucei Variant Surface Glycoprotein Switching by Magnetic Activated Cell Sorting and Flow Cytometry

Protocols for Investigating the Host-tissue Distribution, Transmission-mode, and Effect on the Host Fitness of a Densovirus in the Cotton Bollworm

Detection of Enterohemorrhagic Escherichia Coli Colonization in Murine Host by Non-invasive In Vivo Bioluminescence System

A High-throughput Platform for the Screening of Salmonella spp./Shigella spp.

Temporal Analysis of the Nuclear-to-cytoplasmic Translocation of a Herpes Simplex Virus 1 Protein by Immunofluorescent Confocal Microscopy