S. aureus strains SF8300 WT and SF8300 &#916;fnbA/B were generously gifted by Prof. Binh Diep (University of California, San Francisco, USA). This work was supported by a grant of the FINOVI association (#AO13 FINOVI) under the aegis of the Foundation for the University of Lyon.

1. Culture of human epithelial cells
<ol>
	<li>Prepare complete culture medium with Dulbecco&#39;s modified Eagle medium (DMEM) high glucose with phenol red, supplemented with 10% fetal bovine serum (FBS) without antibiotics.</li>
	<li>Grow A549 epithelial cells in complete culture medium at 36 &#177; 1 &#176;C in 5% CO2. Ensure the use of an appropriately sized culture vessel to have enough cells for subsequent steps (see step 1.10). 
	NOTE: One 75 cm2 (T-75) flask is sufficient to seed two 24-well plates and subculture the cells.</li>
	<li>Two days before infection, prepare a single 24-well plate.</li>
	<li>Remove and discard the spent culture medium from the T-75 flask and wash the cells once with 10 mL of Dulbecco&#8242;s phosphate-buffered saline (DPBS).</li>
	<li>Add 5 mL of trypsin-EDTA and incubate the cells for 5 min at 36 &#177; 1 &#176;C in 5% CO2.</li>
	<li>Add 5 mL of complete culture medium and transfer the cells into a tube.</li>
	<li>Centrifuge the cells for 5 min at 300 &#215; g.</li>
	<li>Discard the supernatant and resuspend the cells in 10 mL of fresh complete culture medium.</li>
	<li>Count the cells with an automatic cell counter (or a counting chamber).</li>
	<li>Dilute the cells in complete culture medium to prepare 30 mL of cell suspension at a concentration of 2.0 &#215; 105 cells/mL.</li>
	<li>Add 1 mL of the cell suspension to each well of a 24-well plate, which corresponds to a cell density of approximately 1.0 &#215; 105 cell/cm&#178; for a well area of 2 cm&#178;.</li>
	<li>Incubate the cells for 48 h at 36 &#177; 1 &#176;C in 5% CO2 until they reach 100% confluence. 
	&#8203;NOTE: In addition to the conditions to be tested, three wells should be reserved for cell counting on the day of infection (see step 3.1.4). According to the number of conditions to be tested, Up to two 24-well plates can be prepared simultaneously. Volumes indicated in the protocol should be increased accordingly.</li>
</ol>
2. Culture of S. aureus strains
<ol>
	<li>Two days before infection, prepare complete infection medium with DMEM high glucose without phenol red, supplemented with 10% FBS without antibiotics.</li>
	<li>Thaw S. aureus strains to be tested on agar plates.</li>
	<li>Incubate the agar plates for 18-24 h at 36 &#177; 1 &#176;C.</li>
	<li>The day before inoculation, inoculate one colony of the S. aureus strain to be tested in 10 mL of complete infection medium.</li>
	<li>Incubate the bacteria for 18-24 h at 36 &#177; 1 &#176;C with shaking at 160 rpm. Use 50 mL tubes held at 45&#176; to avoid the bacteria settling. 
	&#8203;NOTE: Before starting with a new strain, it is recommended to verify its lysostaphin susceptibility in the same conditions of culture that will be used for further experiments (media, bacterial loads, and lysostaphin concentration and incubation time). It is also important to determine the bacterial load corresponding to an OD600nm of 0.5 because it could vary slightly from one strain to another. Culture conditions of bacterial strains could be adapted according to the experimental aim.</li>
</ol>
3. Infection assay with S. aureus
<ol>
	<li>Determination of cell density and viability
	<ol>
		<li>Remove and discard the spent culture medium from the three wells dedicated for counting A549 cells.</li>
		<li>Add 1 mL of complete infection medium containing 5 &#181;g/mL of Hoechst 33342 and 1 &#181;g/mL of propidium iodide. 
		NOTE: Hoechst 33342 is a known mutagen and should be handled with care. Propidium iodide, a potential mutagen, must be handled with care and disposed of safely according to applicable regulations.</li>
		<li>Incubate the cells for 30 min at 36 &#177; 1 &#176;C in 5% CO2.</li>
		<li>Count the cell number and calculate the cell viability using a wield-field fluorescence microscope. 
		NOTE: If a fluorescence microscope is not available, the cell density and viability can be calculated with trypan blue staining by using a cell counting chamber.</li>
	</ol>
	</li>
	<li>Preparation of the bacterial suspension
	<ol>
		<li>Dispense 25 mL of complete infection medium in a tube and pre-warm at 36 &#177; 1 &#176;C.</li>
		<li>Adjust the S. aureus suspension to anOD600nm of 0.5 in complete infection medium using a cell density meter.</li>
		<li>Prepare 20 mL of bacterial suspension for cell inoculation by diluting the 0.5 OD600nm in complete infection medium to achieve a multiplicity of infection (MOI) of 1 according to the number of cells per well. 
		NOTE: The MOI corresponds to the number of bacteria added per cell in each well. For example, to achieve an MOI of 1 with 1.0 &#215; 106 cells per well, prepare a bacterial suspension at 2.0 &#215; 106 CFU/mL so that 106 CFU can be added in a volume of 500 &#181;L (see step 3.3.3). The MOI can be adjusted according to the cell types and bacterial strains to be tested.</li>
		<li>Use an automatic spiral plater to determine the S. aureus load of the diluted bacterial suspension to be used for the cell inoculation step.</li>
		<li>Incubate the agar plates for 18-24 h at 36 &#177; 1 &#176;C.</li>
		<li>The next day, count the number of colonies with a colony counter to calculate the accurate MOI for each strain tested. 
		NOTE: If no automatic spiral plater is available, the bacterial load could be determined by serial dilution on an agar plate. See the bacteriological analytical manual for details14.</li>
	</ol>
	</li>
	<li>Cell inoculation
	<ol>
		<li>Observe every well of the 24-well plate by low magnification microscopy to ensure that the cells are healthy and growing as expected.</li>
		<li>Remove and discard the spent cell culture medium from the 24-well plate.</li>
		<li>Add 500 &#181;L of the bacterial suspension for inoculation to each well with 100% confluent cells.</li>
		<li>Incubate the cells for 2 h at 36 &#177; 1 &#176;C and 5% CO2. 
		NOTE: it is recommended to use three wells of the plate for each condition to be tested (triplicate) and to perform at least three independent experiments. The delay of incubation can be adapted according to the experimental aim.</li>
	</ol>
	</li>
	<li>Quantification of intracellular bacteria with improved enzyme protection assay (iEPA)
	<ol>
		<li>Prepare 7 mL of 4x lysis buffer with 3.5 mL of 2% Triton X-100 in sterile water and 3.5 mL of trypsin-EDTA.</li>
		<li>Prepare a lysostaphin stock solution at 10 mg/mL in acetate buffer and aliquot 25 &#181;L into cryovials. Store at -80 &#176;C for up to 6 months.</li>
		<li>Prepare 250 &#181;L of a fresh lysostaphin working solution at 1 mg/mL by mixing 25 &#181;L of the lysostaphin stock solution (10 mg/mL) and 225 &#181;L of 0.1 M Tris-HCl. Store at 4 &#176;C for up to 48 h.</li>
		<li>Prepare 6.25 mL of complete infection medium supplemented with lysostaphin by adding 6 mL of complete infection medium to 250 &#181;L of the lysostaphin working solution.</li>
		<li>Add 250 &#181;L of complete infection medium supplemented with lysostaphin into each well and gently agitate the plate by swiveling the plate by hand.</li>
		<li>Incubate the cells for 1 h at 36 &#177; 1 &#176;C in 5% CO2 to let the lysostaphin kill the extracellular bacteria.</li>
		<li>At the end of the incubation time, add 10 &#181;L of proteinase K at 20 mg/mL into each well to inactivate the lysostaphin.</li>
		<li>Incubate the cells for 2 min at room temperature.</li>
		<li>Add 250 &#181;L of 4x lysis buffer to lyse the cells by osmotic shock.</li>
		<li>Incubate the cells for 10 min at 36 &#177; 1 &#176;C.</li>
		<li>Mix thoroughly by pipetting up and down ten times all over the bottom of the well to ensure that the cells are fully lysed and homogenized.</li>
		<li>Use an automatic spiral plater to determine the S. aureus load of each well.</li>
		<li>Incubate the agar plates for 18-24 h at 36 &#177; 1 &#176;C.</li>
		<li>The next day, count the number of colonies with a colony counter to calculate the intracellular S. aureus load of each well.</li>
	</ol>
	</li>
	<li>Measurement of intracellular efficacy of antimicrobial compounds with enzyme protection assay (EPA)
	<ol>
		<li>Prepare 25 mL of 1x lysis buffer with 3.125 mL of 2% Triton X-100 in sterile water, 6.25 mL of trypsin-EDTA, and 15.625 mL of sterile water.</li>
		<li>Prepare 250 &#181;L of a fresh lysostaphin working solution at 1 mg/mL by mixing 25 &#181;L of a lysostaphin stock solution (10 mg/mL) and 225 &#181;L of 0.1 M Tris-HCl.</li>
		<li>Prepare 25 mL of complete infection medium supplemented with lysostaphin by adding 24.75 mL of complete infection medium to 250 &#181;L of the lysostaphin working solution.</li>
		<li>For each antimicrobial compound to be tested, prepare 3.1 mL of complete infection medium supplemented with lysostaphin and the antimicrobial compound at the concentration to be studied.</li>
		<li>Remove and discard the spent cell culture medium from the 24-well plate.</li>
		<li>Add 1 mL of complete infection medium supplemented with lysostaphin.</li>
		<li>Incubate the cells for 1 h at 36 &#177; 1 &#176;C in 5% CO2 to let the lysostaphin kill the extracellular bacteria.</li>
		<li>Remove and discard the medium supplemented with lysostaphin from the 24-well plate.</li>
		<li>Fill three wells with 1 mL of medium supplemented with lysostaphin plus the antimicrobial compound to be tested.</li>
		<li>Repeat step 3.5.9 for each antimicrobial compound to be tested.</li>
		<li>For the control condition, fill three wells with 1 mL of medium supplemented with lysostaphin without any antimicrobial compound.</li>
		<li>Incubate the cells for 24 h at 36 &#177; 1 &#176;C in 5% CO2.</li>
		<li>At the end of the incubation period, remove and discard the spent medium and gently wash each well three times with sterile DPBS with CaCl2 and MgCl2.</li>
		<li>Add 1 mL of 1x lysis buffer to each well to detach and lyse the cells by osmotic shock.</li>
		<li>Incubate the cells for 10 min at 36 &#177; 1 &#176;C.</li>
		<li>Mix thoroughly by pipetting up and down ten times all over the well to ensure that the cells are fully lysed and homogenized.</li>
		<li>Use an automatic spiral plater to determine the S. aureus load of each well.</li>
		<li>Incubate the agar plates for 18-24 h at 36 &#177; 1 &#176;C.</li>
		<li>The next day, count the number of colonies with a colony counter to calculate the intracellular S. aureus load of each well. 
		NOTE: The intracellular activity of each antimicrobial compound should be calculated according to the bacterial load of the control condition. It is also important to check the cytotoxicity of all antimicrobial compounds to prove that the differences observed between the control and the compounds are not due to cell death.</li>
	</ol>
	</li>
</ol>

<ol>
	<li>Verhoeven, P. O., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Detection+and+clinical+relevance+of+Staphylococcus+aureus+nasal+carriage:+an+update.">Detection and clinical relevance of Staphylococcus aureus nasal carriage: an update.</a> Expert Review of Anti-Infective Therapy. 12 (1), 75-89 (2014).</li><li>Gagnaire, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Epidemiology+and+clinical+relevance+of+Staphylococcus+aureus+intestinal+carriage:+a+systematic+review+and+meta-analysis.">Epidemiology and clinical relevance of Staphylococcus aureus intestinal carriage: a systematic review and meta-analysis.</a> Expert Review of Anti-Infective Therapy. 15 (8), 767-785 (2017).</li><li>Tong, S. Y. C., Davis, J. S., Eichenberger, E., Holland, T. L., Fowler, V. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Staphylococcus+aureus+infections:+epidemiology,+pathophysiology,+clinical+manifestations,+and+management.">Staphylococcus aureus infections: epidemiology, pathophysiology, clinical manifestations, and management.</a> Clinical Microbiology Reviews. 28 (3), 603-661 (2015).</li><li>Josse, J., Laurent, F., Diot, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Staphylococcal+adhesion+and+host+cell+invasion:+fibronectin-binding+and+other+mechanisms.">Staphylococcal adhesion and host cell invasion: fibronectin-binding and other mechanisms.</a> Frontiers in Microbiology. 8, 2433 (2017).</li><li>Hanssen, A. -. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Localization+of+Staphylococcus+aureus+in+tissue+from+the+nasal+vestibule+in+healthy+carriers.">Localization of Staphylococcus aureus in tissue from the nasal vestibule in healthy carriers.</a> BMC Microbiology. 17 (1), 89 (2017).</li><li>Rigaill, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Evaluation+of+the+intracellular+efficacy+of+antimicrobial+agents+used+for+Staphylococcus+aureus+decolonization+in+a+cell+model+mimicking+nasal+colonization.">Evaluation of the intracellular efficacy of antimicrobial agents used for Staphylococcus aureus decolonization in a cell model mimicking nasal colonization.</a> Journal of Antimicrobial Chemotherapy. 73 (11), 3044-3048 (2018).</li><li>Yang, D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Novel+insights+into+Staphylococcus+aureus+deep+bone+infections:+the+Involvement+of+osteocytes.">Novel insights into Staphylococcus aureus deep bone infections: the Involvement of osteocytes.</a> mBio. 9 (2), 00415-00418 (2018).</li><li>Tuchscherr, L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Staphylococcus+aureus+phenotype+switching:+an+effective+bacterial+strategy+to+escape+host+immune+response+and+establish+a+chronic+infection.">Staphylococcus aureus phenotype switching: an effective bacterial strategy to escape host immune response and establish a chronic infection.</a> EMBO Molecular Medicine. 3 (3), 129-141 (2011).</li><li>Valour, F., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Antimicrobial+activity+against+intraosteoblastic+Staphylococcus+aureus.">Antimicrobial activity against intraosteoblastic Staphylococcus aureus.</a> Antimicrobial Agents and Chemotherapy. 59 (4), 2029-2036 (2015).</li><li>Proctor, R. A., Prendergast, E., Mosher, D. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fibronectin+mediates+attachment+of+Staphylococcus+aureus+to+human+neutrophils.">Fibronectin mediates attachment of Staphylococcus aureus to human neutrophils.</a> Blood. 59 (4), 681-687 (1982).</li><li>Climo, M. W., Ehlert, K., Archer, G. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mechanism+and+suppression+of+lysostaphin+resistance+in+oxacillin-resistant+Staphylococcus+aureus.">Mechanism and suppression of lysostaphin resistance in oxacillin-resistant Staphylococcus aureus.</a> Antimicrobial Agents and Chemotherapy. 45 (5), 1431-1437 (2001).</li><li>Bur, S., Preissner, K. T., Herrmann, M., Bischoff, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+Staphylococcus+aureus+extracellular+adherence+protein+promotes+bacterial+internalisation+by+keratinocytes+independent+of+fibronectin-binding+proteins.">The Staphylococcus aureus extracellular adherence protein promotes bacterial internalisation by keratinocytes independent of fibronectin-binding proteins.</a> Journal of Investigative Dermatology. 133 (8), 2004-2012 (2013).</li><li>Kim, J. -. H., Chaurasia, A. K., Batool, N., Ko, K. S., Kim, K. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Alternative+enzyme+protection+assay+to+overcome+the+drawbacks+of+the+gentamicin+protection+assay+for+measuring+entry+and+intracellular+survival+of+Staphylococci.">Alternative enzyme protection assay to overcome the drawbacks of the gentamicin protection assay for measuring entry and intracellular survival of Staphylococci.</a> Infection and Immunity. 87 (5), 00119 (2019).</li><li>Aerobic plate count. Bacteriological Analytical Manual., Edition 8, Revision A Available from: <a target="_blank" href="https://www.fda.gov/food/laboratory-methods-food/bam-chapter-3-aerobic-plate-count">https://www.fda.gov/food/laboratory-methods-food/bam-chapter-3-aerobic-plate-count</a> (2021)</li><li>Kolenda, C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Evaluation+of+the+activity+of+a+combination+of+three+bacteriophages+alone+or+in+association+with+antibiotics+on+Staphylococcus+aureus+embedded+in+biofilm+or+internalized+in+Osteoblasts.">Evaluation of the activity of a combination of three bacteriophages alone or in association with antibiotics on Staphylococcus aureus embedded in biofilm or internalized in Osteoblasts.</a> Antimicrobial Agents and Chemotherapy. 64 (3), 02231 (2020).</li><li>Abad, L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Antibiofilm+and+intraosteoblastic+activities+of+rifamycins+against+Staphylococcus+aureus:+promising+in+vitro+profile+of+rifabutin.">Antibiofilm and intraosteoblastic activities of rifamycins against Staphylococcus aureus: promising in vitro profile of rifabutin.</a> Journal of Antimicrobial Chemotherapy. 75 (6), 1466-1473 (2020).</li></ol>

The authors have no conflicts of interest to declare.

The assays described here are valuable for studying the extent of internalization and the intracellular survival of S. aureus in NPPCs, as well as the intracellular efficacy of antimicrobial compounds6,15,16. Some steps in both assay protocols can be critical. The health condition and the density of the cells must be perfectly controlled and consistent between independent experiments. The bacterial inoculum must be carefully standardized to obtain a real MOI close to the targeted theoretical MOI. In general, care must be taken not to detach any of the cells while pipetting. The washes to remove lysostaphin and antibiotics are critical steps in the EPA. The use of proteinase K has been found to improve this step when no antibiotic is used (see below). Last but not least, the cells should be fully detached in each well and thoroughly homogenized after the incubation with the lysis buffer to reliably quantify the S. aureus intracellular load.
In some instances, issues may be encountered, and several points must be checked first. In case of a lack of reproducibility, it must be kept in mind that S. aureus can form clumps, making quantification by absorbance inaccurate. The clumping of bacteria can be increased by centrifugation and washing steps if the culture medium is to be replaced (e.g., for eliminating a secreted protein). The bacterial suspension should be used rapidly because bacteria continue to grow at room temperature. The lysostaphin efficacy could decrease because of incorrect storage conditions, suboptimal pH for enzyme activity in the culture media, variability in the enzymatic activity between batches and providers, and lack of lysostaphin sensitivity of some strains in specific growth conditions. Phenol red could have a slight bacteriostatic effect, especially when the culture medium is relatively poor in nutrients compared to the typical broths used for growing bacteria. Thus, it is advisable to use a cell culture medium without phenol red, which also improves fluorescence microscopic observations by reducing the background noise.
Although this method is a valuable tool to study the intracellular fate of different strains, some limits of the method should be considered. The use of a very high MOI can overload the capability of internalization by NPPCs and level out the differences between the different strains tested. The extent of internalization of the most cytotoxic strains may be underestimated because lysostaphin (or antibiotics) rapidly destroys S. aureus that is released by damaged cells. Thus, experiments with extended durations (i.e., to study intracellular survival or intracellular activity of antibiotics) are easier to set up with strains with low cytotoxicity. Therefore, the incubation time and the MOI should be accurately adjusted according to the strain virulence, the cell type, and the experimental aim.
The method described here with the use of lysostaphin is more reliable than those based on gentamicin because, unlike lysostaphin, gentamicin tends to be internalized by host cells13. The other advantage is the possibility to inactivate the lysostaphin. Inhibition of lysostaphin activity was reported by Kim et al.13 with the use of EDTA to chelate zinc ions or 1,10-phenanthroline; however, intensive washes are still required to remove the enzyme before plating of the bacteria. Here, proteinase K enables rapid inactivation of lysostaphin. We observed that cells tend to detach from the culture plate when they become heavily infected because of the multiplication of intracellular S. aureus. By skipping the final washing step, the iEPA method greatly simplified technical handling and enabled the recovery of the internalized bacteria in loosely adherent or already detached cells. 
The more concentrated reagents and buffers used in iEPA also helped reduce pipetting effort and minimize the loss of cells. In addition, iEPA can be used with cells in suspension, as well as with organoids that are difficult to wash. In conclusion, enzyme protection assays enable the study of the extent of internalization and the intracellular fate of S. aureus, as well as the intracellular activity of antimicrobials drugs with different in vitro models. Improvements should be made to better characterize the relationship between internalization and cytotoxicity to better appreciate the importance of developing drugs capable of reaching S. aureus inside the cell.

An erratum was issued for:&#160;<a href="https://www.jove.com/t/62903">Improved Enzyme Protection Assay to Study Staphylococcus aureus Internalization and Intracellular Efficacy of Antimicrobial Compounds</a>. One of the affiliations was updated.
The fifth affiliation was updated from:
D&#233;partement de Bact&#233;riologie, Institut des Agents Infectieux, Hospices Civils de Lyon, Lyon, France
to:
Department of Bacteriology, Institute for Infectious Agents, Hospices Civils de Lyon, Lyon, France&#160;

An erratum was issued for:&#160;<a href="https://www.jove.com/t/62903">Improved Enzyme Protection Assay to Study Staphylococcus aureus Internalization and Intracellular Efficacy of Antimicrobial Compounds</a>. One of the affiliations was updated.

Erratum: Improved Enzyme Protection Assay to Study Staphylococcus aureus Internalization and Intracellular Efficacy of Antimicrobial Compounds

Staphylococcus aureus is both a life-threatening pathogen and a commensal bacterium of the skin and the mucosa that colonizes two billion individuals around the world1. In humans, nasal carriers of S. aureus have an increased risk of infection with their own strain of carriage; however, the multifactorial determinants of S. aureus mucosal carriage are still unclear1,2. In addition to acute infections, patients can also develop chronic S. aureus infections that are often challenging to cure3. A better understanding of host-pathogen interactions during colonization and infection is crucial for developing novel therapeutic strategies and improving patient management.
In vitro, S. aureus can trigger its internalization into host cells expressing the &#945;5&#946;1 integrin4. The tripartite interaction between the staphylococcal fibronectin-binding proteins anchored to the cell wall of S. aureus, the fibronectin, and the &#946;1 integrin expressed at the host cell surface is well known as the main pathway of S. aureus internalization in NPPCs such as keratinocytes, osteoblasts, fibroblasts, and epithelial and endothelial cells4. Recent studies show that S. aureus can be found inside human cells during nasal colonization5,6 and infection7. However, the role of the intracellular reservoir in the pathogenesis of S. aureus infection remains unclear. The host cells could act as a shelter for S. aureus, which is protected from both the immune system8 and most antimicrobial compounds6,9.
The lysostaphin protection assay, described by Proctor10 earlier in the 1980s, enables the study of bacterial and host factors involved in the internalization of S. aureus isolates. Lysostaphin is a bacteriocin produced by Staphylococcus simulans, which&#160;exhibits potent activity against almost all S. aureus isolates, including antibiotic-resistant strains11. Lysostaphin has been used to destroy only extracellular S. aureus to enable the counting of only viable intracellular bacteria12. This technique has been widely used and has contributed to the discovery of several virulence factors of S. aureus. Gentamycin, alone and combined with lysostaphin, is also widely used to study intracellular bacteria.
However, a recent study showed that gentamycin enters eukaryotic cells and reaches internalized bacteria in a time- and concentration-dependent manner13. This study also demonstrated that lysostaphin does not enter eukaryotic cells, confirming that a lysostaphin-based enzyme protection assay (EPA) is the most accurate assay for quantifying intracellular S. aureus load by culture13. Regardless of which compound is used to destroy extracellular bacteria (e.g., lysostaphin or gentamycin), it should be removed by washing the cells before plating intracellular S. aureus on agar plates. Successive washes may result in the detachment of cells, especially poorly adherent cells (e.g., heavily infected cells), which would lead to an underestimation of the intracellular S. aureus load. This paper describes in detail how EPA can be used to quantify the intracellular S. aureus load&#160;and to measure the intracellular efficacy of antimicrobials compounds using an in vitro model. Of note, a simple method has been proposed to improve the reliability of intracellular load quantification by avoiding intensive washes.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>24-well plate</td>
			<td>CORNING-FALCON</td>
			<td>353047</td>
			<td></td>
		</tr>
		<tr>
			<td>A549 cell line</td>
			<td>ATCC</td>
			<td>CCL-185</td>
			<td></td>
		</tr>
		<tr>
			<td>Acetate buffer solution pH 4.6</td>
			<td>Fluka</td>
			<td>31048</td>
			<td>Used to prepare lysostpahine stock solution at 10 mg/mL in 20 mM sodium acetate.</td>
		</tr>
		<tr>
			<td>AMBICIN (Recombinant lysostaphin)</td>
			<td>AMBI</td>
			<td>LSPN-50</td>
			<td>Lyophilized recominant lysostaphin. Freeze at -80 &#176;C for long-term storage.</td>
		</tr>
		<tr>
			<td>COS - Colombia agar + 5% sheep blood</td>
			<td>Biomerieux</td>
			<td>43049</td>
			<td>Any agar plate suitable for growing staphylococci can be used instead.</td>
		</tr>
		<tr>
			<td>Densitometer WPA CO8000</td>
			<td>Biochrom Ltd.</td>
			<td>80-3000-45</td>
			<td>Cell density meter</td>
		</tr>
		<tr>
			<td>Dulbecco&#8217;s Modified Eagle&#8217;s Medium, high glucose with phenol red</td>
			<td>Sigma-Aldrich</td>
			<td>D6429</td>
			<td></td>
		</tr>
		<tr>
			<td>Dulbecco&#8217;s Modified Eagle&#8217;s Medium, high glucose without phenol red</td>
			<td>Sigma-Aldrich</td>
			<td>D1145</td>
			<td></td>
		</tr>
		<tr>
			<td>Dulbecco&#8217;s Phosphate Buffered Saline</td>
			<td>Sigma-Aldrich</td>
			<td>D8537</td>
			<td></td>
		</tr>
		<tr>
			<td>Dulbecco&#8242;s Phosphate-buffered Saline with MgCl2 and CaCl2, sterile-filtered</td>
			<td>Sigma-Aldrich</td>
			<td>D8662</td>
			<td></td>
		</tr>
		<tr>
			<td>Dulbecco&#8242;s Phosphate-buffered Saline, sterile-filtered</td>
			<td>Sigma-Aldrich</td>
			<td>D8537</td>
			<td></td>
		</tr>
		<tr>
			<td>Easyspiral dilute</td>
			<td>Interscience</td>
			<td>414000</td>
			<td>Automatic diluter and spiral plater</td>
		</tr>
		<tr>
			<td>Fetal bovine serum</td>
			<td>Gibco</td>
			<td>10270-106</td>
			<td></td>
		</tr>
		<tr>
			<td>HaCaT cell line</td>
			<td>Cell lines service (CLS)</td>
			<td>300493</td>
			<td></td>
		</tr>
		<tr>
			<td>Hoechst 33342, Trihydrochloride, Trihydrate, 10 mg/mL Solution in Water</td>
			<td>Fisher scientific</td>
			<td>11534886</td>
			<td></td>
		</tr>
		<tr>
			<td>Propidium iodide, 1.0 mg/mL solution in water</td>
			<td>Invitrogen</td>
			<td>P3566</td>
			<td></td>
		</tr>
		<tr>
			<td>Proteinase K, recombinant 20 mg/mL</td>
			<td>Eurobio</td>
			<td>GEXPRK01-B5</td>
			<td>&#62; 30 U/mg, lot 901727</td>
		</tr>
		<tr>
			<td>Scan 4000</td>
			<td>Interscience</td>
			<td>438000</td>
			<td>Automatic colony counter</td>
		</tr>
		<tr>
			<td>Sterile water</td>
			<td>OTEC</td>
			<td>600500</td>
			<td></td>
		</tr>
		<tr>
			<td>T-75 culture flask</td>
			<td>CORNING-FALCON</td>
			<td>353136</td>
			<td></td>
		</tr>
		<tr>
			<td>TC20 Automated cell counter</td>
			<td>Biorad</td>
			<td>1450102</td>
			<td>Automatic cell counter</td>
		</tr>
		<tr>
			<td>Tris 1 M pH 8.0</td>
			<td>Invitrogen</td>
			<td>AM9855G</td>
			<td>Used to prepare lysostaphine working solution at 1 mg/mL in 0.1 M Tris-HCl.</td>
		</tr>
		<tr>
			<td>Triton X-100</td>
			<td>Sigma-Aldrich</td>
			<td>T8787</td>
			<td></td>
		</tr>
		<tr>
			<td>Trypsin - EDTA solution</td>
			<td>Sigma-Aldrich</td>
			<td>T3924</td>
			<td>0.05% porcine trypsin and 0.02% EDTA in Hanks&#8242; Balanced Salt Solution with phenol red</td>
		</tr>
		<tr>
			<td>Wide-field fluorescence microscope</td>
			<td>Nikon</td>
			<td>Ti2</td>
			<td></td>
		</tr>
	</tbody>
</table>

improved enzyme protection assay to study staphylococcus aureus internalization and intracellular efficacy of antimicrobial compounds

Staphylococcus aureus expresses virulence factors to trigger its internalization into eukaryote cells and to survive inside different subcellular compartments. This paper describes an enzyme protection assay to study the extent of S. aureus internalization and its intracellular survival in adherent non-professional phagocytic cells (NPPCs) as well as the intracellular efficacy of antimicrobial compounds. NPPCs are grown in a multi-well plate until they reach 100% confluence. S. aureus cultures are grown overnight in cell culture medium. The bacterial suspension is diluted according to the number of cells per well to inoculate the cells at a controlled multiplicity of infection. Inoculated cells are incubated for 2 h to allow the bacteria to be internalized by the NPPCs, following which lysostaphin is added to the culture medium to selectively kill extracellular bacteria. Lysostaphin is present in the culture medium for the rest of the experiment. 
At this point, the infected cells could be incubated with antimicrobial compounds to assess their intracellular activities against S. aureus. Next, the cells are washed three times to remove the drugs, and intracellular S. aureus load is then quantified by culturing on agar plates. Alternatively, for studying staphylococcal virulence factors involved in intracellular survival and cell toxicity, lysostaphin could be inactivated with proteinase K to eliminate the need for washing steps. This tip improves the reliability of the intracellular bacterial load quantification, especially if cells tend to detach from the culture plate when they become heavily infected because of the multiplication of intracellular S. aureus. These protocols can be used with virtually all types of adherent NPPCs and with 3D cell culture models such as organoids.

The results of S. aureus internalization by A549 epithelial cells are depicted in Figure 1A. A549 cells were inoculated with S. aureus SF8300 WT and SF8300 &#916;fnbA/B, which lacks fibronectin-binding proteins A and B, at an MOI of 1 for 2 h. To destroy extracellular S. aureus, lysostaphin was added to the culture medium, and the cells were incubated for 1 h. Next, lysostaphin was either removed by washing for EPA or inactivated with proteinase K for iEPA. Then, the cells were disrupted in lysis buffer, and the bacterial load was quantified by culture. By using EPA, the mean intracellular loads were 4.46 and 0.49 Log CFU/mL for SF8300 WT and SF8300 &#916;fnbA/B, respectively (Figure 1A, green bars). Using iEPA, the mean intracellular loads were 4.53 and 0.56 Log CFU/mL for SF8300 WT and SF8300 &#916;fnbA/B, respectively (Figure 1A,&#160;red bars). It is interesting to note that both EPA and iEPA showed similar results, which can be explained by the ease of performing the washes when the cells are in good condition and because the S. aureus-induced cytotoxicity is very low in these experimental settings (data not shown).
The results of intracellular activity of vancomycin, rifampicin, and levofloxacin against S. aureus are depicted in Figure 1B. To measure the intracellular activity of these antibiotics, HaCaT cells were inoculated with S. aureus ATCC 29213 at an MOI of 1 for 2 h. The cells were incubated with lysostaphin, with or without the antimicrobial compounds to be tested, for 24 h. Next, lysostaphin and the antimicrobial compounds were removed by washing. The cells were disrupted in lysis buffer, and the bacterial load was quantified by culture. The mean intracellular loads were 4.57, 4.51, 3.03, and 2.91 log CFU/mL for control, vancomycin (50 &#181;g/mL), rifampicin (7 &#181;g/mL), and levofloxacin (10 &#181;g/mL), respectively (Figure 1B).
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/62903/62903fig01.jpg" /> 
Figure 1: Intracellular Staphylococcus aureus load in epithelial cells. (A) Enzyme protection assay (green bars) and improved enzyme protection assay (red bars) in A549 cells infected with S. aureus SF8300 WT and &#916;fnbA/B. (B) Intracellular activity of antimicrobial compounds in HaCaT cells infected with S. aureus ATCC 29213. Bars represent the mean values of three independent experiments performed in triplicate. Error bars represent the standard deviations. **** p &#60; 0.0001. Abbreviations: Ctrl = control; cfu = colony-forming units. <a href="https://www.jove.com/files/ftp_upload/62903/62903fig01large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

Watch this Scientific Journal Video about Improved Enzyme Protection Assay to Study Staphylococcus aureus Internalization and Intracellular Efficacy of Antimicrobial Compounds at JoVE.com

Improved Enzyme Protection Assay to Study Staphylococcus aureus Internalization and Intracellular Efficacy of Antimicrobial Compounds

This protocol aims to describe how to study the extent of Staphylococcus aureus internalization and its ability to survive inside the human host cell, as well as the intracellular efficacy of antimicrobial compounds.

Improved Enzyme Protection Assay to Study Staphylococcus aureus Internalization and Intracellular Efficacy of Antimicrobial Compounds

Staphylococcus aureus expresses virulence factors to trigger its internalization into eukaryote cells and to survive inside different subcellular ...

improved-enzyme-protection-assay-to-study-staphylococcus-aureus

Research

JoVE Journal

Biology

2.6K Views.  University of Lyon, INSERM U1111, CNRS, UMR5308, ENS Lyon, UCBL1, University of St-Etienne, France. This protocol aims to describe how to study the extent of Staphylococcus aureus internalization and its ability to survive inside the human host cell, as well as the intracellular efficacy of antimicrobial compounds.

Video: Improved Enzyme Protection Assay to Study Staphylococcus aureus Internalization and Intracellular Efficacy of Antimicrobial Compounds

Testing Visual Sensitivity to the Speed and Direction of Motion in Lizards

Testing visual sensitivity in any species provides basic information regarding behaviour, evolution, and ecology. However, testing specific features of the visual system provide more empirical evidence for functional applications. Investigation into the sensory system provides information about the sensory capacity, learning and memory ability, and establishes known baseline behaviour in which to gauge deviations (Burghardt, 1977). However, unlike mammalian or avian systems, testing for learning and memory in a reptile species is difficult. Furthermore, using an operant paradigm as a psychophysical measure of sensory ability is likewise as difficult. Historically, reptilian species have responded poorly to conditioning trials because of issues related to motivation, physiology, metabolism, and basic biological characteristics. Here, I demonstrate an operant paradigm used a novel model lizard species, the Jacky dragon (Amphibolurus muricatus) and describe how to test peripheral sensitivity to salient speed and motion characteristics. This method uses an innovative approach to assessing learning and sensory capacity in lizards. I employ the use of random-dot kinematograms (RDKs) to measure sensitivity to speed, and manipulate the level of signal strength by changing the proportion of dots moving in a coherent direction. RDKs do not represent a biologically meaningful stimulus, engages the visual system, and is a classic psychophysical tool used to measure sensitivity in humans and other animals. Here, RDKs are displayed to lizards using three video playback systems. Lizards are to select the direction (left or right) in which they perceive dots to be moving. Selection of the appropriate direction is reinforced by biologically important prey stimuli, simulated by computer-animated invertebrates.

Testing visual sensitivity in lizards using an operant conditioning paradigm that employs video playback of random-dot kinematograms and computer-generated invertebrates.

Testing visual sensitivity in any species provides basic information regarding behaviour, evolution, and ecology. However, testing specific features of ...

Intracellular Refolding Assay

This protocol describes a method to measure the enzymatic activity of molecular chaperones in a cell-based system and the possible effects of compounds with inhibitory/stimulating activity. Molecular chaperones are proteins involved in regulation of protein folding1 and have a crucial role in promoting cell survival upon stress insults like heat shock2, nutrient starvation and exposure to chemicals/poisons3. For this reason chaperones are found to be involved in events like tumor development, chemioresistance of cancer cells4 as well as neurodegeneration5. Design of small molecules able to inhibit or stimulate the activity of these enzymes is therefore one of the most studied strategies for cancer therapy7 and neurodegenerative disorders9. The assay here described offers the possibility to measure the refolding activity of a particular molecular chaperone and to study the effect of compounds on its activity. In this method the gene of the molecular chaperone investigated is transfected together with an expression vector encoding for the firefly luciferase gene. It has been already described that denaturated firefly luciferase can be refolded by molecular chaperones10,11. As normalizing transfection control, a vector encoding for the renilla luciferase gene is transfected. All transfections described in this protocol are performed with X-treme Gene 11 (Roche) in HEK-293 cells. In the first step, protein synthesis is inhibited by treating the cells with cycloheximide. Thereafter protein unfolding is induced by heat shock at 45&deg;C for 30 minutes. Upon recovery at 37&deg;C, proteins are re-folded into their active conformation and the activity of the firefly luciferase is used as read-out: the more light will be produced, the more protein will have re-gained the original conformation. Non-heat shocked cells are set as reference (100% of refolded luciferase).

In this protocol a method to measure intracellular protein refolding after heat shock is described. This method can be used to study foldases like molecular chaperones and their co-factors or compounds able to influence their activity. Firefly luciferase activity is used as reporter to measure chaperone refolding activity.

This protocol describes a method to measure the enzymatic activity of molecular chaperones in a cell-based system and the possible effects of compounds ...

Application of an In vitro DNA Protection Assay to Visualize Stress Mediation Properties of the Dps Protein

Oxidative stress is an unavoidable byproduct of aerobic life. Molecular oxygen is essential for terrestrial metabolism, but it also takes part in many damaging reactions within living organisms. The combination of aerobic metabolism and iron, which is another vital compound for life, is enough to produce radicals through Fenton chemistry and degrade cellular components. DNA degradation is arguably the most damaging process involving intracellular radicals, as DNA repair is far from trivial. The assay presented in this article offers a quantitative technique to measure and visualize the effect of molecules and enzymes on radical-mediated DNA damage.
The DNA protection assay is a simple, quick, and robust tool for the in vitro characterization of the protective properties of proteins or chemicals. It involves exposing DNA to a damaging oxidative reaction and adding varying concentrations of the compound of interest. The reduction or increase of DNA damage as a function of compound concentration is then visualized using gel electrophoresis. In this article we demonstrate the technique of the DNA protection assay by measuring the protective properties of the DNA-binding protein from starved cells (Dps). Dps is a mini-ferritin that is utilized by more than 300 bacterial species to powerfully combat environmental stressors. Here we present the Dps purification protocol and the optimized assay conditions for evaluating DNA protection by Dps.

Application of an In vitro DNA Protection Assay to Visualize Stress Mediation Properties of the Dps Protein

The DNA-binding protein from starved cells (Dps) plays a crucial role in combating bacterial stress. This article discusses the purification of E. coli Dps and the protocol for an in vitro assay demonstrating Dps-mediated protection of DNA from degradation by reactive oxygen species.

Oxidative stress is an unavoidable byproduct of  aerobic life.  Molecular oxygen is  essential for terrestrial metabolism, but it also takes part in many ...

The Cell-based L-Glutathione Protection Assays to Study Endocytosis and Recycling of Plasma Membrane Proteins

Membrane trafficking involves transport of proteins from the plasma membrane to the cell interior (i.e. endocytosis) followed by trafficking to lysosomes for degradation or to the plasma membrane for recycling. The cell based L-glutathione protection assays can be used to study endocytosis and recycling of protein receptors, channels, transporters, and adhesion molecules localized at the cell surface. The endocytic assay requires labeling of cell surface proteins with a cell membrane impermeable biotin containing a disulfide bond and the N-hydroxysuccinimide (NHS) ester at 4 &#186;C -&#160;a temperature at which membrane trafficking does not occur. Endocytosis of biotinylated plasma membrane proteins is induced by incubation at 37 &#186;C. Next, the temperature is decreased again to 4 &#186;C to stop endocytic trafficking and the disulfide bond in biotin covalently attached to proteins that have remained at the plasma membrane is reduced with L-glutathione. At this point, only proteins that were endocytosed remain protected from L-glutathione and thus remain biotinylated. After cell lysis, biotinylated proteins are isolated with streptavidin agarose, eluted from agarose, and the biotinylated protein of interest is detected by western blotting. During the recycling assay, after biotinylation cells are incubated at 37 &#176;C to load endocytic vesicles with biotinylated proteins and the disulfide bond in biotin covalently attached to proteins remaining at the plasma membrane is reduced with L-glutathione at 4 &#186;C as in the endocytic assay. Next, cells are incubated again at 37 &#176;C to allow biotinylated proteins from endocytic vesicles to recycle to the plasma membrane. Cells are then incubated at 4 &#186;C, and the disulfide bond in biotin attached to proteins that recycled to the plasma membranes is reduced with L-glutathione. The biotinylated proteins protected from L-glutathione are those that did not recycle to the plasma membrane.

Membrane trafficking involves transport of proteins from the plasma membrane to the cell interior (i.e. endocytosis) followed by trafficking to lysosomes for degradation or to the plasma membrane for recycling. Methods described in this article are designed to study endocytosis and recycling of plasma membrane proteins.

Membrane trafficking involves transport of proteins from the plasma membrane to the cell interior (i.e. endocytosis) followed by trafficking to lysosomes ...

In vitro Methylation Assay to Study Protein Arginine Methylation

Protein arginine methylation is one of the most abundant post-translational modifications in the nucleus. Protein arginine methylation can be identified and/or determined via proteomic approaches, and/or immunoblotting with methyl-arginine specific antibodies. However, these techniques sometimes can be misleading and often provide false positive results. Most importantly, these techniques cannot provide direct evidence in support of the PRMT substrate specificity. In vitro methylation assays, on the other hand, are useful biochemical assays, which are sensitive, and consistently reveal if the identified proteins are indeed PRMT substrates. A typical in vitro methylation assay includes purified, active PRMTs, purified substrate and a radioisotope labeled methyl donor (S-adenosyl-L-[methyl-3H] methionine). Here we describe a step-by-step protocol to isolate catalytically active PRMT1, a ubiquitously expressed PRMT family member. The methyl transferase activities of the purified PRMT1 were later tested on Ras-GTPase activating protein binding protein 1 (G3BP1), a known PRMT substrate, in the presence of S-adenosyl-L-[methyl-3H] methionine as the methyl donor. This protocol can be employed not only for establishing the methylation status of novel physiological PRMT1 substrates, but also for understanding the basic mechanism of protein arginine methylation.

In vitro Methylation Assay to Study Protein Arginine Methylation

Protein arginine methylation, catalyzed by a class of enzymes viz., protein arginine methyl transferases (PRMTs), is the process of enzymatic addition of methyl group(s) to arginines within proteins. The in vitro methylation assay is the most dependable tool for assessing the methylation status of known or novel PRMT substrates.

Protein arginine methylation is one of the most abundant post-translational modifications in the nucleus. Protein arginine methylation can be identified ...

Tracking Drug-induced Changes in Receptor Post-internalization Trafficking by Colocalizational Analysis

The intracellular trafficking of receptors is a collection of complex and highly controlled processes. Receptor trafficking modulates signaling and overall cell responsiveness to ligands and is, itself, influenced by intra- and extracellular conditions, including ligand-induced signaling. Optimized for use with monolayer-plated cultured cells, but extendable to free-floating tissue slices, this protocol uses immunolabelling and colocalizational analysis to track changes in intracellular receptor trafficking following both chronic/prolonged and acute interventions, including exogenous drug treatment. After drug treatment, cells are double-immunolabelled for the receptor and for markers for the intracellular compartments of interest. Sequential confocal microscopy is then used to capture two-channel photomicrographs of individual cells, which are subjected to computerized colocalizational analysis to yield quantitative colocalization scores. These scores are normalized to permit pooling of independent replicates prior to statistical analysis. Representative photomicrographs may also be processed to generate illustrative figures. Here, we describe a powerful and flexible technique for quantitatively assessing induced receptor trafficking.

Receptor trafficking modulates signaling and cell responsiveness to ligands and is, itself, responsive to cell conditions, including ligand-induced signaling. Here, we describe a powerful and flexible technique for quantitatively assessing drug-induced receptor trafficking using immunolabeling and colocalizational analysis.

The intracellular trafficking of receptors is a collection of complex and highly controlled processes. Receptor trafficking modulates signaling and ...

Gap Junctional Intercellular Communication: A Functional Biomarker to Assess Adverse Effects of Toxicants and Toxins, and Health Benefits of Natural Products

This protocol describes a scalpel loading-fluorescent dye transfer (SL-DT) technique that measures intercellular communication through gap junction channels, which is a major intercellular process by which tissue homeostasis is maintained. Interruption of gap junctional intercellular communication (GJIC) by toxicants, toxins, drugs, etc. has been linked to numerous adverse health effects. Many genetic-based human diseases have been linked to mutations in gap junction genes. The SL-DT technique is a simple functional assay for the simultaneous assessment of GJIC in a large population of cells. The assay involves pre-loading cells with a fluorescent dye by briefly perturbing the cell membrane with a scalpel blade through a population of cells. The fluorescent dye is then allowed to traverse through gap junction channels to neighboring cells for a designated time. The assay is then terminated by the addition of formalin to the cells. The spread of the fluorescent dye through a population of cells is assessed with an epifluorescence microscope and the images are analyzed with any number of morphometric software packages that are available, including free software packages found on the public domain. This assay has also been adapted for in vivo studies using tissue slices from various organs from treated animals. Overall, the SL-DT assay can serve a broad range of in vitro pharmacological and toxicological needs, and can be potentially adapted for high throughput set-up systems with automated fluorescence microscopy imaging and analysis to elucidate more samples in a shorter time.

This protocol describes a scalpel loading-fluorescent dye transfer technique that measures intercellular communication through gap junction channels. Gap junctional intercellular communication is a major cellular process by which tissue homeostasis is maintained and disruption of this cell signaling has adverse health effects.

This protocol describes a scalpel loading-fluorescent dye transfer (SL-DT) technique that measures intercellular communication through gap junction ...

In Vitro SUMOylation Assay to Study SUMO E3 Ligase Activity

Small ubiquitin-like modifier (SUMO) modification is an important post-translational modification (PTM) that mediates signal transduction primarily through modulating protein-protein interactions. Similar to ubiquitin modification, SUMOylation is directed by a sequential enzyme cascade including E1-activating enzyme (SAE1/SAE2), E2-conjugation enzyme (Ubc9), and E3-ligase (i.e., PIAS family, RanBP2, and Pc2). However, different from ubiquitination, an E3 ligase is non-essential for the reaction but does provide precision and efficacy for SUMO conjugation. Proteins modified by SUMOylation can be identified by in vivo assay via immunoprecipitation with substrate-specific antibodies and immunoblotting with SUMO-specific antibodies. However, the demonstration of protein SUMO E3 ligase activity requires in vitro reconstitution of SUMOylation assays using purified enzymes, substrate, and SUMO proteins. Since in the in vitro reactions, usually SAE1/SAE2 and Ubc9, alone are sufficient for SUMO conjugation, enhancement of SUMOylation by a putative E3 ligase is not always easy to detect. Here, we describe a modified in vitro SUMOylation protocol that consistently identifies SUMO modification using an in vitro reconstituted system. A step-by-step protocol to purify catalytically active K-bZIP, a viral SUMO-2/3 E3 ligase, is also presented. The SUMOylation activities of the purified K-bZIP are shown on p53, a well-known target of SUMO. This protocol can not only be employed for elucidating novel SUMO E3 ligases, but also for revealing their SUMO paralog specificity.

In Vitro SUMOylation Assay to Study SUMO E3 Ligase Activity

Unlike ubiquitin ligases, few E3 SUMO ligases have been identified. This modified in vitro SUMOylation protocol is able to identify novel SUMO E3 ligases by an in vitro reconstitution assay.

Small ubiquitin-like modifier (SUMO) modification is an important post-translational modification (PTM) that mediates signal transduction primarily ...

An In Vitro Model to Study the Effect of 5-Aminolevulinic Acid-mediated Photodynamic Therapy on Staphylococcus aureus Biofilm

Staphylococcus aureus (S. aureus) is a common human pathogen, which causes pyogenic and systemic infections. S. aureus infections are difficult to eradicate not only due to the emergence of antibiotic-resistant strains but also its ability to form biofilms. Recently, photodynamic therapy (PDT) has been indicated as one of the potential treatments for controlling biofilm infections. However, further studies are required to improve our knowledge of its effect on bacterial biofilms, as well as the underlying mechanisms. This manuscript describes an in vitro model of PDT with 5-aminolevulinic acid (5-ALA), a precursor of the actual photosensitizer, protoporphyrin IX (PpIX). Briefly, mature S. aureus biofilms were incubated with ALA and then exposed to light. Subsequently, the antibacterial effect of ALA-PDT on S. aureus biofilm was quantified by calculating the colony forming units (CFUs) and visualized by viability fluorescent staining via confocal laser scanning microscopy (CLSM). Representative results demonstrated a strong antibacterial effect of ALA-PDT on S. aureus biofilms. This protocol is simple and can be used to develop an in vitro model to study the treatment of S. aureus biofilms with ALA-PDT. In the future, it could also be referenced in PDT studies utilizing other photosensitizers for different bacterial strains with minimal adjustments.

An In Vitro Model to Study the Effect of 5-Aminolevulinic Acid-mediated Photodynamic Therapy on Staphylococcus aureus Biofilm

This manuscript describes a protocol to study the antimicrobial effect of 5-aminolevulinic acid-mediated photodynamic therapy (ALA-PDT) on a Staphylococcus aureus biofilm. This protocol can be used to develop an in vitro model to study the treatment of bacterial biofilms with PDT in the future.

Staphylococcus aureus (S. aureus) is a common human pathogen, which causes pyogenic and systemic infections. S. aureus infections are difficult to ...

Production and Visualization of Bacterial Spheroplasts and Protoplasts to Characterize Antimicrobial Peptide Localization

The use of confocal microscopy as a method to assess peptide localization patterns within bacteria is commonly inhibited by the resolution limits of conventional light microscopes. As the resolution for a given microscope cannot be easily enhanced, we present protocols to transform the small rod-shaped gram-negative Escherichia coli (E. coli) and gram-positive Bacillus megaterium (B. megaterium) into larger, easily imaged spherical forms called spheroplasts or protoplasts. This transformation allows observers to rapidly and clearly determine whether peptides lodge themselves into the bacterial membrane (i.e., membrane localizing) or cross the membrane to enter the cell (i.e., translocating). With this approach, we also present a systematic method to characterize peptides as membrane localizing or translocating. While this method can be used for a variety of membrane-active peptides and bacterial strains, we demonstrate the utility of this protocol by observing the interaction of Buforin II P11A (BF2 P11A), an antimicrobial peptide (AMP), with E. coli spheroplasts and B. megaterium protoplasts.

Here we present a protocol to produce gram-negative Escherichia coli (E. coli) spheroplasts and gram-positive Bacillus megaterium (B. megaterium) protoplasts to clearly visualize and rapidly characterize peptide-bacteria interactions. This provides a systematic method to define membrane localizing and translocating peptides.

The use of confocal microscopy as a method to assess peptide localization patterns within bacteria is commonly inhibited by the resolution limits of ...