This work was supported by NIH grants R01 AI125421, R01 AI127793, and U19 AI144182. The writing author would like to thank all lab members who contributed to proofreading and review of this method.

1. Preparation of Chelex-treated Defined Medium (CDM) Stock Solutions
<ol>
	<li>Stock solution I
	<ol>
		<li>Combine NaCl (233.8 g), K2SO4 (40.0 g), NH4Cl (8.8 g), K2HPO4 (13.9 g), and KH2PO4 (10.9 g) in deionized water to a final volume of 1 L.</li>
		<li>Filter sterilize the solution and aliquot into 50 mL conical tubes.</li>
		<li>Store at -20 &#176;C.</li>
	</ol>
	</li>
	<li>Stock solution II
	<ol>
		<li>Combine thiamine HCl (0.2 g), thiamine pyrophosphate-Cl (0.05 g), calcium pantothenate (0.19 g), and biotin (0.3 g) in 50% (vol/vol) ethanol to a final volume of 1 L.</li>
		<li>Aliquot into 50 mL conical tubes and store at -20 &#176;C.</li>
	</ol>
	</li>
	<li>Stock solution III
	<ol>
		<li>Combine L-aspartate (4.0 g), L-glutamate (10.4 g), L-arginine (1.2 g), glycine (0.2 g), L-serine (0.4 g), L-leucine (0.72 g), L-isoleucine (0.24 g), L-valine (0.48 g), L-tyrosine (0.56 g), L-proline (0.4 g), L-tryptophan (0.64 g), L-threonine (0.4 g), L-phenylalanine (0.2 g), L-asparagine-H2O (0.2 g), L-glutamine (0.4 g), L-histidine-HCl (0.2 g), L-methionine (0.12 g), L-alanine (0.8 g), L-lysine (0.4 g), and reduced glutathione (0.36 g) in 500 mL deionized water.</li>
		<li>Dissolve L-cysteine (0.44 g) and L-cystine (0.28 g) in a minimal volume (~1 mL) of 1 M HCl and add to the above amino acid solution.</li>
		<li>Adjust the pH of the solution with 10 N NaOH until all particulate is dissolved. The final pH will be 10.0&#8211;11.0.</li>
		<li>Bring the final volume to 1 L with deionized water.</li>
		<li>Filter sterilize the solution and aliquot into 250 mL volumes.</li>
		<li>Store at -20 &#176;C.</li>
	</ol>
	</li>
	<li>Stock solution IV
	<ol>
		<li>Dissolve glucose (200 g) in deionized water to a final volume of 1 L. 
		NOTE: The solution may have to be heated to dissolve the glucose.</li>
		<li>Filter sterilize and aliquot the solution into 50 mL conical tubes.</li>
		<li>Store at -20 &#176;C.</li>
	</ol>
	</li>
	<li>Stock solution V
	<ol>
		<li>Combine hypoxanthine (5.0 g), uracil (5.0 g), and NaOH (4.0 g) in deionized water to a final volume of 1 L.</li>
		<li>Filter sterilize and aliquot into 50 mL conical tubes.</li>
		<li>Store at -20 &#176;C.</li>
	</ol>
	</li>
	<li>Solution VI
	<ol>
		<li>Prepare a 1 M CaCl2-2H2O (147 g/L) solution in deionized water. Filter sterilize and store at room temperature (RT).</li>
	</ol>
	</li>
	<li>Stock solution VII
	<ol>
		<li>Prepare a 1 M anhydrous MgSO4 solution in deionized water. Filter sterilize and store at RT.</li>
	</ol>
	</li>
	<li>Stock solution VIII
	<ol>
		<li>Prepare a 1 M NaHCO3 solution in deionized water. Filter sterilize and store at RT.</li>
	</ol>
	</li>
</ol>
2. Preparation of 4x Sterile Concentrate and 1x CDM
NOTE: This procedure is to be performed in either acid treated sterile glassware or plastic to prevent leaching of metals into the solutions.
<ol>
	<li>Combine stock solutions I (50 mL), II (20 mL), III (250 mL), IV (50 mL), and V (20 mL) with 20.0 g of HEPES and stir to mix.</li>
	<li>Adjust pH to 7.4, then bring the final volume to 500 mL with deionized water.</li>
	<li>Wash Chelex-100 resin in 1 L deionized water to remove preservatives prior to adding it to the 4x sterile concentrate. Do this by adding 50 g of resin to 1 L deionized water and stirring for at least 1 h. Remove the water by vacuum filtration and use this washed resin for step 2.4.</li>
	<li>Add 50 g of resin and slowly stir for exactly 90 min.</li>
	<li>Remove resin by filter sterilization and store the 4x sterile concentrate at 4 &#176;C.</li>
	<li>To prepare a 1x working concentration of CDM, first dilute the 4x concentrate with sterile deionized water, then add solution VI (125 &#181;L per 500 mL of 1x solution), solution VII (535 &#181;L per 500 mL), and solution VIII (10 mL per 500 mL). 
	NOTE: Although all stock solutions have already been sterilized, it is recommended to filter sterilize the 1x solution again after preparation.</li>
</ol>
3. Preparation of CDM Plates
NOTE: The recipe below makes 1 L media for plates, but it is best to prepare these in smaller volumes. Everything scales down proportionally.
<ol>
	<li>Washed agarose
	<ol>
		<li>Dissolve 50 g of agarose in 1 L deionized water, stirring for 1 h.</li>
		<li>Transfer the solution to a centrifuge bottle and centrifuge at 1,200 x g for 15 min at RT. Carefully pour off the supernatant and discard.</li>
		<li>Add sufficient deionized water to resuspend the agarose pellet, then transfer to a 1 L flask. Bring to a final volume of 1 L with deionized water and then stir and centrifuge as in step 3.1.2. Discard the supernatant.</li>
		<li>Add sufficient 100% ethanol to resuspend the pellet, then transfer to a new 1 L flask. Bring to a final volume of 1 L with ethanol. Stir and centrifuge as in step 3.1.2.</li>
		<li>Repeat step 3.1.4.</li>
		<li>Add methanol to the agarose pellet to resuspend, then transfer to a 1 L flask. Bring to a final volume of 1 L with methanol. Stir and centrifuge as in step 3.1.2.</li>
		<li>Repeat step 3.1.6.</li>
		<li>Transfer washed agarose to a tray lined with aluminum foil and allow to dry in a fume hood. When dry, transfer to a metal-free container for long-term storage.</li>
	</ol>
	</li>
	<li>Add 10 g of washed agarose and 5 g of potato starch to 750 mL of deionized water.</li>
	<li>Autoclave for 30 min at 121 &#176;C, 100 kPa above atmospheric pressure.</li>
	<li>Allow media to cool to ~65 &#176;C, then add 250 mL of 4X CDM, 250 &#181;L of solution VI, 1.07 mL of solution VII, and 20 mL of solution VIII.</li>
	<li>If desired, add the metals of choice before pouring plates. The addition of chelators is not necessary to maintain metal-free conditions.</li>
	<li>Pour into Petri dishes and allow to solidify.</li>
</ol>
4. Metal Limited Growth of Neisseria gonorrhoeae
NOTE: For most applications, it is not necessary to metal stress the bacteria prior to inoculation of CDM. The initial doubling step in CDM and the subsequent dilution is sufficient to deplete the gonococci of their internal iron and zinc stores. As such, the first two steps of the following procedure are conducted using agar plates made from GC medium base that have been supplemented with Kellogg&#39;s supplement I14 and 12.5 &#956;M Fe(NO3)3. If early metal stress is desired, we recommend preparing GC medium base plates without Fe(NO3)3 and with 5 &#956;M TPEN (N,N,N&#39;,N&#39;-tetrakis (2-pyridylmethyl) ethylenediamine) for zinc chelation or 10 &#956;M deferoxamine for iron chelation. All incubation is conducted at 37 &#176;C with 5% CO2.
<ol>
	<li>Two days prior to the experiment, on day -2, streak gonococci from freezer stocks onto GC medium plates and incubate for no more than 24 h.</li>
	<li>On day -1, streak single colonies onto fresh GC medium plates. Try to do this 14&#8211;16 h prior to the growth experiment.</li>
	<li>On the day of the experiment, add 5&#8211;10 mL 1x CDM to an acid washed 125 mL baffled sidearm flask (Supplemental Figure 1) and use this to blank a Klett colorimeter.</li>
	<li>Use a sterile, cotton-tipped swab to inoculate CDM from healthy, single colonies. Aim for 20 Klett units. 
	NOTE: If a Klett colorimeter is not available, a spectrophotometer is suitable as well. There is no universal conversion formula for Klett units to OD, but a rough guide is available (<a href="https://support.hunterlab.com/hc/en-us/articles/214490283-Klett-Color-Scales" target="_blank">https://support.hunterlab.com/hc/en-us/articles/214490283-Klett-Color-Scales</a>).</li>
	<li>Incubate with shaking at 250 rpm until approximately one mass doubling (40 Klett units). This should take between 1&#8211;2 h.</li>
	<li>At this point, the cultures are back diluted by addition of a sufficient volume of CDM to reach half of the initial culture density (e.g., if 5 mL of culture has gone from 20 to 40 Klett units, 15 mL of CDM will bring it back down to 10 Klett units) and growth will continue as in step 4.5. The specific amount of back dilution, metal treatments, etc. depend on downstream applications. We give three examples below (sections 5, 6, or 7).</li>
</ol>
5. Western Analysis of Metal Responsive Gene Products
<ol>
	<li>Beginning at step 4.6, back dilute the cultures with three volumes of CDM (e.g., 15 mL of CDM if starting with 5 mL). At this point, add metal treatments if desired.
	<ol>
		<li>Iron-sensing genes can be derepressed with 12.5 &#956;M Fe(NO3)3. Zinc-responsive genes may be derepressed with 10 &#956;M ZnSO4.</li>
		<li>Additional iron or zinc stress is not necessary, as the media is already depleted, so responsive genes will already be expressed. If further stress is desired, we recommend no more than 1&#8211;2 &#956;M of deferoxamine or TPEN, as excessive stress will prevent cultures from growing.</li>
	</ol>
	</li>
	<li>Grow cultures as in step 4.5 for 4 h and record the final cell density of the samples. Less metal-stressed cultures will grow to higher final densities.</li>
	<li>Standardize cultures to a suitable density and prepare lysates.
	<ol>
		<li>Standardize whole-cell lysates to a density equivalent to 100 Klett units in 1 mL of culture. To accomplish this, divide 100 by the Klett unit density of your sample. The number you get is the volume, in mL, of culture that will be used to make the cell pellet.</li>
	</ol>
	</li>
	<li>Follow standard SDS-PAGE and Western blotting procedures15 to probe for proteins of interest.</li>
</ol>
6. Metal-limited Growth Assays
NOTE: These assays describe premade growth premixes. The preparation of these mixes is described in section 8.
<ol>
	<li>During the mass doubling in step 4.5, pretreat the wells of a 96 well microplate with 10x premixes. Additionally, designate three wells to serve as blanks. To these wells, add 10 &#181;L of 10x premix and 90 &#181;L of CDM.</li>
	<li>Once the cultures in the sidearm flasks have doubled, add 100 &#181;L of each culture to an unused well in the microplate and measure the optical density at 600 nm (OD600). This may be done with cuvettes in a spectrophotometer, but with larger numbers of strains within an assay this can become cumbersome and is generally not advised unless your spectrophotometer can measure directly from the arm of the side arm flask (Supplemental Figure 2).
	<ol>
		<li>While measuring the OD, place the flasks back in the incubator to ensure the gonococci remain viable.</li>
	</ol>
	</li>
	<li>Calculate the correct amount of dilution required to bring the cultures to OD600 = 0.02. The 10x premixes have a negligible effect on OD and can be omitted from calculation.</li>
	<li>Dilute cultures with CDM in small culture tubes and add sufficient volume to dilute the 10x premixes to 1x in the plate. If a plate warmer is available, keep the plate at 37 &#176;C while working.</li>
	<li>Incubate the plate for 8&#8211;12 h with shaking in a plate reader, taking OD600 measurements at desired intervals.</li>
</ol>
7. Detection of Ligand Binding by Outer Membrane Metal Transporters
<ol>
	<li>Treat cultures as desired as in step 5.1, then incubate with shaking for 4 h.</li>
	<li>Shortly before the 4 h mark, cut three pieces of filter paper and a piece of nitrocellulose to the approximate size needed to fit into a dot blot apparatus (Supplemental Figure 3). Presoak the nitrocellulose in deionized water, then assemble the apparatus with filter paper below the nitrocellulose.</li>
	<li>At 4 h, record the cell densities and standardize to an appropriate final density. It is recommended to use ~10% of the density used for the preparation of the cell lysates in step 5. For example, if using 100 KU in 1 mL for the lysates, this means ~10 KU in 1 mL for these blots. The calculation is otherwise the same as described in 5.3.1, and cultures are added directly to the nitrocellulose in the calculated volumes rather than made into lysates.</li>
	<li>Pipet cell cultures onto the nitrocellulose and allow sufficient time for the filter paper to absorb all the liquid.</li>
	<li>Disassemble the apparatus, allow the blot to dry, and block the nitrocellulose membrane for 1 h in 5% bovine serum albumin or nonfat milk (w/v) in Tris-buffered saline.</li>
	<li>Reassemble the dot blot apparatus, replacing the filter paper with paraffin film to create a leak-proof seal under the nitrocellulose.</li>
	<li>Dilute the metal-binding ligand of interest to 0.2 &#956;M in blocker and probe cells for 1 h.</li>
	<li>Siphon off the liquid with a vacuum, wash the blot, then follow standard immunological procedures to develop the signal16. The wash steps may be done in or out of the apparatus.</li>
</ol>
8. Metal Loading of Transferrin, S100A7, and Calprotectin, and Preparation of 10x Premixes
NOTE: As with CDM preparation, use acid washed glass or plastic for solution preparation.
<ol>
	<li>Dissolve human transferrin at 10 mg/mL (125 &#956;M) in initial buffer (100 mM Tris, 150 mM NaCl, 20 mM NaHCO3, pH = 8.4). S100A7 and calprotectin are suspended in buffer consisting of 20 mM Tris, 100 mM NaCl, 10 mM 2-Mercaptoethanol, and 1 mM CaCl2, pH = 8.0).
	<ol>
		<li>Add ferration solution (100 mM sodium citrate, 100 mM NaHCO3, 5 mM FeCl3-6H2O, pH = 8.4) to the transferrin solution to achieve 30% iron saturation (e.g., 75 &#181;L of ferration solution is suitable for 5 mL transferrin if made at 10 mg/mL).</li>
		<li>Add ZnSO4 to S100A7 or calprotectin at a 50% molar ratio to the protein to create 25% saturation (each protein molecule has two metal binding sites). Preparations of 100 &#956;M S100A7 or calprotectin with 50 &#956;M ZnSO4 can be used.</li>
		<li>For both cases, allow end-over-end mixing for at least 1 h for metal loading.</li>
	</ol>
	</li>
	<li>Prepare 4 L of dialysis buffer (40 mM Tris, 150 mM NaCl, 20 mM NaHCO3, pH = 7.4). Split this into two separate 2 L volumes and place one at 4 &#176;C.</li>
	<li>Add the metal loaded proteins to a dialysis cassette using a syringe and dialyze against the first buffer volume for 4 h at RT.</li>
	<li>Move the cassette to the second buffer volume and dialyze overnight at 4 &#176;C. After these steps, any unbound metals should be removed. 
	NOTE: We advise a bicinchoninic acid assay to determine protein concentrations after dialysis.</li>
	<li>Use a 10x transferrin premix for transferrin utilization as a sole iron source.
	<ol>
		<li>Prepare this premix by diluting 30% human Fe-transferrin and bovine apo-transferrin (prepared at 125 &#956;M as described for human transferrin, without the iron loading step) to 75 &#956;M and 30 &#956;M, respectively, in PBS. A positive control premix replaces 30% Fe-transferrin with 75 &#956;M Fe(NO3)3, and a negative control premix omits any added iron, retaining only the bovine apo-transferrin. In the growth assay, dilute 10 &#181;L of these concentrates with 90 &#181;L of culture. Final concentrations are 7.5 &#956;M 30% human Fe-transferrin and 3 &#956;M bovine apo-transferrin.</li>
	</ol>
	</li>
	<li>Use a modified version of the transferrin 10x premix for S100A7 or calprotectin utilization as a sole zinc source.
	<ol>
		<li>For a positive control premix, incorporate 50 &#956;M ZnSO4 into the transferrin premix. For a negative control, incorporate 50 &#956;M TPEN and omit the zinc. Then, take 10 &#181;L of each of these for every sample well needed, and move that volume to a new tube. Add to this half as much volume of sterile PBS. For example, if 10 wells will receive the positive control premix, take 100 &#181;L of premix, move it to a new tube, and add 50 &#181;L of PBS. Do the same for the negative control.</li>
		<li>To make the S100A7 or calprotectin premix, take 10 &#181;L of the negative control premix per well needed, move to a new tube, and add half that volume of the 25% Zn-S100A7 or calprotectin. In the growth assay, use 15 &#181;L of premix and dilute with 85 &#181;L of culture. Final concentrations for the transferrins remain the same as in step 8.5, with an added 5 &#956;M of Zn, TPEN, or S100A7/calprotectin.</li>
	</ol>
	</li>
</ol>

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J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Carbon+dioxide+requirement+of+Neisseria+gonorrhoeae+growing+on+a+solid+medium.">Carbon dioxide requirement of Neisseria gonorrhoeae growing on a solid medium.</a> Journal of Clinical Microbiology. 4 (2), 129-132 (1976).</li><li>Grim, K. P., 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=The+Metallophore+Staphylopine+Enables+Staphylococcus+aureus+To+Compete+with+the+Host+for+Zinc+and+Overcome+Nutritional+Immunity.">The Metallophore Staphylopine Enables Staphylococcus aureus To Compete with the Host for Zinc and Overcome Nutritional Immunity.</a> MBio. 8 (5), 01281-01317 (2017).</li><li>Helbig, K., Bleuel, C., Krauss, G. J., Nies, D. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Glutathione+and+transition-metal+homeostasis+in+Escherichia+coli.">Glutathione and transition-metal homeostasis in Escherichia coli.</a> Journal of Bacteriology. 190 (15), 5431-5438 (2008).</li><li>Calmettes, 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=The+molecular+mechanism+of+Zinc+acquisition+by+the+neisserial+outer-membrane+transporter+ZnuD.">The molecular mechanism of Zinc acquisition by the neisserial outer-membrane transporter ZnuD.</a> Nature Communications. 6, 7996 (2015).</li><li>Hubert, K., 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=ZnuD,+a+potential+candidate+for+a+simple+and+universal+Neisseria+meningitidis+vaccine.">ZnuD, a potential candidate for a simple and universal Neisseria meningitidis vaccine.</a> Infection and Immunity. 81 (6), 1915-1927 (2013).</li><li>Kumar, P., Sannigrahi, S., Tzeng, Y. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+Neisseria+meningitidis+ZnuD+zinc+receptor+contributes+to+interactions+with+epithelial+cells+and+supports+heme+utilization+when+expressed+in+Escherichia+coli.">The Neisseria meningitidis ZnuD zinc receptor contributes to interactions with epithelial cells and supports heme utilization when expressed in Escherichia coli.</a> Infection and Immunity. 80 (2), 657-667 (2012).</li><li>Stork, 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=An+outer+membrane+receptor+of+Neisseria+meningitidis+involved+in+zinc+acquisition+with+vaccine+potential.">An outer membrane receptor of Neisseria meningitidis involved in zinc acquisition with vaccine potential.</a> PLoS Pathogens. 6, 1000969 (2010).</li><li>Rosadini, C. V., Gawronski, J. D., Raimunda, D., Arg&#252;ello, J. M., Akerley, B. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+novel+zinc+binding+system,+ZevAB,+is+critical+for+survival+of+nontypeable+Haemophilus+influenzae+in+a+murine+lung+infection+model.">A novel zinc binding system, ZevAB, is critical for survival of nontypeable Haemophilus influenzae in a murine lung infection model.</a> Infection and Immunity. 79 (8), 3366-3376 (2011).</li><li>Ammendola, S., 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=High-affinity+Zn2++uptake+system+ZnuABC+is+required+for+bacterial+zinc+homeostasis+in+intracellular+environments+and+contributes+to+the+virulence+of+Salmonella+enterica.">High-affinity Zn2+ uptake system ZnuABC is required for bacterial zinc homeostasis in intracellular environments and contributes to the virulence of Salmonella enterica.</a> Infection and Immunity. 75 (12), 5867-5876 (2007).</li><li>Gabbianelli, R., 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=Role+of+ZnuABC+and+ZinT+in+Escherichia+coli+O157:H7+zinc+acquisition+and+interaction+with+epithelial+cells.">Role of ZnuABC and ZinT in Escherichia coli O157:H7 zinc acquisition and interaction with epithelial cells.</a> BMC Microbiology. 11, 36 (2011).</li><li>Biswas, G. D., Anderson, J. E., Chen, C. J., Cornelissen, C. N., Sparling, P. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Identification+and+functional+characterization+of+the+Neisseria+gonorrhoeae+lbpB+gene+product.">Identification and functional characterization of the Neisseria gonorrhoeae lbpB gene product.</a> Infection and Immunity. 67 (1), 455-459 (1999).</li><li>Biswas, G. D., Sparling, P. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Characterization+of+lbpA,+the+structural+gene+for+a+lactoferrin+receptor+in+Neisseria+gonorrhoeae.">Characterization of lbpA, the structural gene for a lactoferrin receptor in Neisseria gonorrhoeae.</a> Infection and Immunity. 63 (8), 2958-2967 (1995).</li><li>Chen, C. J., Sparling, P. F., Lewis, L. A., Dyer, D. W., Elkins, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Identification+and+purification+of+a+hemoglobin-binding+outer+membrane+protein+from+Neisseria+gonorrhoeae.">Identification and purification of a hemoglobin-binding outer membrane protein from Neisseria gonorrhoeae.</a> Infection and Immunity. 64 (12), 5008-5014 (1996).</li><li>Wong, C. T., 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=Structural+analysis+of+haemoglobin+binding+by+HpuA+from+the+Neisseriaceae+family.">Structural analysis of haemoglobin binding by HpuA from the Neisseriaceae family.</a> Nature Communications. 6, 10172 (2015).</li><li>Carson, S. D., Klebba, P. E., Newton, S. M., Sparling, P. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ferric+enterobactin+binding+and+utilization+by+Neisseria+gonorrhoeae.">Ferric enterobactin binding and utilization by Neisseria gonorrhoeae.</a> Journal of Bacteriology. 181 (9), 2895-2901 (1999).</li><li>Tseng, H. J., Srikhanta, Y., McEwan, A. G., Jennings, M. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Accumulation+of+manganese+in+Neisseria+gonorrhoeae+correlates+with+resistance+to+oxidative+killing+by+superoxide+anion+and+is+independent+of+superoxide+dismutase+activity.">Accumulation of manganese in Neisseria gonorrhoeae correlates with resistance to oxidative killing by superoxide anion and is independent of superoxide dismutase activity.</a> Molecular Microbiology. 40 (5), 1175-1186 (2001).</li></ol>

The authors have nothing to declare.

Growth media serves a variety of roles in microbiological research. Specialized media are used for selection, enrichment, and various other applications for many unique types of study. One such application is the induction of metal-responsive genes, which is typically accomplished by addition of a specific chelator that targets a particular metal ion. This method is limited, as the amount of chelation necessary for various trace metals is likely to be variable due to different water sources containing unique metal profil...

Neisseria gonorrhoeae causes the common sexually-transmitted infection gonorrhea. During infection, pathogenic Neisseria express a repertoire of metal-responsive genes that allow the bacteria to overcome metal restriction efforts by the human host1,2,3. Trace metals like iron and zinc play key roles in many cellular processes, such as binding to enzymes in catalytic sites, participation in redox reactions, and as structural factors in various proteins4,5. In metal-limited conditions, metal-responsive loci are derepressed and their resultant proteins can aid the acquisition of these nutrients. Characterization of these genes and proteins presents a unique technical challenge for the investigator. Metal ions must be withheld from bacteria in order to induce transcription of these genes from their native loci, but effective chelation of these ions from metal-laden media can be difficult to optimize. The different metal profiles of source water and inherent lot-to-lot variation6 of powdered ingredients means that the amount of chelator required to remove a specific metal from a rich medium will vary between different locations, ingredient vendors, and even over time within a single laboratory as chemical inventory is replaced.
To circumvent this challenge, we describe the preparation of a defined medium that is treated with Chelex-100 resin during preparation to remove trace metals from the solution. This medium is sufficiently nutrient dense to allow for the growth of gonococcus, which is difficult to culture outside of the human host, and allows the investigator to introduce a specific metal profile by addition of their own defined sources and concentrations of metals. The method of controlled add-back of desired metals to depleted medium increases experimental consistency and allows for robust, replicable experiments regardless of factors such as water source and chemical lot numbers. Moreover, this media can be deployed as either a liquid or solid with only minor modifications, making it quite versatile.
In order to demonstrate the utility of the this medium, we outline a protocol for its use for gonococcal growth and describe three successful experiments to characterize metal-responsive Neisseria genes. First, we prepare gonococcal whole-cell lysates from metal-depleted or supplemented cultures and demonstrate variable levels of protein production from metal-responsive loci. We then outline a zinc-restricted growth assay in which gonococcal growth is controlled by supplementation of specific, useable zinc sources. Finally, we show binding assays that demonstrate whole gonococcal cells expressing metal-responsive surface receptors binding to their respective metal-containing ligands. Successful surface presentation of these receptors requires growth in metal-depleted medium.
The present protocol was optimized specifically for Neisseria gonorrhoeae, but numerous other bacterial pathogens employ metal acquisition strategies during infection7, so this protocol may be adapted for the study of metal homeostasis in other bacteria. Optimizing this media and these experimental protocols for use in other bacteria will likely require slight modification of metal chelator concentrations and/or treatment time with Chelex-100, as other bacteria may have slightly different metal requirements than gonococcus. Iron and zinc are the primary metals of concern for the described investigations, but other metals (e.g., manganese) have been demonstrated as critical for bacteria, including Neisseria8,9,10,11,12. Furthermore, similar methods have been described for metal characterizations in eukaryotic cell culture work, which may also be considered.13

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>125 mL sidearm flasks</td>
			<td>Bellco</td>
			<td>2578-S0030</td>
			<td>Must be custom ordered</td>
		</tr>
		<tr>
			<td>2-Mercaptoethanol</td>
			<td>VWR</td>
			<td>M131</td>
			<td>Open in fume hood</td>
		</tr>
		<tr>
			<td>3MM Paper</td>
			<td>GE Health</td>
			<td>3030-6461</td>
			<td>Called &#34;filter paper&#34; in text</td>
		</tr>
		<tr>
			<td>Agarose</td>
			<td>Biolone</td>
			<td>BIO-41025</td>
			<td>Powder</td>
		</tr>
		<tr>
			<td>Ammonium chloride</td>
			<td>Sigma-Aldrich</td>
			<td>A9434</td>
			<td>Powder</td>
		</tr>
		<tr>
			<td>Biotin</td>
			<td>Sigma-Aldrich</td>
			<td>B4501</td>
			<td>Powder</td>
		</tr>
		<tr>
			<td>Blotting grade blocker</td>
			<td>Bio-Rad</td>
			<td>170-6404</td>
			<td>Nonfat dry milk</td>
		</tr>
		<tr>
			<td>Bovine serum albumin</td>
			<td>Roche</td>
			<td>3116964001</td>
			<td>Powder</td>
		</tr>
		<tr>
			<td>Bovine transferrin</td>
			<td>Sigma-Aldrich</td>
			<td>T1428</td>
			<td>Powder</td>
		</tr>
		<tr>
			<td>Calcium chloride dihydrate</td>
			<td>Sigma-Aldrich</td>
			<td>C5080</td>
			<td>Powder</td>
		</tr>
		<tr>
			<td>Calcium pantothenate</td>
			<td>Sigma-Aldrich</td>
			<td>C8731</td>
			<td>Powder</td>
		</tr>
		<tr>
			<td>Calprotectin</td>
			<td>N/A</td>
			<td>N/A</td>
			<td>We are supplied with this by a collaborator</td>
		</tr>
		<tr>
			<td>Chelex-100 Resin</td>
			<td>Bio-Rad</td>
			<td>142-2832</td>
			<td>Wash with deionized water prior to use</td>
		</tr>
		<tr>
			<td>Cotton-tipped sterile swab</td>
			<td>Puritan</td>
			<td>25-806</td>
			<td>Cotton is better than polyester for this application</td>
		</tr>
		<tr>
			<td>Deferoxamine</td>
			<td>Sigma-Aldrich</td>
			<td>D9533</td>
			<td>Powder</td>
		</tr>
		<tr>
			<td>D-glucose</td>
			<td>Sigma-Aldrich</td>
			<td>G8270</td>
			<td>Powder</td>
		</tr>
		<tr>
			<td>Dialysis cassette</td>
			<td>Thermo</td>
			<td>66380</td>
			<td>Presoak in buffer prior to use</td>
		</tr>
		<tr>
			<td>Dot blot apparatus</td>
			<td>Schleicher &#38; Schwell</td>
			<td>10484138</td>
			<td>Lock down lid as tightly as possible before sample loading</td>
		</tr>
		<tr>
			<td>Ethanol</td>
			<td>Koptec</td>
			<td>V1016</td>
			<td>Flammable liquid, store in flammables cabinet</td>
		</tr>
		<tr>
			<td>Ferric chloride</td>
			<td>Sigma-Aldrich</td>
			<td>F7134</td>
			<td>Irritant, do not inhale</td>
		</tr>
		<tr>
			<td>Ferric nitrate nonahydrate</td>
			<td>Sigma-Aldrich</td>
			<td>F1143</td>
			<td>Irritant, do not inhale</td>
		</tr>
		<tr>
			<td>GC medium base</td>
			<td>Difco</td>
			<td>228950</td>
			<td>Powder, already contains agar</td>
		</tr>
		<tr>
			<td>Glycine</td>
			<td>Sigma-Aldrich</td>
			<td>G8898</td>
			<td>Powder</td>
		</tr>
		<tr>
			<td>HEPES</td>
			<td>Fisher</td>
			<td>L-15694</td>
			<td>Powder</td>
		</tr>
		<tr>
			<td>Human transferrin</td>
			<td>Sigma-Aldrich</td>
			<td>T2030</td>
			<td>Powder</td>
		</tr>
		<tr>
			<td>Hypoxanthine</td>
			<td>Sigma-Aldrich</td>
			<td>H9377</td>
			<td>Powder</td>
		</tr>
		<tr>
			<td>Klett colorimeter</td>
			<td>Manostat</td>
			<td>37012-0000</td>
			<td>Uses color transmission to assess culture density</td>
		</tr>
		<tr>
			<td>L-alanine</td>
			<td>Sigma-Aldrich</td>
			<td>A7627</td>
			<td>Powder</td>
		</tr>
		<tr>
			<td>L-arginine</td>
			<td>Sigma-Aldrich</td>
			<td>A5006</td>
			<td>Powder</td>
		</tr>
		<tr>
			<td>L-asparagine monohydrate</td>
			<td>Sigma-Aldrich</td>
			<td>A8381</td>
			<td>Powder</td>
		</tr>
		<tr>
			<td>L-aspartate</td>
			<td>Sigma-Aldrich</td>
			<td>A9256</td>
			<td>Powder</td>
		</tr>
		<tr>
			<td>L-cysteine hydrochloride</td>
			<td>Sigma-Aldrich</td>
			<td>C1276</td>
			<td>Powder</td>
		</tr>
		<tr>
			<td>L-cystine</td>
			<td>Sigma-Aldrich</td>
			<td>C8755</td>
			<td>Powder</td>
		</tr>
		<tr>
			<td>L-glutamate</td>
			<td>Sigma-Aldrich</td>
			<td>G1251</td>
			<td>Powder</td>
		</tr>
		<tr>
			<td>L-glutamine</td>
			<td>Sigma-Aldrich</td>
			<td>G3126</td>
			<td>Powder</td>
		</tr>
		<tr>
			<td>L-histidine monohydrochloride</td>
			<td>Sigma-Aldrich</td>
			<td>H8125</td>
			<td>Powder</td>
		</tr>
		<tr>
			<td>L-isoleucine</td>
			<td>Sigma-Aldrich</td>
			<td>I2752</td>
			<td>Powder</td>
		</tr>
		<tr>
			<td>L-leucine</td>
			<td>Sigma-Aldrich</td>
			<td>L8000</td>
			<td>Powder</td>
		</tr>
		<tr>
			<td>L-lysine</td>
			<td>Sigma-Aldrich</td>
			<td>L5501</td>
			<td>Powder</td>
		</tr>
		<tr>
			<td>L-methionine</td>
			<td>Sigma-Aldrich</td>
			<td>M9625</td>
			<td>Powder</td>
		</tr>
		<tr>
			<td>L-phenylalanine</td>
			<td>Sigma-Aldrich</td>
			<td>P2126</td>
			<td>Powder</td>
		</tr>
		<tr>
			<td>L-proline</td>
			<td>Sigma-Aldrich</td>
			<td>P0380</td>
			<td>Powder</td>
		</tr>
		<tr>
			<td>L-serine</td>
			<td>Sigma-Aldrich</td>
			<td>S4500</td>
			<td>Powder</td>
		</tr>
		<tr>
			<td>L-threonine</td>
			<td>Sigma-Aldrich</td>
			<td>T8625</td>
			<td>Powder</td>
		</tr>
		<tr>
			<td>L-tryptophan</td>
			<td>Sigma-Aldrich</td>
			<td>T0254</td>
			<td>Powder</td>
		</tr>
		<tr>
			<td>L-tyrosine</td>
			<td>Sigma-Aldrich</td>
			<td>T3754</td>
			<td>Powder</td>
		</tr>
		<tr>
			<td>L-valine</td>
			<td>Sigma-Aldrich</td>
			<td>V0500</td>
			<td>Powder</td>
		</tr>
		<tr>
			<td>Magnesium sulfate</td>
			<td>Sigma-Aldrich</td>
			<td>M7506</td>
			<td>Powder</td>
		</tr>
		<tr>
			<td>Methanol</td>
			<td>VWR</td>
			<td>BDH1135-4LP</td>
			<td>Flammable liquid, store in flammables cabinet</td>
		</tr>
		<tr>
			<td>Nitrocellulose</td>
			<td>GE Health</td>
			<td>10600002</td>
			<td>Keep in protective sheath until use</td>
		</tr>
		<tr>
			<td>Potassium phosphate dibasic</td>
			<td>Sigma-Aldrich</td>
			<td>60356</td>
			<td>Powder</td>
		</tr>
		<tr>
			<td>Potassium phosphate monobasic</td>
			<td>Sigma-Aldrich</td>
			<td>P9791</td>
			<td>Powder</td>
		</tr>
		<tr>
			<td>Potassium sulfate</td>
			<td>Sigma-Aldrich</td>
			<td>P0772</td>
			<td>Powder</td>
		</tr>
		<tr>
			<td>Potato starch</td>
			<td>Sigma-Aldrich</td>
			<td>S4251</td>
			<td>Powder</td>
		</tr>
		<tr>
			<td>Reduced glutathione</td>
			<td>Sigma-Aldrich</td>
			<td>G4251</td>
			<td>Handle carefully. Can oxidize easily.</td>
		</tr>
		<tr>
			<td>S100A7</td>
			<td>N/A</td>
			<td>N/A</td>
			<td>We are supplied with this by a collaborator</td>
		</tr>
		<tr>
			<td>Sodium bicarbonate</td>
			<td>Sigma-Aldrich</td>
			<td>S5761</td>
			<td>Powder</td>
		</tr>
		<tr>
			<td>Sodium chloride</td>
			<td>VWR</td>
			<td>470302</td>
			<td>Powder</td>
		</tr>
		<tr>
			<td>Sodium citrate</td>
			<td>Fisher</td>
			<td>S279</td>
			<td>Powder</td>
		</tr>
		<tr>
			<td>Sodium hydroxide</td>
			<td>Acros Organics</td>
			<td>383040010</td>
			<td>Highly hygroscopic</td>
		</tr>
		<tr>
			<td>Thiamine hydrochloride</td>
			<td>Sigma-Aldrich</td>
			<td>T4625</td>
			<td>Powder</td>
		</tr>
		<tr>
			<td>Thiamine pyrophosphate</td>
			<td>Sigma-Aldrich</td>
			<td>C8754</td>
			<td>Also called cocarboxylase</td>
		</tr>
		<tr>
			<td>TPEN</td>
			<td>Sigma-Aldrich</td>
			<td>P4413</td>
			<td>Powder</td>
		</tr>
		<tr>
			<td>Tris</td>
			<td>VWR</td>
			<td>497</td>
			<td>Powder</td>
		</tr>
		<tr>
			<td>Uracil</td>
			<td>Sigma-Aldrich</td>
			<td>U0750</td>
			<td>Powder</td>
		</tr>
		<tr>
			<td>Zinc sulfte heptahydrate</td>
			<td>Sigma-Aldrich</td>
			<td>204986</td>
			<td>Irritant, do not inhale</td>
		</tr>
	</tbody>
</table>

metal-limited growth of neisseria gonorrhoeae for characterization of metal-responsive genes and metal acquisition from host ligands

Trace metals such as iron and zinc are vital nutrients known to play key roles in prokaryotic processes including gene regulation, catalysis, and protein structure. Metal sequestration by hosts often leads to metal limitation for the bacterium. This limitation induces bacterial gene expression whose protein products allow bacteria to overcome their metal-limited environment. Characterization of such genes is challenging. Bacteria must be grown in meticulously prepared media that allows sufficient access to nutritional metals to permit bacterial growth while maintaining a metal profile conducive to achieving expression of the aforementioned genes. As such, a delicate balance must be established for the concentrations of these metals. Growing a nutritionally fastidious organism such as Neisseria gonorrhoeae, which has evolved to survive only in the human host, adds an additional level of complexity. Here, we describe the preparation of a defined metal-limited medium sufficient to allow gonococcal growth and the desired gene expression. This method allows the investigator to chelate iron and zinc from undesired sources while supplementing the media with defined sources of iron or zinc, whose preparation is also described. Finally, we outline three experiments that utilize this media to help characterize the protein products of metal-regulated gonococcal genes.

A specific defined medium in the absence of trace metals for the growth of Neisseria gonorrhoeae was developed and implemented for the characterization of metal-responsive genes and their gene products. In the optimized protocol, the metal profile of media is controlled by adding metals back at the discretion of the investigator, rather than by titrated chelation of a metal target, allowing for increased control and consistency from lab to lab and experiment to experiment. This m...

Watch this Scientific Journal Video about Metal-Limited Growth of Neisseria gonorrhoeae for Characterization of Metal-Responsive Genes and Metal Acquisition from Host Ligands at JoVE.com

Metal-Limited Growth of Neisseria gonorrhoeae for Characterization of Metal-Responsive Genes and Metal Acquisition from Host Ligands

We describe here a method for growth of Neisseria gonorrhoeae in metal-restricted liquid medium to facilitate the expression of genes for metal uptake. We also outline downstream experiments to characterize the phenotype of gonococci grown in these conditions. These methods may be adapted to be suitable for characterization of metal-responsive genes in other bacteria.

Metal-Limited Growth of Neisseria gonorrhoeae for Characterization of Metal-Responsive Genes and Metal Acquisition from Host Ligands

Trace metals such as iron and zinc are vital nutrients known to play key roles in prokaryotic processes including gene regulation, catalysis, and protein ...

metal-limited-growth-neisseria-gonorrhoeae-for-characterization-metal

Research

JoVE Journal

Immunology and Infection

5.4K Views.  Georgia State University. We describe here a method for growth of Neisseria gonorrhoeae in metal-restricted liquid medium to facilitate the expression of genes for metal uptake. We also outline downstream experiments to characterize the phenotype of gonococci grown in these conditions. These methods may be adapted to be suitable for characterization of metal-responsive genes in other bacteria.

Video: Metal-Limited Growth of Neisseria gonorrhoeae for Characterization of Metal-Responsive Genes and Metal Acquisition from Host Ligands

Dissecting Host-virus Interaction in Lytic Replication of a Model Herpesvirus

In response to viral infection, a host develops various defensive responses, such as activating innate immune signaling pathways that lead to antiviral cytokine production1,2. In order to colonize the host, viruses are obligate to evade host antiviral responses and manipulate signaling pathways. Unraveling the host-virus interaction will shed light on the development of novel therapeutic strategies against viral infection.
Murine &gamma;HV68 is closely related to human oncogenic Kaposi's sarcoma-associated herpesvirus and Epsten-Barr virus3,4. &gamma;HV68 infection in laboratory mice provides a tractable small animal model to examine the entire course of host responses and viral infection in vivo, which are not available for human herpesviruses. In this protocol, we present a panel of methods for phenotypic characterization and molecular dissection of host signaling components in &gamma;HV68 lytic replication both in vivo and ex vivo. The availability of genetically modified mouse strains permits the interrogation of the roles of host signaling pathways during &gamma;HV68 acute infection in vivo. Additionally, mouse embryonic fibroblasts (MEFs) isolated from these deficient mouse strains can be used to further dissect roles of these molecules during &gamma;HV68 lytic replication ex vivo.
Using virological and molecular biology assays, we can pinpoint the molecular mechanism of host-virus interactions and identify host and viral genes essential for viral lytic replication. Finally, a bacterial artificial chromosome (BAC) system facilitates the introduction of mutations into the viral factor(s) that specifically interrupt the host-virus interaction. Recombinant &gamma;HV68 carrying these mutations can be used to recapitulate the phenotypes of &gamma;HV68 lytic replication in MEFs deficient in key host signaling components. This protocol offers an excellent strategy to interrogate host-pathogen interaction at multiple levels of intervention in vivo and ex vivo.
Recently, we have discovered that &gamma;HV68 usurps an innate immune signaling pathway to promote viral lytic replication5. Specifically, &gamma;HV68 de novo infection activates the immune kinase IKK&beta; and activated IKK&beta; phosphorylates the master viral transcription factor, replication and transactivator (RTA), to promote viral transcriptional activation. In doing so, &gamma;HV68 efficiently couples its transcriptional activation to host innate immune activation, thereby facilitating viral transcription and lytic replication. This study provides an excellent example that can be applied to other viruses to interrogate host-virus interaction.

We describe a protocol to identify key roles of host signaling molecules in lytic replication of a model herpesvirus, gamma herpesvirus 68 (&gamma;HV68). Utilizing genetically modified mouse strains and embryonic fibroblasts for &gamma;HV68 lytic replication, the protocol permits both phenotypic characterization and molecular interrogation of virus-host interactions in viral lytic replication.

In response to viral infection, a host develops various defensive responses, such as activating innate immune signaling pathways that lead to antiviral ...

Growth of Mycobacterium tuberculosis Biofilms

Mycobacterium tuberculosis, the etiologic agent of human tuberculosis, has an extraordinary ability to survive against environmental stresses including antibiotics. Although stress tolerance of M. tuberculosis is one of the likely contributors to the 6-month long chemotherapy of tuberculosis 1, the molecular mechanisms underlying this characteristic phenotype of the pathogen remain unclear. Many microbial species have evolved to survive in stressful environments by self-assembling in highly organized, surface attached, and matrix encapsulated structures called biofilms 2-4. Growth in communities appears to be a preferred survival strategy of microbes, and is achieved through genetic components that regulate surface attachment, intercellular communications, and synthesis of extracellular polymeric substances (EPS) 5,6. The tolerance to environmental stress is likely facilitated by EPS, and perhaps by the physiological adaptation of individual bacilli to heterogeneous microenvironments within the complex architecture of biofilms 7.
In a series of recent papers we established that M. tuberculosis and Mycobacterium smegmatis have a strong propensity to grow in organized multicellular structures, called biofilms, which can tolerate more than 50 times the minimal inhibitory concentrations of the anti-tuberculosis drugs isoniazid and rifampicin 8-10. M. tuberculosis, however, intriguingly requires specific conditions to form mature biofilms, in particular 9:1 ratio of headspace: media as well as limited exchange of air with the atmosphere 9. Requirements of specialized environmental conditions could possibly be linked to the fact that M. tuberculosis is an obligate human pathogen and thus has adapted to tissue environments. In this publication we demonstrate methods for culturing M. tuberculosis biofilms in a bottle and a 12-well plate format, which is convenient for bacteriological as well as genetic studies. We have described the protocol for an attenuated strain of M. tuberculosis, mc27000, with deletion in the two loci, panCD and RD1, that are critical for in vivo growth of the pathogen 9. This strain can be safely used in a BSL-2 containment for understanding the basic biology of the tuberculosis pathogen thus avoiding the requirement of an expensive BSL-3 facility. The method can be extended, with appropriate modification in media, to grow biofilm of other culturable mycobacterial species.
Overall, a uniform protocol of culturing mycobacterial biofilms will help the investigators interested in studying the basic resilient characteristics of mycobacteria. In addition, a clear and concise method of growing mycobacterial biofilms will also help the clinical and pharmaceutical investigators to test the efficacy of a potential drug.

Growth of Mycobacterium tuberculosis Biofilms

Mycobacterium tuberculosis forms drug tolerant biofilms when cultured in certain conditions. Here we describe methods for culturing M. tuberculosis biofilms and determining the frequency of drug tolerant persisters. These protocols will be useful for further studies into the mechanisms of drug tolerance in M. tuberculosis.

Mycobacterium tuberculosis, the etiologic agent of human tuberculosis, has an extraordinary ability to survive against environmental stresses including ...

Using RNA-interference to Investigate the Innate Immune Response in Mouse Macrophages

Macrophages are key phagocytic innate immune cells. When macrophages encounter a pathogen, they produce antimicrobial proteins and compounds to kill the pathogen, produce various cytokines and chemokines to recruit and stimulate other immune cells, and present antigens to stimulate the adaptive immune response. Thus, being able to efficiently manipulate macrophages with techniques such as RNA-interference (RNAi) is critical to our ability to investigate this important innate immune cell. However, macrophages can be technically challenging to transfect and can exhibit inefficient RNAi-induced gene knockdown. In this protocol, we describe methods to efficiently transfect two mouse macrophage cell lines (RAW264.7 and J774A.1) with siRNA using the Amaxa Nucleofector 96-well Shuttle System and describe procedures to maximize the effect of siRNA on gene knockdown. Moreover, the described methods are adapted to work in 96-well format, allowing for medium and high-throughput studies. To demonstrate the utility of this approach, we describe experiments that utilize RNAi to inhibit genes that regulate lipopolysaccharide (LPS)-induced cytokine production.

In this protocol, we describe methods to efficiently transfect murine macrophage cell lines with siRNAs using the Amaxa Nucleofector 96-well Shuttle System, stimulate the macrophages with lipopolysaccharide, and monitor the effects on inflammatory cytokine production.

Macrophages are key phagocytic innate immune cells.  When macrophages encounter a pathogen, they produce antimicrobial proteins and compounds to kill the ...

Phenotypic Characterization of Macrophages from Rat Kidney by Flow Cytometry

There is increasing evidence suggesting the important role of inflammation and, subsequently, macrophages in the development and progression of renal disease. Macrophages are heterogeneous cells that have been implicated in kidney injury. Macrophages may be classified into two different phenotypes: classically activated macrophages (M1 macrophages), that release pro-inflammatory cytokines and promote fibrosis; and alternatively activated macrophages (M2 macrophages) that are associated with immunoregulatory and tissue-remodeling functions. These macrophage phenotypes need to be discriminated and analyzed to determine their contribution to renal injury. However, there are scarce studies reporting consistent phenotypic and functional information about macrophage subtypes in inflammatory renal disease models, especially in rats. This fact may be related to the limited macrophage markers used in rats, contrary to mice. Therefore, novel strategies are necessary to quantify and characterize the renal content of these infiltrating cells in a reliable way. This manuscript details a protocol for kidney digestion and further phenotypic and quantitative analysis of macrophages from rat kidneys by flow cytometry. Briefly, kidneys were incubated with collagenase and total macrophages were identified according to the dual presence of CD45 (leukocytes common antigen) and CD68 (PAN macrophage marker) in live cells.This was followed by surface staining of CD86 (M1 marker) and CD163 (M2 marker). Rat peritoneal macrophages were used as positive control for macrophage marker detection by flow cytometry. Our protocol resulted in low cellular mortality and allowed characterization of different intracellular and surface protein markers, thus limiting the loss of cellular integrity observed in other protocols. Moreover, this procedure allows the use of macrophages for further techniques, including cell sorting and mRNA or protein expression studies, among others.

This manuscript describes a detailed protocol for phenotypic and quantitative analysis of resident macrophages from rat kidneys by flow cytometry. The resulting stained cells can be also used for other applications, including cell sorting, gene expression analysis or functional studies, thus increasing the information obtained in the experimental model.

There is increasing evidence suggesting the important role of inflammation and, subsequently, macrophages in the development and progression of renal ...

A Murine Model of Group B Streptococcus Vaginal Colonization

Streptococcus agalactiae (group B Streptococcus, GBS), is a Gram-positive, asymptomatic colonizer of the human gastrointestinal tract and vaginal tract of 10 - 30% of adults. In immune-compromised individuals, including neonates, pregnant women, and the elderly, GBS may switch to an invasive pathogen causing sepsis, arthritis, pneumonia, and meningitis. Because GBS is a leading bacterial pathogen of neonates, current prophylaxis is comprised of late gestation screening for GBS vaginal colonization and subsequent peripartum antibiotic treatment of GBS-positive mothers. Heavy GBS vaginal burden is a risk factor for both neonatal disease and colonization. Unfortunately, little is known about the host and bacterial factors that promote or permit GBS vaginal colonization. This protocol describes a technique for establishing persistent GBS vaginal colonization using a single &#946;-estradiol pre-treatment and daily sampling to determine bacterial load. It further details methods to administer additional therapies or reagents of interest and to collect vaginal lavage fluid and reproductive tract tissues. This mouse model will further the understanding of the GBS-host interaction within the vaginal environment, which will lead to potential therapeutic targets to control maternal vaginal colonization during pregnancy and to prevent transmission to the vulnerable newborn. It will also be of interest to increase our understanding of general bacterial-host interactions in the female vaginal tract.

A Murine Model of Group B Streptococcus Vaginal Colonization

The purpose of this protocol is to imitate human group B Streptococcus (GBS) vaginal colonization in a murine model. This method may be used to investigate host immune responses and bacterial factors contributing to GBS vaginal persistence, as well as to test therapeutic strategies.

Streptococcus agalactiae (group B Streptococcus, GBS), is a Gram-positive, asymptomatic colonizer of the human gastrointestinal tract and vaginal tract of ...

SILAC Based Proteomic Characterization of Exosomes from HIV-1 Infected Cells

Proteomics is the large-scale analysis of proteins. Proteomic techniques, such as liquid chromatography tandem mass spectroscopy (LC-MS/MS), can characterize thousands of proteins at a time. These powerful techniques allow us to have a systemic understanding of cellular changes, especially when cells are subjected to various stimuli, such as infections, stresses, and specific test conditions. Even with recent developments, analyzing the exosomal proteome is time-consuming and often involves complex methodologies. In addition, the resultant large dataset often needs robust and streamlined analysis in order for researchers to perform further downstream studies. Here, we describe a SILAC-based protocol for characterizing the exosomal proteome when cells are infected with HIV-1. The method is based on simple isotope labeling, isolation of exosomes from differentially labeled cells, and mass spectrometry analysis. This is followed by detailed data mining and bioinformatics analysis of the proteomic hits. The resultant datasets and candidates are easy to understand and often offer a wealth of information that is useful for downstream analysis. This protocol is applicable to other subcellular compartments and a wide range of test conditions.

Here, we describe a quantitative proteomics method using the technique of stable isotope labeling by amino acids in cell culture (SILAC) to analyze the effects of HIV-1 infection on host exosomal proteomes. This protocol can be easily adapted to cells under different stress or infection conditions.

Proteomics is the large-scale analysis of proteins. Proteomic techniques, such as liquid chromatography tandem mass spectroscopy (LC-MS/MS), can ...

Rescue and Characterization of Recombinant Virus from a New World Zika Virus Infectious Clone

Infectious cDNA clones allow for genetic manipulation of a virus, thus facilitating work on vaccines, pathogenesis, replication, transmission and viral evolution. Here we describe the construction of an infectious clone for Zika virus (ZIKV), which is currently causing an explosive outbreak in the Americas. To prevent toxicity to bacteria that is commonly observed with flavivirus-derived plasmids, we generated a two-plasmid system which separates the genome at the NS1 gene and is more stable than full-length constructs that could not be successfully recovered without mutations. After digestion and ligation to join the two fragments, full-length viral RNA can be generated by in vitro transcription with T7 RNA polymerase. Following electroporation of transcribed RNA into cells, virus was recovered that exhibited similar in vitro growth kinetics and in vivo virulence and infection phenotypes in mice and mosquitoes, respectively.

This protocol describes the recovery of infectious Zika virus from a two-plasmid infectious cDNA clone.

Infectious cDNA clones allow for genetic manipulation of a virus, thus facilitating work on vaccines, pathogenesis, replication, transmission and viral ...

Isolation and Characterization of Extracellular Vesicles from Adult Schistosoma japonicum

Extracellular vesicles (EVs) are membranous vesicles released by a variety of cells into the extracellular microenvironment. EVs represent a population of heterogeneous vesicles, whose size range between 40 and 1,000 nm. Accumulated evidence indicated that EVs play important regulatory roles in pathogen-host interactions. A deep understanding of schistosome EVs should provide insights into the mechanisms underlying schistosome-host interactions, enabling development of novel strategies against schistosomiasis. Here, we aim to further study EVs functions in schistosomes by presenting a protocol for the isolation and characterization of EVs from adult Schistosoma japonicum (S. japonicum). EVs were isolated from in vitro culture medium using centrifugation combined with a commercial exosome isolation kit. The isolated S. japonicum EVs (SjEVs) typically possess a diameter of 100 - 400 nm, and are characterized by transmission electronic microscopy and western blotting. The usage of PKH67 dye-labeled SjEVs has demonstrated that SjEVs are internalized by the recipient cells. Overall, our protocol provides an alternative method for isolating EVs from adult schistosomes; the isolated SjEVs may be suitable for functional analysis.

Isolation and Characterization of Extracellular Vesicles from Adult Schistosoma japonicum

Here, we present a protocol to describe extracellular vesicles (EVs) isolation in in vitro cultured medium from adult Schistosoma japonicum.

Extracellular vesicles (EVs) are membranous vesicles released by a variety of cells into the extracellular microenvironment. EVs represent a population of ...

Quantitative Examination of Antibiotic Susceptibility of Neisseria gonorrhoeae Aggregates Using ATP-utilization Commercial Assays and Live/Dead Staining

The emergence of antibiotic resistant Neisseria gonorrhoeae (GC) is a worldwide health threat and highlights the need to identify individuals who fail treatment. This Gram-negative bacterium causes gonorrhea exclusively in humans. During infection, it is able to form aggregates and/or biofilms. The minimum inhibitory concentration (MIC) test is used for to determine susceptibility to antibiotics and to define appropriate treatment. However, the mechanism of the eradication in vivo and its relationship to laboratory results are not known. A method that examines how GC aggregation affects antibiotic susceptibility and shows the relationship between aggregate size and antibiotic susceptibility was developed. When GC aggregate, they are more resistant to antibiotic killing, with bacteria in the center surviving ceftriaxone treatment better than those in the periphery. The data indicate that N. gonorrhoeae aggregation can reduce its susceptibility to ceftriaxone, which is not reflected using the standard agar plate-based MIC methods. The method used in this study will allow researchers to test bacterial susceptibility under clinically relevant conditions.

Quantitative Examination of Antibiotic Susceptibility of Neisseria gonorrhoeae Aggregates Using ATP-utilization Commercial Assays and Live/Dead Staining

A simple ATP-measuring assay and live/dead staining method were used to quantify and visualize Neisseria gonorrhoeae survival after treatment with ceftriaxone. This protocol can be extended to examine the antimicrobial effects of any antibiotic and can be used to define the minimal inhibitory concentration of antibiotics in bacterial biofilms.

The emergence of antibiotic resistant Neisseria gonorrhoeae (GC) is a worldwide health threat and highlights the need to identify individuals who fail ...

Isolation of Salivary Epithelial Cells from Human Salivary Glands for In Vitro Growth as Salispheres or Monolayers

The salivary glands are a site of significant interest for researchers interested in multiple aspects of human disease. One goal of researchers is to restore function of glands damaged by radiation therapies or due to pathologies associated with Sj&#246;gren's syndrome. A second goal of researchers is to define the mechanisms by which viruses replicate within glandular tissue where they can then gain access to salivary fluids important for horizontal transmission. These goals highlight the need for a robust and accessible in vitro salivary gland model that can be utilized by researchers interested in the above mentioned as well as related research areas. Here we discuss a simple protocol to isolate epithelial cells from human salivary glands and propagate them in vitro. Our protocol can be further optimized to meet the needs of individual studies. Briefly, salivary tissue is mechanically and enzymatically separated to isolate single cells or small clusters of cells. Selection for epithelial cells occurs by plating onto a basement membrane matrix in the presence of media optimized to promote epithelial cell growth. These resulting cultures can be maintained as three-dimensional clusters, termed "salispheres", or grown as a monolayer on treated plastic tissue culture dishes. This protocol results in the outgrowth of a heterogenous population of mainly epithelial cells that can be propagated for 5-8 passages (15-20 population doublings) before undergoing cellular senescence.

We present a method for isolating and cultivating primary human salivary gland-derived epithelial cells. These cells exhibit gene expression patterns consistent with them being of salivary epithelial origin and can be grown as salispheres on basement membrane matrices derived from Engelbreth-Holm-Swarm tumor cells or as monolayers on treated culture dishes.

The salivary glands are a site of significant interest for researchers interested in multiple aspects of human disease. One goal of researchers is to ...