Name: metal-limited growth of neisseria gonorrhoeae for characterization of metal-responsive genes and metal acquisition from host ligands
Uploaded: 2020-03-04T14:30:00
Description: 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

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), thiam...

<ol>
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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+zinc-responsive+regulon+of+Neisseria+meningitidis+comprises+17+genes+under+control+of+a+Zur+element.">The zinc-responsive regulon of Neisseria meningitidis comprises 17 genes under control of a Zur element.</a> Journal of Bacteriology. 194 (23), 6594-6603 (2012).</li><li>Andreini, C., Banci, L., Bertini, I., Rosato, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Zinc+through+the+three+domains+of+life.">Zinc through the three domains of life.</a> Journal of Proteome Research. 5 (11), 3173-3178 (2006).</li><li>Frawley, E. R., Fang, F. 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P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Nutritional+immunity+beyond+iron:+a+role+for+manganese+and+zinc.">Nutritional immunity beyond iron: a role for manganese and zinc.</a> Current Opinion in Chemical Biology. 14 (2), 218-224 (2010).</li><li>Seib, K. 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=Defenses+against+oxidative+stress+in+Neisseria+gonorrhoeae:+a+system+tailored+for+a+challenging+environment.">Defenses against oxidative stress in Neisseria gonorrhoeae: a system tailored for a challenging environment.</a> Microbiology and Molecular Biology Reviews. 70 (2), 344-361 (2006).</li><li>Wu, H. 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=PerR+controls+Mn-dependent+resistance+to+oxidative+stress+in+Neisseria+gonorrhoeae.">PerR controls Mn-dependent resistance to oxidative stress in Neisseria gonorrhoeae.</a> Molecular Microbiology. 60 (2), 401-416 (2006).</li><li>Rayner, M. H., Suzuki, K. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+simple+and+effective+method+for+the+removal+of+trace+metal+cations+from+a+mammalian+culture+medium+supplemented+with+10%+fetal+calf+serum.">A simple and effective method for the removal of trace metal cations from a mammalian culture medium supplemented with 10% fetal calf serum.</a> Biometals. 8 (3), 188-192 (1995).</li><li>Kellogg, D. S., Peacock, W. L., Deacon, W. E., Brown, L., Pirkle, D. 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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=Iron+piracy:+acquisition+of+transferrin-bound+iron+by+bacterial+pathogens.">Iron piracy: acquisition of transferrin-bound iron by bacterial pathogens.</a> Molecular Microbiology. 14 (5), 843-850 (1994).</li><li>Quillin, S. J., Seifert, H. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neisseria+gonorrhoeae+host+adaptation+and+pathogenesis.">Neisseria gonorrhoeae host adaptation and pathogenesis.</a> Nature Reviews Microbiology. 16 (4), 226-240 (2018).</li><li>Platt, D. 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>

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 ...

This protocol enables researchers to analyze bacteria grown under conditions that more closely mimic those found in a mammalian host, which are metal limited through a process called nutritional immunity. This protocol facilitates generation of consistent reproducible bacterial growth under metal restricted conditions and can be adapted for growth of many different types of bacteria. Two days before beginning the experiment, streak gonococcide from freezer stocks onto GC medium plates for a no more than 24 hour incubation at 37 degrees Celsius.14 to 16 hours before the growth experiment, steak single colonies onto fresh GC medium plates. On the day of the experiment, add five to 10 milliliters CDM to an acid-washed 125 milliliter baffled side-arm flask and use this medium to blank a Klett colorimeter. Use a sterile cotton tipped swab to inoculate 20 Klett units of CDM from healthy single colonies.Generation of a healthy Neisseria gonorrhoeae inoculum for subculture into the 96 well plates for liquid growth assays is critical to the success of all downstream experiments. Incubate the cultures at 37 degrees Celsius in 250 revolutions per minute for one to two hours until approximate a one mass doubling before diluting the cultures with a sufficient volume of CDM to reach half of the initial culture density. Then, return the culture to the shaking incubator.To prepare metal loaded proteins, add phoration solution to a 10 milligram per milliliter human transferrin solution to achieve 30%iron saturation. Add the metal of interest to the protein solution at a 15%molar ratio to obtain a 25%saturation. Use a syringe to add the metal loaded protein to a dialysis cassette and dialyze against two liters of dialysis buffer for four hours at room temperature.Then, move the cassette to two liters of four degrees Celsius dialysis buffer and dialyze overnight at four degrees Celsius to remove any unbound metals. During the mass doubling, slightly dilute 10X pre-mixes of interest with PBS. Pre-treat the wells of a 96 well microplate with 15 microliters of the diluted pre-mixes and add 10 microliters of 10X pre-mix and 90 microliters of CDM to each of three wells for the blank controls.Once the cultures in the side-arm flask have doubled, add 100 microliters of each culture to a unused well in the microplate and measure the optical density at 600 nanometers. After calculating the correct amount of dilution required to bring the cultures to an optical density at 600 nanometers of 0.02, dilute the cultures with CDM in small culture tubes and add a sufficient volume to dilute the 10X pre-mixes to 1X in the plate. Then, place the plate in a plate reader for eight to 12 hours with shaking to obtain optical density at 600 nanometers readings at the desired experimental intervals.After the mass doubling incubation, back dilute the cultures with three volumes of CDM and add the metal treatments of interest. Then, incubate the cultures at 37 degrees Celsius with shaking for four hours. Shortly before the four hour mark, cut three pieces of filter paper and a piece of nitrocellulose to the approximate size needed to fit into a dot-blot apparatus.Pre-soak the nitrocellulose in deionized water for about 10 minutes before assembling the apparatus with filter paper below the nitrocellulose. At the end of the incubation, record the cell densities and standardize the densities to an appropriate final density. Then, pipette the cell cultures onto the nitrocellulose.When the filter paper has absorbed all of the liquid disassemble the apparatus and allow the blot to dry. Block the nitrocellulose membrane for one hour with an appropriate blocking solution and reassemble the dot-blot apparatus, replacing the filter paper with paraffin film to create a leak proof seal under the nitrocellulose. Dilute the metal binding ligand of interest to 0.2 micromolar in blocker and probe the cells for one hour with moderate shaking.At the end of the incubation, siphon off the liquid with a vacuum, wash the blot, and follow standard immunological procedures to develop the signal. Western blot analysis of protein production in variable metal concentrations reveals up regulation of the zinc responsive outer membrane tdfJ in response to zinc chelation by TPEN. TdfJ is essentially undetectable when zinc is added back to the medium.N.gonorrhoeae grows in the presence of zinc loaded calprotectin, which requires the action of the zinc responsive tdfH. When no usable zinc source is available, growth is restricted. Similar results are observed in the presence of zinc S100 A7, which can serve as a sole zinc source when the outer membrane transporter, tdfJ, is produced.Gonococcide grown in CDM are able to bind calprotectin when producing tdfH and as a result of zinc scarcity. Increased binding of transferrin is also observed in cultures grown in CDM, indicative of higher levels of protein expression due to the more iron depleted nature of CDM compared to chelated GC broth. This protocol's been optimized for use with the described water sources and additional titration with other chelators or metals will be needed if other types of water are used.For additional analysis, one could perform quantitative metal uptake assays or quantitative RTPCR for gene expression studies under metal deplete or metal replete conditions.

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

Research

JoVE Journal

Immunology and Infection

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 ...

Use of Image Cytometry for Quantification of Pathogenic Fungi in Association with Host Cells

Studies of the cellular pathogenesis mechanisms of pathogenic yeasts such as Candida albicans, Histoplasma capsulatum, and Cryptococcus neoformans commonly employ infection of mammalian hosts or host cells (i.e. macrophages) followed by yeast quantification using colony forming unit analysis or flow cytometry. While colony forming unit enumeration has been the most commonly used method in the field, this technique has disadvantages and limitations, including slow growth of some fungal species on solid media and low and/or variable plating efficiencies, which is of particular concern when comparing growth of wild-type and mutant strains. Flow cytometry can provide rapid quantitative information regarding yeast viability, however, adoption of flow cytometric detection for pathogenic yeasts has been limited for a number of practical reasons including its high cost and biosafety considerations. Here, we demonstrate an image-based cytometric methodology using the Cellometer Vision (Nexcelom Bioscience, LLC) for the quantification of viable pathogenic yeasts in co-culture with macrophages. Our studies focus on detection of two human fungal pathogens: Histoplasma capsulatum and Candida albicans. H. capsulatum colonizes alveolar macrophages by replicating within the macrophage phagosome, and here, we quantitatively assess the growth of H. capsulatum yeasts in RAW 264.7 macrophages using acridine orange/propidium iodide staining in combination with image cytometry. Our method faithfully recapitulates growth trends as measured by traditional colony forming unit enumeration, but with significantly increased sensitivity. Additionally, we directly assess infection of live macrophages with a GFP-expressing strain of C. albicans. Our methodology offers a rapid, accurate, and economical means for detection and quantification of important human fungal pathogens in association with host cells.

Here, we demonstrate how image cytometry can be used for quantification of pathogenic fungi in association with host cells in culture. This technique can be used as an alternative to CFU enumeration.

Studies of the cellular pathogenesis mechanisms of pathogenic yeasts such as Candida albicans, Histoplasma capsulatum, and Cryptococcus neoformans ...

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 ...