The authors thank Dara Frank and Carrie Harwood for their generous contribution of strains. University of Wisconsin Milwaukee Research Foundation holds patent no. 8,354,451; with claims broadly directed to treating or inhibiting the progression of infection of a microorganism in a patient by administering a 4-hydroxyphenylpyruvate dioxygenase-inhibiting compound such as 2-[2-nitro-4-(trifluoromethyl)benzoyl]-1,3-cyclohexanedione (NTBC). Inventors are Graham Moran and Pang He. This research was supported by the National Institutes of Health (R00-GM083147). The University of Washington P. aeruginosa transposon mutant library is supported by NIH P30 DK089507.

1. Preparation of Culture Media, Antibiotics, and 2-[2-nitro-4-(trifluoromethyl)benzoyl]-1,3-cyclohexanedione (NTBC)
<ol>
	<li>Make LB broth (1% tryptone, 0.5% yeast extract, 0.25% NaCl in H2O) and aliquot into appropriate volumes. Sterilize by autoclave. Store at room temperature.</li>
	<li>Make 100 ml LB agar (1% tryptone, 0.5% yeast extract, 0.25% NaCl, 1.5% agar in H2O) in 250 ml flasks. Sterilize by autoclave and store at room temperature. Ensure that the agar is melted before pouring into plates. 
	NOTE: Flasks containing 100 ml of LB agar will yield 4 plates. The amount of LB agar can be altered to correspond to the number of plates needed for the assay.</li>
	<li>Make PBS (137 mM NaCl, 2.7 mM KCl, 10 mM Na2HPO4, 2 mM KH2PO4)16. Sterilize by autoclave or filtration and store at room temperature.</li>
	<li>Prepare the antibiotic stock solutions for gentamicin, kanamycin, and tobramycin.
	<ol>
		<li>Prepare appropriate antibiotic stock concentrations for P. aeruginosa strains containing 100 mg/ml gentamicin, 30 mg/ml kanamycin, and 10 mg/ml tobramycin. Dissolve the antibiotics in water, filter sterilize (0.2 &#181;m), and store at 4 &#176;C. Alter the antibiotics and concentrations depending on the bacterium studied.</li>
	</ol>
	</li>
	<li>Prepare the NTBC stock solutions. Dissolve 10 mg of NTBC in 400 &#181;l of DMSO. This yields a concentration of 75.9 mM NTBC. Store NTBC stock solutions at -20 &#176;C. Thaw solutions at room temperature as needed. 
	NOTE: Different sources of NTBC have differences in solubility. Determine the appropriate vehicle in which to dissolve the NTBC based on the manufacturer&#8217;s recommendations and adjust Step 1.5 accordingly.</li>
</ol>
2. NTBC Titrations of Bacterial Strains
<ol>
	<li>Set up overnight cultures of the strains to be tested. Add 2 ml LB broth to 16 x 150 mm test tubes (one per strain) and inoculate with 1 isolated colony from each strain. Incubate overnight at 37 &#176;C with aeration on a tissue culture rotator in an air incubator.</li>
	<li>The following day, prepare titrations of NTBC in LB broth. Use an initial range from 0 to 900 &#181;M NTBC since different strains have differences in sensitivity to NTBC.
	<ol>
		<li>Add 1 ml LB broth to 4 to 5 test tubes (16 x 150 mm) per strain.</li>
		<li>Add the NTBC stock solution (75.9 mM) to the test tubes (16 x 150 mm) in a range of concentrations. See Table 1 for NTBC concentrations and corresponding stock volumes to add to 1 ml of LB broth.</li>
	</ol>
	</li>
	<li>Measure the OD600 of the overnight cultures. Wash cultures before taking OD600 readings to eliminate pyomelanin present in the media.
	<ol>
		<li>Wash the cultures by centrifuging 1 ml of culture in a microcentrifuge at 16,000 x g for 2 min. Remove the supernatant and any loosely pelleted cells with a micropipettor and resuspend the solid cell pellet in 1 ml LB.</li>
	</ol>
	</li>
	<li>Inoculate titration tubes at OD600 0.05. Calculate the amount of washed culture needed to inoculate the tubes. 
	NOTE: Use the washed cultures for inoculations since pyomelanin should not be present.</li>
	<li>Incubate the titration tubes for approximately 24 hr&#160;at 37 &#176;C with aeration using a tissue culture rotator in an air incubator.</li>
	<li>Photograph the titration tubes and compare pigment production within and between strains to determine the amount of NTBC to use for MIC and oxidative stress assays. Use OD600 readings to determine the amount of pyomelanin in cell free culture supernatant and to determine cell density. 
	NOTE: The OD600 ratio of pyomelanin in culture supernatant to cells can be calculated to quantify differences in pyomelanin production after treatment with NTBC.</li>
</ol>
3. Antibiotic Minimum Inhibitory Concentration (MIC) Assay in 96-well Plates
<ol>
	<li>Set up overnight cultures of the strains to be tested in LB with and without NTBC. 
	NOTE: This protocol is described using the representative level of 300 &#181;M NTBC. The appropriate level of NTBC to be used is determined in Step 2.6.
	<ol>
		<li>Add 300 &#181;M NTBC to 2 ml LB. Add an equivalent volume of vehicle (DMSO) to 2 ml LB for the no NTBC condition.</li>
		<li>Using sterile toothpicks, inoculate tubes with one isolated colony of bacteria. There will be one culture with NTBC and one culture without NTBC for each strain. Incubate overnight at 37 &#176;C with aeration using a tissue culture rotator in an air incubator.</li>
	</ol>
	</li>
	<li>The following day, make LB + NTBC and LB + DMSO master solutions for setting up the MIC assay. Add NTBC at a concentration of 600 &#181;M as this will be diluted two fold when inoculum is added, yielding a final concentration of 300 &#181;M.
	<ol>
		<li>To test one antibiotic for one strain, add 600 &#181;M NTBC to 2 ml LB and mix to make the NTBC master solution. Add an equivalent volume of vehicle (DMSO) to 2 ml LB and mix to make the no NTBC master solution. Use these solutions for creating antibiotic stock solutions as well as for setting up the dilution series in 96-well plates. 
		NOTE: The master solution formulations will yield extra solution to account for pipetting errors. The master solutions can be scaled up or down as required depending on the number of antibiotics and strains tested.</li>
	</ol>
	</li>
	<li>Prepare the antibiotic solutions in the LB + NTBC or LB + DMSO master solutions. 
	NOTE: The antibiotic concentration in these solutions should be double the final desired concentration. Enough solution should be made to transfer 100 &#181;l to four wells in a 96-well plate.
	<ol>
		<li>Prepare gentamicin +/- NTBC stock solution at 64 &#181;g/ml. To make this solution, add 0.288 &#181;l of gentamicin stock (100 mg/ml) to 450 &#181;l LB + NTBC or LB + DMSO master solution. 
		NOTE: The maximum concentration of gentamicin for P. aeruginosa PAO1 is 32 &#181;g/ml.</li>
		<li>Make the kanamycin +/- NTBC stock solution at 256 &#181;g/ml. To make this solution, add 3.84 &#181;l of kanamycin stock (30 mg/ml) to 450 &#181;l LB + NTBC or LB + DMSO master solutions. 
		NOTE: The maximum concentration of kanamycin for P. aeruginosa PAO1 is 128 &#181;g/ml.</li>
		<li>Prepare tobramycin +/- NTBC stock solution at 8 &#181;g/ml. To make this solution, add 0.36 &#181;l of tobramycin stock (10 mg/ml) to 450 &#181;l LB + NTBC or LB + DMSO master solutions. 
		NOTE: For P. aeruginosa PAO1, the maximum concentration of tobramycin is 4 &#181;g/ml. 
		NOTE: The antibiotics and concentrations can be adjusted for the bacteria to be tested.</li>
	</ol>
	</li>
	<li>Add 100 &#181;l of each 2x antibiotic solution to four wells in a 96-well plate. Place these solutions in row A. For example, gentamicin should be placed in A1 through A4, kanamycin should be placed in A5 through A8, and tobramycin should be placed in A9 through A12. See Figure 1A for a diagram of a 96-well plate set up. 
	NOTE: Multiple antibiotics can be tested in one plate, but only one strain should be tested per plate to eliminate the potential for cross-contamination from other strains.</li>
	<li>Add 50 &#181;l of the LB + NTBC or LB + DMSO master solution to rows B through H of the 96-well plate. Ensure that one plate is LB + NTBC and one plate is LB + DMSO. See Figure 1A.
	<ol>
		<li>Use LB + NTBC for the antibiotics in LB + NTBC. Use LB + DMSO for the antibiotics in LB + DMSO.</li>
	</ol>
	</li>
	<li>Using a micropipettor perform two fold serial dilutions of the antibiotics by transferring 50 &#181;l of the solution from row A to row B. Mix the solution, change the pipet tips, and transfer 50 &#181;l of the solution from row B to row C. Repeat for the remaining rows. After diluting row G, remove 50 &#181;l of the solution from that row and discard. Use row H as a no antibiotic control for bacterial growth. See Figure 1B. 
	NOTE: Each well in rows A through G now contains 50 &#181;l of antibiotic in LB + NTBC or LB + DMSO at 2X the final desired concentration. Row H contains LB + NTBC or LB + DMSO with no antibiotics.</li>
	<li>Measure the OD600 of the overnight cultures. Wash all cultures before taking OD600 readings to eliminate pyomelanin present in the media.
	<ol>
		<li>Wash the cultures by centrifuging 1 ml of culture in a microcentrifuge at 16,000 x g for 2 min. Remove the supernatant with a micropipettor and resuspend the cell pellet in 1 ml LB.</li>
	</ol>
	</li>
	<li>Dilute the overnight cultures to 2.75x105 CFU/ml in LB. 
	NOTE: Assume that one OD600 unit is the equivalent of 1x109 CFU/ml for P. aeruginosa. OD to CFU/ml conversions may be different in other bacteria.</li>
	<li>Add 50 &#181;l of the diluted bacterial culture to the appropriate well. 
	NOTE: Cultures grown in NTBC should be added to the wells containing NTBC and cultures grown in DMSO should be added to the wells containing DMSO. See Figure 1B.
	<ol>
		<li>Add bacteria to three wells for each strain and antibiotic concentration. Add 50 &#181;l of LB to the fourth well to act as a control for bacterial contamination. See Figure 1B.</li>
		<li>Use a multi-channel micropipettor to inoculate the wells. Ensure that pipet tips are near the bottom of the wells when adding inoculum to prevent contamination of neighboring wells. 
		NOTE: Adding bacterial culture to the wells will dilute the antibiotic and NTBC concentrations two fold.</li>
	</ol>
	</li>
	<li>Cover the 96-well plates with parafilm and incubate approximately 24 hr&#160;at 37 &#176;C. Incubate 96-well plates statically, in an air incubator.</li>
	<li>Examine the plates for bacterial growth in the wells. The MIC is the lowest concentration of antibiotic in which no bacterial growth is seen for all three replicates of each strain.
	<ol>
		<li>Visually examine the plate for growth or read using a plate reader set to OD600.</li>
	</ol>
	</li>
</ol>
4. Spot Plate Assay for Oxidative Stress Response
<ol>
	<li>Set up overnight cultures of the strains to be tested in LB with and without NTBC as described in step 3.1.</li>
	<li>The next day, prepare LB agar plates containing H2O2 as an oxidative stressor. A range of H2O2 concentrations from 0 to 1 mM is a good starting point.
	<ol>
		<li>Melt the LB agar flasks. Cool media to approximately 50 &#176;C at room temperature.</li>
		<li>Add H2O2 directly to the cooled media at the desired concentrations. Swirl flasks to mix. See Table 2 for concentrations of H2O2 and volumes of concentrated H2O2 to add. These values are based on 100 ml of LB agar.</li>
		<li>Pour plates immediately after adding H2O2 and flame the surface to remove bubbles. The yield is 4 plates per 100 ml of LB agar. Mark the plates with the H2O2 concentration.</li>
		<li>Place the plates uncovered in a biological flow hood with the fan running for 30 min to remove excess moisture from the plates. 
		NOTE: Use the plates the same day they are prepared. Failure to do so may result in inconsistent data. 
		NOTE: Oxidative stressors such as paraquat can be substituted for H2O2 in this assay. The concentrations used for other oxidative stressors may be different than those used for H2O2.</li>
	</ol>
	</li>
	<li>Wash and measure the OD600 of the overnight cultures as described in step 3.7.</li>
	<li>Normalize the OD600 of all the overnight cultures to the lowest value for the set of strains being tested. P. aeruginosa generally has an OD600 of approximately 2.5 when grown overnight in LB + NTBC or LB + DMSO.
	<ol>
		<li>Determine the volume of culture needed to dilute the culture to the lowest OD600 in a total volume of 1 ml. For example, if a culture has an OD600 of 3 and the lowest OD600 for the set of strains is 2.5, perform the following calculation: (2.5)(1 ml) = (3)(x). x = 0.833 ml. 0.833 ml of culture will be placed in a microfuge tube.</li>
		<li>Calculate the amount of LB + NTBC (300 &#181;M) or LB + DMSO needed to bring the culture volume to 1 ml. For the example in step 4.4.1, the amount of LB + NTBC or LB + DMSO added to the culture would be 0.167 ml (1 ml total volume &#8211; 0.833 ml culture). Make stock solutions of LB + NTBC and LB + DMSO to use for these dilutions based on the volume needed for diluting all strains.</li>
		<li>Mix the culture and LB + NTBC or LB + DMSO by vortexing.</li>
	</ol>
	</li>
	<li>To maintain cultures in a constant concentration of NTBC or DMSO, perform ten fold serial dilutions of the normalized overnight cultures in PBS + NTBC or PBS + DMSO.
	<ol>
		<li>Make stock solutions of PBS + NTBC and PBS + DMSO. For one set of dilutions for one strain, mix 300 &#181;M NTBC or an equivalent volume of DMSO with PBS to yield a total volume of 720 &#181;l. Scale these stocks up or down depending on how many strains are tested.</li>
		<li>Label microfuge tubes for 10-1 through 10-7 serial dilutions. Add 90 &#181;l of PBS + NTBC or PBS + DMSO to the appropriate tubes. Use PBS + NTBC for strains grown in LB + NTBC and use PBS + DMSO for strains grown in LB + DMSO.</li>
		<li>Add 10 &#181;l of culture to the appropriate 10-1 dilution tube. Mix by vortexing and transfer 10 &#181;l of the 10-1 dilution to the 10-2 dilution tube. Repeat until all dilutions have been performed. Change pipet tips between dilutions.</li>
	</ol>
	</li>
	<li>Spot 5 &#181;l of the 10-3 through 10-7 dilutions on LB + H2O2 plates in duplicate for each strain. Use one pipet tip if spots are plated from most dilute to least dilute (10-7 to 10-3). Do not tip or tilt the plate until the liquid has dried into the plate.</li>
	<li>Incubate the plates for 24-48 hr&#160;at 37 &#176;C (air incubator), depending on the strain. 
	NOTE: P. aeruginosa PAO1 will have good sized colonies on LB after 24 hr&#160;of incubation. Incubate strains until they have colonies approximately the same size as PAO1.</li>
	<li>Photograph the plates using a CCD camera above a transluminator. Optionally, edit the photos for contrast and crop to the same size. Count the number of colonies in each spot to determine changes in sensitivity to oxidative stress.</li>
</ol>

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The authors declare that they have no competing financial interests.

The NTBC titration method described in this protocol will allow the user to determine if NTBC can reduce or eliminate pyomelanin production in bacteria, and determine the concentration of NTBC required. The most critical step in the NTBC titration assay is determining the range of NTBC concentrations to use in the assay. Different strains of P. aeruginosa have different sensitivities to NTBC, and laboratory strains may be more sensitive to NTBC than clinical isolates12 (Figure 2). The...

Pseudomonas aeruginosa is a Gram negative bacterium that produces a variety of pigments including pyomelanin, a red-brown pigment that helps provide protection from oxidative stress1-4 and binds a variety of compounds, including aminoglycoside antibiotics5-7. Pyomelanin production is caused by a defect in the tyrosine catabolism pathway4,8, either through deletions or mutations of the gene encoding homogentisate 1,2-dioxygenase (HmgA)1,9 or through imbalances in the various enzymes in the pathway10. Homogentisate accumulates due to inactivation of HmgA, and is secreted and oxidized to form pyomelanin11. Production of pyomelanin can be abolished or reduced in a dose dependent manner through treatment with the herbicide 2-[2-nitro-4-(trifluoromethyl)benzoyl]-1,3-cyclohexanedione (NTBC)12, which inhibits 4-hydroxyphenylpyruvate dioxygenase (Hpd) in the tyrosine catabolism pathway13. Hpd is required for the formation of homogentisate, and therefore pyomelanin11. 
We describe in detail three techniques that were important in our studies of NTBC treatment of pyomelanin producing strains of P. aeruginosa. These techniques include titration of NTBC to determine the concentrations that will abolish or reduce pyomelanin production in laboratory and clinical pyomelanin producing strains, determination of the minimum inhibitory concentration (MIC) of antibiotics when bacteria are treated with NTBC, and the resulting sensitivity to oxidative stress with NTBC treatment.
The titration assay we developed serves two purposes. First, the assay will allow the user to determine if NTBC can abolish or reduce pyomelanin production in the bacterium being studied and at which concentrations. This will allow the user to determine sensitivity to NTBC, since different strains of bacteria may have different sensitivities to this compound, as observed in P. aeruginosa12. Second, the NTBC titration assay will allow the user to determine the appropriate concentration of NTBC to use in subsequent assays, such as antibiotic MIC and oxidative stress response assays, if the goal is to abolish or reduce pyomelanin production and determine the effects of pigment reduction. 
The titration assay works because a visible difference in pyomelanin production can be seen in strains treated with NTBC and the differences in pyomelanin production are dose dependent12. Additionally, this technique can be applied to the study of other compounds that may eliminate or enhance pigment production in bacteria.
Antibiotic MICs are used to determine the sensitivity of bacteria to antibiotics. There are several methods to determine MICs, including agar dilution plates and broth dilutions14. Broth dilutions can be performed in small test tubes or in a 96-well microtiter plate. The microtiter plate method of MIC determination described herein will allow the user to test a wide range of antibiotics using a minimum of resources. The assay provides reproducibility as well as flexibility in the number of antibiotics and strains tested by this method. Additionally, with the incorporation of NTBC in the assay, the user can determine if elimination or reduction of pyomelanin production alters antibiotic sensitivity in bacteria that produce pyomelanin.
Bacterial response to oxidative stress can be tested in several ways. The most common methods described are either viable counts of bacteria subjected to oxidative stress for a period of time1, or oxidative stress disc diffusion assays15. These methods tend to use high concentrations of oxidative stressors to examine the effects of oxidative stress in bacteria and results can be quite variable between biological replicates. The viable count assay also tends to use more agar plates than the other methods. The spot plate assay we describe uses low concentrations of H2O2 and allows the user to test the oxidative stress response of multiple strains using a minimum of plates. The assay is also consistently reproducible between technical and biological replicates. As pyomelanin is involved in resistance to oxidative stress, the incorporation of NTBC in the assay allows the user to determine the effects of elimination of pyomelanin production on oxidative stress resistance.

<table border="0" cellpadding="0" cellspacing="0">
	<tbody>
		<tr>
			<td>2-[2-nitro-4-(trifluoromethyl)benzoyl]-1,3-cyclohexanedione (NTBC)</td>
			<td>Sigma-Aldrich</td>
			<td>SML0269-50mg</td>
			<td>Also called nitisinone.&#160; Soluble in DMSO.</td>
		</tr>
		<tr>
			<td>H2O2</td>
			<td>Sigma-Aldrich</td>
			<td>216763-100ML</td>
			<td>30 wt. % in H2O.&#160; Stabilized.</td>
		</tr>
		<tr>
			<td>Gentamycin</td>
			<td>Gold Bio</td>
			<td>G-400-100</td>
			<td>Soluble in H2O.&#160; Filter sterilize.</td>
		</tr>
		<tr>
			<td>Kanamycin</td>
			<td>Fisher Scientific</td>
			<td>BP906-5</td>
			<td>Soluble in H2O.&#160; Filter sterilize.</td>
		</tr>
		<tr>
			<td>Tobramycin</td>
			<td>Sigma-Aldrich</td>
			<td>T4014-100MG</td>
			<td>Soluble in H2O.&#160; Filter sterilize.</td>
		</tr>
	</tbody>
</table>

methods to inhibit bacterial pyomelanin production and determine the corresponding increase in sensitivity to oxidative stress

Pyomelanin is an extracellular red-brown pigment produced by several bacterial and fungal species. This pigment is derived from the tyrosine catabolism pathway and contributes to increased oxidative stress resistance. Pyomelanin production in Pseudomonas aeruginosa is reduced in a dose dependent manner through treatment with 2-[2-nitro-4-(trifluoromethyl)benzoyl]-1,3-cyclohexanedione (NTBC). We describe a titration method using multiple concentrations of NTBC to determine the concentration of drug that will reduce or abolish pyomelanin production in bacteria. The titration method has an easily quantifiable outcome, a visible reduction in pigment production with increasing drug concentrations. We also describe a microtiter plate method to assay antibiotic minimum inhibitory concentration (MIC) in bacteria. This method uses a minimum of resources and can easily be scaled up to test multiple antibiotics in one microtiter plate for one strain of bacteria. The MIC assay can be adapted to test the affects of non-antibiotic compounds on bacterial growth at specific concentrations. Finally, we describe a method for testing bacterial sensitivity to oxidative stress by incorporating H2O2 into agar plates and spotting multiple dilutions of bacteria onto the plates. Sensitivity to oxidative stress is indicated by reductions in colony number and size for the different dilutions on plates containing H2O2 compared to a no H2O2 control. The oxidative stress spot plate assay uses a minimum of resources and low concentrations of H2O2. Importantly, it also has good reproducibility. This spot plate assay could be adapted to test bacterial sensitivity to various compounds by incorporating the compounds in agar plates and characterizing the resulting bacterial growth.

NTBC titrations
The NTBC titrations were used to determine if NTBC was able to reduce pyomelanin production in P. aeruginosa, and also identify the concentration of NTBC that eliminates or reduces pyomelanin production for use in additional assays. There may be variations in the levels of pyomelanin produced in different replications, but general trends remain constant. The NTBC titration assay could also be modified to test other compounds that may affect pigment production in other ...

Watch this Scientific Journal Video about Methods to Inhibit Bacterial Pyomelanin Production and Determine the Corresponding Increase in Sensitivity to Oxidative Stress at JoVE.com

Methods to Inhibit Bacterial Pyomelanin Production and Determine the Corresponding Increase in Sensitivity to Oxidative Stress

Bacterial pyomelanin production results in increased resistance to oxidative stress and virulence. We report on techniques that can be used to determine inhibition of pyomelanin production and assay the resulting increase in sensitivity to oxidative stress in bacteria, as well as determine antibiotic minimum inhibitory concentration (MIC).

Pyomelanin is an extracellular red-brown pigment produced by several bacterial and fungal species. This pigment is derived from the tyrosine catabolism ...

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Research

JoVE Journal

Immunology and Infection

8.1K Views.  University of Wisconsin Milwaukee. Bacterial pyomelanin production results in increased resistance to oxidative stress and virulence. We report on techniques that can be used to determine inhibition of pyomelanin production and assay the resulting increase in sensitivity to oxidative stress in bacteria, as well as determine antibiotic minimum inhibitory concentration (MIC).

Video: Methods to Inhibit Bacterial Pyomelanin Production and Determine the Corresponding Increase in Sensitivity to Oxidative Stress

Introducing Shear Stress in the Study of Bacterial Adhesion

During bacterial infections a sequence of interactions occur between the pathogen and its host. Bacterial adhesion to the host cell surface is often the initial and determining step of the pathogenesis. Although experimentally adhesion is mostly studied in static conditions adhesion actually takes place in the presence of flowing liquid. First encounters between bacteria and their host often occur at the mucosal level, mouth, lung, gut, eye, etc. where mucus flows along the surface of epithelial cells. Later in infection, pathogens occasionally access the blood circulation causing life-threatening illnesses such as septicemia, sepsis and meningitis. A defining feature of these infections is the ability of these pathogens to interact with endothelial cells in presence of circulating blood. The presence of flowing liquid, mucus or blood for instance, determines adhesion because it generates a mechanical force on the pathogen. To characterize the effect of flowing liquid one usually refers to the notion of shear stress, which is the tangential force exerted per unit area by a fluid moving near a stationary wall, expressed in dynes/cm2. Intensities of shear stress vary widely according to the different vessels type, size, organ, location etc. (0-100 dynes/cm2). Circulation in capillaries can reach very low shear stress values and even temporarily stop during periods ranging between a few seconds to several minutes 1. On the other end of the spectrum shear stress in arterioles can reach 100 dynes/cm2 2. The impact of shear stress on different biological processes has been clearly demonstrated as for instance during the interaction of leukocytes with the endothelium 3. To take into account this mechanical parameter in the process of bacterial adhesion we took advantage of an experimental procedure based on the use of a disposable flow chamber 4. Host cells are grown in the flow chamber and fluorescent bacteria are introduced in the flow controlled by a syringe pump. We initially focused our investigations on the bacterial pathogen Neisseria meningitidis, a Gram-negative bacterium responsible for septicemia and meningitis. The procedure described here allowed us to study the impact of shear stress on the ability of the bacteria to: adhere to cells 1, to proliferate on the cell surface 5and to detach to colonize new sites 6 (Figure 1). Complementary technical information can be found in reference 7. Shear stress values presented here were chosen based on our previous experience1 and to represent values found in the literature. The protocol should be applicable to a wide range of pathogens with specific adjustments depending on the objectives of the study.

During the infection process, a key step is the adhesion of pathogens with host cells. In most instances this adhesion step occurs in the presence of mechanical stress generated by flowing liquid. We describe a technique that introduces shear stress as an important parameter in the study of bacterial adhesion.

During bacterial infections a sequence of interactions occur between the pathogen and its host. Bacterial adhesion to the host cell surface is often the ...

Using the Overlay Assay to Qualitatively Measure Bacterial Production of and Sensitivity to Pneumococcal Bacteriocins

Streptococcus pneumoniae colonizes the highly diverse polymicrobial community of the nasopharynx where it must compete with resident organisms. We have shown that bacterially produced antimicrobial peptides (bacteriocins) dictate the outcome of these competitive interactions. All fully-sequenced pneumococcal strains harbor a bacteriocin-like peptide (blp) locus. The blp locus encodes for a range of diverse bacteriocins and all of the highly conserved components needed for their regulation, processing, and secretion. The diversity of the bacteriocins found in the bacteriocin immunity region (BIR) of the locus is a major contributor of pneumococcal competition. Along with the bacteriocins, immunity genes are found in the BIR and are needed to protect the producer cell from the effects of its own bacteriocin. The overlay assay is a quick method for examining a large number of strains for competitive interactions mediated by bacteriocins. The overlay assay also allows for the characterization of bacteriocin-specific immunity, and detection of secreted quorum sensing peptides. The assay is performed by pre-inoculating an agar plate with a strain to be tested for bacteriocin production followed by application of a soft agar overlay containing a strain to be tested for bacteriocin sensitivity. A zone of clearance surrounding the stab indicates that the overlay strain is sensitive to the bacteriocins produced by the pre-inoculated strain. If no zone of clearance is observed, either the overlay strain is immune to the bacteriocins being produced or the pre-inoculated strain does not produce bacteriocins. To determine if the blp locus is functional in a given strain, the overlay assay can be adapted to evaluate for peptide pheromone secretion by the pre-inoculated strain. In this case, a series of four lacZ-reporter strains with different pheromone specificity are used in the overlay.

Competition in Streptococcus pneumoniae is mediated by bacteriocins, small antimicrobial peptides with inhibitory activity towards pneumococcus and other related species.&#160; Here we describe an optimized bacterial overlay assay that allows for the characterization of bacteriocin activity and inhibitory spectrum, bacteriocin-specific immunity, and detection of secreted quorum sensing peptides.

Streptococcus pneumoniae colonizes the highly diverse polymicrobial community of the nasopharynx where it must compete with resident organisms.  We have ...

Methods to Increase the Sensitivity of High Resolution Melting Single Nucleotide Polymorphism Genotyping in Malaria

Despite decades of eradication efforts, malaria remains a global burden. Recent renewed interest in regional elimination and global eradication has been accompanied by increased genomic information about Plasmodium parasite species responsible for malaria, including characteristics of geographical populations as well as variations associated with reduced susceptibility to anti-malarial drugs.
One common genetic variation, single-nucleotide polymorphisms (SNPs), offers attractive targets for parasite genotyping. These markers are useful not only for tracking drug resistance markers but also for tracking parasite populations using markers not under drug or other selective pressures.
SNP genotyping methods offer the ability to track drug resistance as well as to fingerprint individual parasites for population surveillance, particularly in response to malaria control efforts in regions nearing elimination status.
While informative SNPs have been identified that are agnostic to specific genotyping technologies, high-resolution melting (HRM) analysis is particularly suited to field-based studies. Compared to standard fluorescent-probe based methods that require individual SNPs in a single labeled probe and offer at best 10% sensitivity to detect SNPs in samples that contain multiple genomes (polygenomic), HRM offers 2-5% sensitivity. Modifications to HRM, such as blocked probes and asymmetric primer concentrations as well as optimization of amplification annealing temperatures to bias PCR towards amplification of the minor allele, further increase the sensitivity of HRM. While the sensitivity improvement depends on the specific assay, we have increased detection sensitivities to less than 1% of the minor allele.
In regions approaching malaria eradication, early detection of emerging or imported drug resistance is essential for prompt response. Similarly, the ability to detect polygenomic infections and differentiate imported parasite types from cryptic local reservoirs can inform control programs.
This manuscript describes modifications to high resolution melting technology that further increase its sensitivity to identify polygenomic infections in patient samples.

While high resolution melting analysis offers the ability to differentiate between single nucleotide polymorphisms in a heterogeneous population, mutant allele amplification bias can increase its ability to detect alleles present at relatively low percentages within a sample. This protocol describes improvements that improve the sensitivity of high resolution melting analysis.

Despite decades of eradication efforts, malaria remains a global burden. Recent renewed interest in regional elimination and global eradication has been ...

In Vitro and In Vivo Model to Study Bacterial Adhesion to the Vessel Wall Under Flow Conditions

In order to cause endovascular infections and infective endocarditis, bacteria need to be able to adhere to the vessel wall while being exposed to the shear stress of flowing blood.
To identify the bacterial and host factors that contribute to vascular adhesion of microorganisms, appropriate models that study these interactions under physiological shear conditions are needed. Here, we describe an in vitro flow chamber model that allows to investigate bacterial adhesion to different components of the extracellular matrix or to endothelial cells, and an intravital microscopy model that was developed to directly visualize the initial adhesion of bacteria to the splanchnic circulation in vivo. These methods can be used to identify the bacterial and host factors required for the adhesion of bacteria under flow. We illustrate the relevance of shear stress and the role of von Willebrand factor for the adhesion of Staphylococcus aureus using both the in vitro and in vivo model.

In Vitro and In Vivo Model to Study Bacterial Adhesion to the Vessel Wall Under Flow Conditions

To study the interaction of bacteria with the blood vessels under shear stress, a flow chamber and an in vivo mesenteric intravital microscopy model are described that allow to dissect the bacterial and host factors contributing to vascular adhesion.

In order to cause endovascular infections and infective endocarditis, bacteria need to be able to adhere to the vessel wall while being exposed to the ...

Neutrophil Isolation and Analysis to Determine their Role in Lymphoma Cell Sensitivity to Therapeutic Agents

Neutrophils are the most abundant (40% to 75%) type of white blood cells and among the first inflammatory cells to migrate towards the site of inflammation. They are key players in the innate immune system and play major roles in cancer biology. Neutrophils have been proposed as key mediators of malignant transformation, tumor progression, angiogenesis and in the modulation of the antitumor immunity; through their release of soluble factors or their interaction with tumor cells. To characterize the specific functions of neutrophils, a fast and reliable method is coveted for in vitro isolation of neutrophils from human blood. Here, a density gradient separation method is demonstrated to isolate neutrophils as well as mononuclear cells from the blood. The procedure consists of layering the density gradient solution such as Ficoll carefully above the diluted blood obtained from patients diagnosed with chronic lymphocytic leukemia (CLL), followed by centrifugation, isolation of mononuclear layer, separation of neutrophils from RBCsby dextran then lysis of residual erythrocytes. This method has been shown to isolate neutrophils &#8805; 90 % pure. To mimic the tumor microenvironment, 3-dimensional (3D) experiments were performed using basement membrane matrix such as Matrigel. Given the short half-life of neutrophils in vitro, 3D experiments with fresh human neutrophils cannot be performed. For this reason promyelocytic HL60 cells are differentiated along the granulocytic pathway using the differentiation inducers dimethyl sulfoxide (DMSO) and retinoic acid (RA). The aim of our experiments is to study the role of neutrophils on the sensitivity of lymphoma cells to anti-lymphoma agents. However these methods can be generalized to study the interactions of neutrophils or neutrophil-like cells with a large range of cell types in different situations.

Neutrophils are the most abundant type of white blood cells. The isolation of neutrophils from human blood by density gradient separation method and the differentiation of human promyelocytic (HL60) cells along the granulocytic pathway are described here; to test their role on sensitivity of lymphoma cells to anti-lymphoma agents.

Neutrophils are the most abundant (40% to 75%) type of white blood cells and among the first inflammatory cells to migrate towards the site of ...

A RAPID Method for Blood Processing to Increase the Yield of Plasma Peptide Levels in Human Blood

Research in the field of food intake regulation is gaining importance. This often includes the measurement of peptides regulating food intake. For the correct determination of a peptide&#39;s concentration, it should be stable during blood processing. However, this is not the case for several peptides which are quickly degraded by endogenous peptidases. Recently, we developed a blood processing method employing Reduced temperatures, Acidification, Protease inhibition, Isotopic exogenous controls and Dilution (RAPID) for the use in rats. Here, we have established this technique for the use in humans and investigated recovery, molecular form and circulating concentration of food intake regulatory hormones. The RAPID method significantly improved the recovery for 125I-labeled somatostatin-28 (+39%), glucagon-like peptide-1 (+35%), acyl ghrelin and glucagon (+32%), insulin and kisspeptin (+29%), nesfatin-1 (+28%), leptin (+21%) and peptide YY3-36 (+19%) compared to standard processing (EDTA blood on ice, p&#160;&#60;0.001). High performance liquid chromatography showed the elution of endogenous acyl ghrelin at the expected position after RAPID processing, while after standard processing 62% of acyl ghrelin were degraded resulting in an earlier peak likely representing desacyl ghrelin. After RAPID processing the acyl/desacyl ghrelin ratio in blood of normal weight subjects was 1:3 compared to 1:23 following standard processing (p&#160;= 0.03). Also endogenous kisspeptin levels were higher after RAPID compared to standard processing (+99%, p&#160;= 0.02). The RAPID blood processing method can be used in humans, yields higher peptide levels and allows for assessment of the correct molecular form.

The RAPID blood processing method can be used in humans and yields higher peptide levels as well as allows for assessment of the correct molecular form. Therefore, this method will be a valuable tool in peptide research.

Research in the field of food intake regulation is gaining importance. This often includes the measurement of peptides regulating food intake. For the ...

Overexpression and Purification of Human Cis-prenyltransferase in Escherichia coli

Prenyltransferases (PT) are a group of enzymes that catalyze chain elongation of allylic diphosphate using isopentenyl diphosphate (IPP) via multiple condensation reactions. DHDDS (dehydrodolichyl diphosphate synthase) is a eukaryotic long-chain cis-PT (forming cis double bonds from the condensation reaction) that catalyzes chain elongation of farnesyl diphosphate (FPP, an allylic diphosphate) via multiple condensations with isopentenyl diphosphate (IPP). DHDDS is of biomedical importance, as a non-conservative mutation (K42E) in the enzyme results in retinitis pigmentosa, ultimately leading to blindness. Therefore, the present protocol was developed in order to acquire large quantities of purified DHDDS, suitable for mechanistic studies. Here, the usage of protein fusion, optimized culture conditions and codon-optimization were used to allow the overexpression and purification of functionally active human DHDDS in E. coli. The described protocol is simple, cost-effective and time sparing. The homology of cis-PT among different species suggests that this protocol may be applied for other eukaryotic cis-PT as well, such as those involved in natural rubber synthesis.

Overexpression and Purification of Human Cis-prenyltransferase in Escherichia coli

A simple protocol for overexpression and purification of codon-optimized, human cis-prenyltransferase, under non-denaturing conditions, from Escherichia coli, is described, along with an enzymatic activity assay. This protocol can be generalized for production of other cis- prenyltransferase proteins in quantity and quality suitable for mechanistic studies.

Prenyltransferases (PT) are a group of enzymes that catalyze chain elongation of allylic diphosphate using isopentenyl diphosphate (IPP) via multiple ...

Isolation of Salmonella typhimurium-containing Phagosomes from Macrophages

Salmonella typhimurium is a facultative intracellular bacterium that causes gastroenteritis in humans. After invasion of the lamina propria, S. typhimurium bacteria are quickly detected and phagocytized by macrophages, and contained in vesicles known as phagosomes in order to be degraded. Isolation of S. typhimurium-containing phagosomes have been widely used to study how S. typhimurium infection alters the process of phagosome maturation to prevent bacterial degradation. Classically, the isolation of bacteria-containing phagosomes has been performed by sucrose gradient centrifugation. However, this process is time-consuming, and requires specialized equipment and a certain degree of dexterity. Described here is a simple and quick method for the isolation of S. typhimurium-containing phagosomes from macrophages by coating the bacteria with biotin-streptavidin-conjugated magnetic beads. Phagosomes obtained by this method can be suspended in any buffer of choice, allowing the utilization of isolated phagosomes for a broad range of assays, such as protein, metabolite, and lipid analysis. In summary, this method for the isolation of S. typhimurium-containing phagosomes is specific, efficient, rapid, requires minimum equipment, and is more versatile than the classical method of isolation by sucrose gradient-ultracentrifugation.

Isolation of Salmonella typhimurium-containing Phagosomes from Macrophages

We describe here a simple and quick method for the isolation of Salmonella typhimurium-containing phagosomes from macrophages by coating the bacteria with biotin and streptavidin.

Salmonella typhimurium is a facultative intracellular bacterium that causes gastroenteritis in humans. After invasion of the lamina propria, S. ...

A Novel Feeder-free System for Mass Production of Murine Natural Killer Cells In Vitro

Natural killer (NK) cells belong to the innate immune system and are a first-line anti-cancer immune defense; however, they are suppressed in the tumor microenvironment and the underlying mechanism is still largely unknown. The lack of a consistent and reliable source of NK cells limits the research progress of NK cell immunity. Here, we report an in vitro system that can provide high quality and quantity of bone marrow-derived murine NK cells under a feeder-free condition. More importantly, we also demonstrate that siRNA-mediated gene silencing successfully inhibits the E4bp4-dependent NK cell maturation by using this system. Thus, this novel in vitro NK cell differentiating system is a biomaterial solution for immunity research.

A Novel Feeder-free System for Mass Production of Murine Natural Killer Cells In Vitro

Here, we present a protocol to mass-produce gene-silencing murine NK cells by using a feeder-free differentiation system for mechanistic study in vitro and in vivo.

Natural killer (NK) cells belong to the innate immune system and are a first-line anti-cancer immune defense; however, they are suppressed in the tumor ...

Metabolic Mapping: Quantitative Enzyme Cytochemistry and Histochemistry to Determine the Activity of Dehydrogenases in Cells and Tissues

Altered cellular metabolism is a hallmark of many diseases, including cancer, cardiovascular diseases and infection. The metabolic motor units of cells are enzymes and their activity is heavily regulated at many levels, including the transcriptional, mRNA stability, translational, post-translational and functional level. This complex regulation means that conventional quantitative or imaging assays, such as quantitative mRNA experiments, Western Blots and immunohistochemistry, yield incomplete information regarding the ultimate activity of enzymes, their function and/or their subcellular localization. Quantitative enzyme cytochemistry and histochemistry (i.e., metabolic mapping) show in-depth information on in situ enzymatic activity and its kinetics, function and subcellular localization in an almost true-to-nature situation.
We describe a protocol to detect the activity of dehydrogenases, which are enzymes that perform redox reactions to reduce cofactors such as NAD(P)+ and FAD. Cells and tissue sections are incubated in a medium that is specific for the enzymatic activity of one dehydrogenase. Subsequently, the dehydrogenase that is the subject of investigation performs its enzymatic activity in its subcellular site. In a chemical reaction with the reaction medium, this ultimately generates blue-colored formazan at the site of the dehydrogenase&#39;s activity. The formazan&#39;s absorbance is therefore a direct measure of the dehydrogenase&#39;s activity and can be quantified using monochromatic light microscopy and image analysis. The quantitative aspect of this protocol enables researchers to draw statistical conclusions from these assays.
Besides observational studies, this technique can be used for inhibition studies of specific enzymes. In this context, studies benefit from the true-to-nature advantages of metabolic mapping, giving in situ results that may be physiologically more relevant than in vitro enzyme inhibition studies. In all, metabolic mapping is an indispensable technique to study metabolism at the cellular or tissue level. The technique is easy to adopt, provides in-depth, comprehensive and integrated metabolic information and enables rapid quantitative analysis.

Here, we present a protocol that can be used to microscopically visualize and quantify activity of dehydrogenases in cells and tissues and its kinetics, function and subcellular localization.

Altered cellular metabolism is a hallmark of many diseases, including cancer, cardiovascular diseases and infection. The metabolic motor units of cells ...