This work was supported by National Institutes of Health grant RO1 AI067979 to NWA. 
YK is recipient of undergraduate fellowship from the Howard Hughes Medical Institute/University of Maryland College Park.

All experimental procedures were conducted in accordance with the recommendations in the Guide for the Care and Use of Laboratory Animals by the National Institutes of Health and were approved by University of Maryland&#39;s IACUC. All steps described in sections 1 through 4 should be carried out aseptically inside biological laminar flow cabinets. Personal protection should be used, and caution should be exercised during handling of live Leishmania parasites throughout all stages of experimentation.
1. Isolation and Differentiation of Bone Marrow-derived Macrophages (BMMs)8,30,31
<ol>
	<li>Day 0: Sacrifice 4 - 6-week-old female C57BL/6 or BALB/c mouse in a CO2 chamber and confirm death via cervical dislocation. Ensure sterilization of scissors and forceps by keeping them soaked in 70% ethanol. Spray gloves with 70% ethanol to allow sterile handling throughout the bone marrow cell isolation process.</li>
	<li>Secure sacrificed mouse to dissection board by pinning its forelimbs and disinfect by spraying copiously with 70% ethanol.</li>
	<li>Make a small incision (about 1 cm) with scissors near the hip joint. Carefully insert scissors under the skin to separate the underlying muscle and cut the skin all the way around the hip joint. 
	NOTE: The dissection board may be rotated to allow improved access to the hip joint.</li>
	<li>Carefully deglove skin from the leg by pulling towards the ankle and remove. Cut the foot off at the ankle, being very careful to cut at or below the ankle joint to leave the tibia fully intact. 
	NOTE: The fibula at this stage is loosely attached and can be easily separated with the forceps.</li>
	<li>Manually locate the hip joint by manipulating the femur to identify the point of rotation. Carefully use scissors to sever the leg above the hip joint to leave the proximal head of the femur intact.</li>
	<li>Remove as much muscle and connective tissue as possible from the leg with scissors. Remove any remaining skin from the ankle and any excess bone material above the hip joint.</li>
	<li>Place the cleaned leg bones in sterile RPMI supplemented with penicillin/streptomycin (1% final concentration) in a Petri dish on ice.</li>
	<li>Repeat steps 1.4 through 1.6 to isolate tibia and femur from the other hind leg.</li>
	<li>Remove any remaining muscle and connective tissue from the bones soaking in RPMI using sterilized forceps and sterile #10 blade.</li>
	<li>Place bones on the Petri dish lid. Cut the tibia carefully with the blade immediately above the ankle joint to access the marrow. Remove the tibia from the rest of the leg by cutting immediately below the knee joint.</li>
	<li>Draw 10 mL sterile RPMI supplemented with penicillin/streptomycin (1% final concentration) into a 10-mL syringe and attach a 25G needle.</li>
	<li>Hold the tibia firmly with forceps, vertically over a 50-mL conical tube on ice. Use the syringe to gently inject approximately 5 mL RPMI into the end of the bone to flush the bone marrow out of the other end into the conical tube. Flush the bone marrow with a total of 10 mL RPMI until the bone turns white. 
	NOTE: After thorough flushing, the bone should turn white. If not, continue flushing to remove marrow. The distal end of the tibia may need to be trimmed back with the blade if it is too narrow to insert the needle.</li>
	<li>Cut the femur immediately above the knee joint and immediately below the hip joint to expose both ends of the bone to access the marrow. Flush bone marrow from the femur with a total of 10 mL RPMI in the same fashion as the tibia.</li>
	<li>Repeat cleaning and flushing steps 1.6 through 1.13 with the remaining bones and collect the bone marrow flushed cells in a single 50 mL conical tube 
	NOTE: If a bone breaks at any point during dissection prior to bone flushing, do not use the marrow from this bone.</li>
	<li>Centrifuge the conical tube used to collect bone marrow flushed from all four bones for 10 min at 4 &#176;C at 300 x g.</li>
	<li>Place 60 mL of sterile BMM medium (RPMI with a final concentration of 20% FBS, 1% penicillin/streptomycin, 1.2 mM Na-pyruvate, 25 &#181;g/mL human M-CSF) into a 175 cm2 cell culture flask with filter cap.</li>
	<li>Discard the supernatant following centrifugation and resuspend the cell pellet by gentle up and down pipetting using 5 mL of BMM medium from the flask.</li>
	<li>Transfer cell suspension to the culture flask and rinse the conical tube twice with BMM medium from the culture flask to ensure maximal cell recovery. Ensure cell suspension is thoroughly mixed into medium and distribute 30 mL from the initial 175 cm2 cell culture flask to a second 175 cm2 cell culture flask with filter cap.</li>
	<li>Incubate both flasks containing 30 mL cell suspension each overnight at 37 &#176;C, 5% CO2 to allow separation of any contaminating fibroblasts, resident macrophages, and other adherent cells.</li>
	<li>DAY 1: Transfer the supernatant containing undifferentiated bone marrow cells from flasks to 100 mm x 15 mm non-TC-treated polystyrene Petri dishes at approximately 10 mL/dish and incubate at 37 &#176;C, 5% CO2. Discard the flasks.</li>
	<li>DAY 3 or 4: Add an additional 5 mL BMM medium (pre-warmed to 37 &#176;C) to each Petri dish and continue incubation at 37 &#176;C, 5% CO2.</li>
</ol>
2. Plating BMMs on Coverslips for Infection (DAY 7 or 8)
<ol>
	<li>Prepare 6-well tissue culture plates by placing four (4) sterile 12 mm glass coverslips (autoclaved and stored in a closed glass container) in the bottom of each empty well. 
	NOTE: Pick each sterile 12 mm coverslip up with an aspirating tip fitted to a vacuum line. Place coverslips into the wells without overlap, releasing at the desired position by pinching the tube to interrupt vacuum.</li>
	<li>Aspirate media (and any non-adherent cells) from Petri dishes coated with adherent bone marrow derived macrophages and gently rinse adherent cells 2x with sterile Dulbecco&#39;s Phosphate buffered saline without calcium and magnesium chloride (PBS -/-) pre-warmed to 37 &#176;C.</li>
	<li>Add dropwise, 5 mL sterile 1x PBS -/- with a final concentration of 1 mM EDTA to each Petri dish and incubate for 5 min at 37 &#176;C to detach adherent BMMs. Verify that the cells are detached using a microscope.</li>
	<li>Collect all detached cells suspended in 1x PBS -/- supplemented with 1 mM EDTA in a 50 mL conical tube. Rinse each dish 2x with 5 mL PBS -/- and add to cell suspension pool. 
	NOTE: Cell suspension may be diluted with additional PBS -/- to protect macrophages from EDTA toxicity during the collection process.</li>
	<li>Centrifuge at 300 x g for 10 min at 4 &#176;C. Discard supernatant and resuspend macrophages in 5 - 10 mL BMM medium by pipetting. Place resuspended cells on ice to prevent attachment to tubes and clumping.</li>
	<li>Count viable cells using Trypan blue exclusion in the hemocytometer. 
	NOTE: To count BMM cells resuspended in 5 mL medium, a dilution-mix of 25 &#181;L cell suspension in 445 &#181;L PBS -/- and 30 &#181;L 0.4% Trypan blue solution typically allows easy quantification in a hemocytometer (taking into account a 20x dilution factor for the final calculation). Count only cells that did not get stained with Trypan blue (viable cells). The volume of cell suspension may be altered if this mixture is too concentrated or diluted. In general, the yield of BMM per preparation varies between 2 x 107 and 5 x 107 cells from a single mouse.</li>
	<li>Prepare the cell suspension in BMM medium at desired plating concentration (5 x 105/mL) and disperse suspension to 6 well plates in 2 mL medium per well so that each well receives 1 x 106 macrophages. 
	NOTE: This is a critical step that ensures every well gets the adequate number of macrophages to cover the coverslips uniformly. Check that coverslips in wells are not overlapping or floating in the medium. If so, gently adjust with a sterile pipette tip.</li>
	<li>Incubate overnight at 37 &#176;C, 5% CO2.</li>
</ol>
3. Purification of Infective Forms of L. amazonensis
NOTE: Prepare Leishmania for infections- purify metacyclic promastigotes from stationary promastigote cultures8,13, or differentiate promastigotes in culture into amastigote form using standard L. amazonensis axenic differentiation protocol6,8.
<ol>
	<li>Purification of Leishmania metacyclic promastigotes using density gradient media(Materials)33
	<ol>
		<li>Prepare 40% stock solution of density gradient media in sterile, endotoxin-free water.</li>
		<li>Dilute density gradient solution in 10x M199 medium (without serum) to prepare 10% concentration in the M199 medium.</li>
		<li>Filter all solutions through 0.22 &#181;m filter. 
		NOTE: Stock solutions can be stored at 4 oC in darkness for no longer than 1 month.</li>
		<li>Add 2 mL of 40% density gradient media solution in the bottom of a 15-mL conical centrifuge tube.</li>
		<li>Layer 2 mL of 10% density gradient media solution in M199 on top of the 40% density gradient media layer carefully, using a Pasteur pipette to avoid any mixing between the two layers.</li>
		<li>Collect 1 x 109 parasites from stationary-phase culture by centrifugation at 1,900 x g for 10 min.</li>
		<li>Resuspend cells in 6 mL Dulbecco&#39;s Modified Essential Medium (DMEM).</li>
		<li>Layer 2 mL of the cell suspension directly on top of the 10% density gradient media layer, gently, using a Pasteur pipette to avoid mixing between layers.</li>
		<li>Centrifuge the gradient for 10 min at 1,300 x g at room temperature. 
		NOTE: Due to differences in physical properties between Leishmania species, centrifugation conditions for each might have to be slightly adjusted to ensure maximum yield.</li>
		<li>Collect parasites (enriched in metacyclic promastigote form) from the band formed at the upper 10% density gradient media boundary (interface between the 0% and 10% density gradient media layers).</li>
		<li>Dilute parasites with one volume of DMEM and collect by centrifugation (1,900 x g for 10 min at room temperature).</li>
		<li>Resuspend in 500 &#181;L DMEM medium and count in a hemocytometer to quantify yield.</li>
	</ol>
	</li>
	<li>Generation of L. amazonensis amastigotes under axenic culture conditions (low pH / elevated temperature)6,8,13
	<ol>
		<li>Mix 5 mL of log-phase promastigote culture (pH 7.4 at 26 oC) with an equal volume of amastigote medium (pH 4.5), using 25 cm2 flasks (total 10 mL medium) and incubate at 26 &#176;C overnight.</li>
		<li>Shift the flask from 26 &#176;C to 32 &#176;C.</li>
		<li>After 3 or 4 days, split culture 1:5 in amastigote media at 32 &#176;C.</li>
		<li>Check the parasites in the next 3 - 4 days (maximum 7 days) to see if they are ready for use in infections. 
		NOTE: Healthy axenic amastigotes should have an oval shape, without visible flagella. Partially differentiated amastigotes have a large oval shape with short flagella. The culture should not have a lot of clumps - many clumps are an indication of non-growing, dying parasites. The amastigote culture can be split 1:5 and maintained for a maximum of 3 weeks.</li>
	</ol>
	</li>
</ol>
4. Infection with L. amazonensis
<ol>
	<li>Dilute parasite suspensions according to desired MOI (usually 3-5 metacyclic promastigotes per macrophage [MOI of 1:3 or 1:5] and 1 amastigote per macrophage [MOI 1:1]). Add Leishmania in PBS -/- in a volume of 50 - 100 &#181;L to each well already containing 2 mL medium.</li>
	<li>Incubate BMMs for 1 h with amastigotes and 3 h with metacyclic promastigotes at 34 oC for infection. 
	NOTE: Optimal infection temperature may vary by species.</li>
	<li>Following incubation, thoroughly wash away free parasites in each well 3x with 2 mL PBS -/- pre-warmed to 37 &#176;C. 
	NOTE: Disperse PBS gently to ensure that coverslips do not float over each other. Gently swirl the plate and then aspirate out the liquid. Use separate aspirating tip if using multiple strains of parasites to avoid cross-contamination.</li>
	<li>Fix 1 h or 3 h time-point samples by incubating each well with 1.5 - 2 mL of 2% PFA in PBS -/- for 10 min. Wash 3x with PBS -/-. Do not aspirate last wash and refrigerate plates containing coverslips in PBS until staining.</li>
	<li>Add 2 mL fresh BMM medium to each well on plates for further time-points and incubate at 34 &#176;C.</li>
	<li>Fix remaining time-points as desired as described in step 4.3, and refrigerate plates until staining.</li>
</ol>
5. DAPI Staining and Coverslip Mounting
<ol>
	<li>Aspirate PBS from wells containing coverslips and add 1.5 mL PBS -/- with 0.1% non-ionic detergent. Incubate for 10 min at room temperature, followed by 3x wash with 2 mL PBS -/-.</li>
	<li>Add 1 mL PBS -/- with 2 &#181;g/mL DAPI (diluted from 5 mg/mL stock solution in water) to wells containing coverslips and incubate for another 1 h at room temperature. 
	NOTE: The incubation time with DAPI is critical for proper visualization of intracellular Leishmania parasites. Macrophage nuclei stain rapidly, due to their larger size and DNA content but staining of nuclei of the intracellular parasites is slower. Thus, extending incubation time beyond what is required to visualize the BMM nuclei is necessary to allow DAPI to permeate through and the parasite plasma membrane, and the parasite nuclear membrane. With proper DAPI stain, the mitochondrial kinetoplast DNA near the flagellar pocket of the parasite can also be visualized, in addition to the nuclear DNA.</li>
	<li>Wash each well containing coverslips 3x with 2 mL PBS -/- and then lift and flip coverslips with forceps to place the cell side down and mount on glass microscope slides with a commercially available antifade mounting reagent. 
	NOTE: Coverslips are extremely fragile and need cautious handling. Avoid putting pressure on them, especially against the sidewall of the wells when lifting. Adding a few drops of PBS -/- reduces surface tension between the coverslip and well and facilitates the lifting process. A fine gauge needle may be used to assist in prying the coverslips from the bottom of the well. After placing a coverslip on the slide, gently press down with forceps to push out any air bubbles in the mounting media. If a coverslip is broken or has possibly flipped during the lifting process, do not remount; simply use the spare fourth coverslip.</li>
	<li>Refrigerate slides until quantification.</li>
</ol>
6. Infection Quantification
<ol>
	<li>Examine DAPI-stained slides under a fluorescence microscope by focusing on macrophages using 100X&#160;objective lens with immersion oil (excitation at 358 nm; optimal emission at 461 nm). 
	Ensure that focus is on the layer of macrophages between the glass slide and coverslip.</li>
	<li>Quantify the number of macrophages (large nuclei stained with DAPI) and the number of smaller amastigote nuclei clustered around each macrophage nucleus (see Figure 2A, DAPI) for each field of vision, using a manual counter. 
	NOTE: Amastigote nuclei may not all be visible in one plane of focus due to their small size. Count those that are visible when focused on macrophage nuclei, and then use fine focus to check for parasite nuclei in planes above and below the macrophage nuclei.</li>
	<li>Move to another field of vision to repeat quantification. Use a separate counter key to track the number of fields counted. Move through visual fields in parallel rows across each coverslip, to prevent counting overlap.</li>
	<li>Quantify a minimum of 200 macrophages per coverslip. Quantify each time-point of infection in triplicate (3 coverslips). Count the fourth coverslip if one of the previous is not suitable for counting due to coverslip overlap, poor mounting, glass breakage, etc. 
	NOTE: If 200 macrophages cannot be counted on any one coverslip, count the spare fourth.</li>
	<li>Calculate infection rates as amastigotes/macrophage or amastigotes/100 macrophages and determine percent infected macrophages from the raw quantification data.</li>
</ol>

The authors declare they have no competing financial interests,

The quantitative data produced by the BMM infection assay described above, allows investigators to obtain rates of infection and a reliable determination of changes in virulence properties in a relatively shorter time period (maximum 2 weeks, compared to the 2 months required for in vivo experiments). This method relies on the DNA specific dye DAPI, which specifically stains macrophage and parasite nuclei, and allows rapid identification and quantification of infected cells. In comparison, other stains such as G...

Leishmaniasis refers to a broad spectrum of human diseases caused by protozoan parasite species of the genus Leishmania. Approximately 12 million people are currently infected with Leishmania worldwide, and more than 350 million are at risk. The disease pathology depends on the Leishmania species and on host factors, and symptoms vary from innocuous self-healing skin lesions to lethal visceralizing forms. If untreated, visceral leishmaniasis is fatal, ranking only after malaria as the deadliest human disease caused by infection with a protozoan parasite1. In spite of the wide-ranging differences in disease pathology and symptoms, all Leishmania species have a digenic life-cycle alternating between promastigote and amastigote stages inside insect and vertebrate hosts, respectively. Inside vertebrates, Leishmania target host macrophages for invasion and induce the formation of parasitophorous vacuoles (PVs), acidic compartments with properties of phagolysosomes where the highly virulent amastigote forms replicate. Amastigotes persist in host tissues during chronic infections and can be passed forward to uninfected sandflies, completing the transmission cycle. Therefore, in the context of human disease development, amastigotes are the most important Leishmania lifecycle form2. Investigating how amastigotes replicate inside macrophage PVs is critical for understanding Leishmania virulence3,4,5,6,7 and for the development of novel efficacious therapies.
We describe here a method regularly used by our laboratory to study Leishmania infection and replication in bone marrow-derived macrophages (BMMs), which involves quantitative assessment of the number of intracellular Leishmania over time. The process involves harvesting of monocytes from mouse bone marrow and differentiation to macrophages in culture, in vitro infection with infective forms (metacyclic promastigotes or amastigotes) of Leishmania and quantification of the number of intracellular parasites at every 24 h interval for a period of 72 - 96 h following infection. This assay has been used in our laboratory to determine the impact of several environmental factors and parasite genes, including identification of the critical role of iron in promoting L. amazonensis virulence that was further validated by footpad lesion development studies in mice6,8,9,10,11,12,13,14,15. Since all pathogenic Leishmania species establish their replicative niche inside host macrophages, this assay can be used universally for virulence determinations in all Leishmania species.
Performing BMM infections allows analysis of host-parasite interactions at the single cell level, and thus a more extensive understanding of how Leishmania parasites interact with their preferred host microenvironment, the PVs of macrophages. Macrophage infection assays have been successfully used by multiple groups16,17,18,19,20,21,22 to explore functions of both the host macrophage and Leishmania specific genes, and their potential involvement in the complex interplay that characterizes intracellular infection. BMM infections allow quantification of parasite growth as a read-out of the impact of host factors that influence intracellular survival, such as microbicidal nitric oxide production, generation of reactive oxygen species and other adverse conditions encountered inside the lysosome-like PVs23. Macrophage infection assays have also been utilized to identify potential anti-leishmanial drug leads for therapeutic development13,24.
The in vitro nature of BMM infections provides several advantages over other methods to assess Leishmania virulence. However, several previous studies examining mechanisms of intracellular parasite survival over time did not quantify infection as a rate20,21,24. Furthermore, many studies focused on following in vivo infections over time did so by measuring cutaneous lesion size and other physiological symptoms that are only indirectly related to parasite replication25,26,27. In vivo infection is a stringent approach to assess parasite virulence, but lesion size measurements based on footpad swelling alone are often inadequate, as they reflect the inflammatory response in infected tissues and not the absolute number of parasites. For this reason, footpad lesion development assays have to be followed by quantification of the parasite load in infected tissues, a procedure that requires lengthy limiting dilution assays28. Additionally, in vivo studies often involve sacrificing multiple animals at different points in time to extract tissues of interest6,8,9,10,11,13. In contrast, large numbers of BMMs can be obtained from just one animal, and these cells can be plated under conditions that allow assessment of infection at various points in time. Furthermore, compared to in vivo studies, performing in vitro BMM infections allows greater control over experimental conditions. Quantifying the macrophages to be infected along with the parasites themselves allows precise control of the multiplicity of infection (MOI) and of culture conditions. Fine control over these factors can be key in identifying characteristics of discrete cellular pathways and in understanding their impact on the course of infection.
Given these advantages, it is somewhat surprising that very few groups studying Leishmania virulence have so far taken full advantage of quantitative assessment of intracellular replication in macrophages. In this article, we discuss common pitfalls that may be hampering the more extensive utilization of this assay, and provide a step-by-step protocol to facilitate its proper implementation. Considering its precision and versatility, the BMM infection assay we describe here can not only be utilized to explore host-pathogen interactions influencing Leishmania virulence, but also to study other microorganisms that replicate inside macrophages29. Importantly, this assay can also be developed as a rapid and economical pre-clinical screening method for anti-leishmanial drug development.

<table>
	<tbody>
		<tr>
			<td>6 well cell culture plate</td>
			<td>Cellstar</td>
			<td>657160</td>
			<td></td>
		</tr>
		<tr>
			<td>Adenine</td>
			<td>Acros Organics</td>
			<td>AC147440250</td>
			<td></td>
		</tr>
		<tr>
			<td>Aerosol Barrier Pipet Tips (100-1000 &#956;L)</td>
			<td>Fisherbrand</td>
			<td>02-707-404</td>
			<td></td>
		</tr>
		<tr>
			<td>Aerosol Barrier Pipet Tips (20-200 &#956;L)</td>
			<td>Fisherbrand</td>
			<td>02-707-430</td>
			<td></td>
		</tr>
		<tr>
			<td>Aerosol Barrier Pipet Tips (2-20 &#956;L)</td>
			<td>Fisherbrand</td>
			<td>02-707-432</td>
			<td></td>
		</tr>
		<tr>
			<td>Bard-Parker Rib-Back Carbon Steel Surgical Blade #10</td>
			<td>Aspen Surgical</td>
			<td>371110</td>
			<td></td>
		</tr>
		<tr>
			<td>BD Luer-Lok Tip 10 mL Syringe</td>
			<td>Becton Dickinson (BD)</td>
			<td>309604</td>
			<td></td>
		</tr>
		<tr>
			<td>BD Precisionglide Needle, 25G&#160;</td>
			<td>Becton Dickinson (BD)</td>
			<td>305124</td>
			<td></td>
		</tr>
		<tr>
			<td>Cell Culture Dish 35 mm x 10 mm</td>
			<td>Cellstar</td>
			<td>627 160</td>
			<td></td>
		</tr>
		<tr>
			<td>Cell Culture Flask</td>
			<td>Cellstar</td>
			<td>660175</td>
			<td></td>
		</tr>
		<tr>
			<td>Cover Glasses: 12 mm circles</td>
			<td>Fisherbrand</td>
			<td>12-545-80</td>
			<td></td>
		</tr>
		<tr>
			<td>DAPI (4&#39;,6-Diamidino-2-Phenylindole, Dihydrochloride)</td>
			<td>Invitrogen</td>
			<td>D1306</td>
			<td></td>
		</tr>
		<tr>
			<td>D-Biotin</td>
			<td>J.T. Baker</td>
			<td>C272-00</td>
			<td></td>
		</tr>
		<tr>
			<td>EDTA</td>
			<td>Sigma Aldrich</td>
			<td>EDS</td>
			<td></td>
		</tr>
		<tr>
			<td>Ethyl alcohol 200 proof</td>
			<td>Pharmco-AAPER</td>
			<td>111000200</td>
			<td></td>
		</tr>
		<tr>
			<td>Falcon 100 mm x 15 mm non-TC-treated polystyrene Petri dish</td>
			<td>Corning</td>
			<td>351029</td>
			<td></td>
		</tr>
		<tr>
			<td>Fetal Bovine Serum</td>
			<td>Seradigm</td>
			<td>1500-500</td>
			<td></td>
		</tr>
		<tr>
			<td>Ficoll400</td>
			<td>Sigma Aldrich</td>
			<td>F8016</td>
			<td></td>
		</tr>
		<tr>
			<td>Fluorescence Microscope</td>
			<td>Nikon</td>
			<td>E200</td>
			<td></td>
		</tr>
		<tr>
			<td>Goat anti-mouse IgG Texas red</td>
			<td>Invitrogen</td>
			<td>T-862</td>
			<td></td>
		</tr>
		<tr>
			<td>Goat anti-rabbit IgG AlexaFluor488</td>
			<td>Invitrogen</td>
			<td>A-11034</td>
			<td></td>
		</tr>
		<tr>
			<td>Hemin</td>
			<td>Tokyo Chemical Industry Co. LTD</td>
			<td>H0008</td>
			<td></td>
		</tr>
		<tr>
			<td>HEPES (1M)</td>
			<td>Gibco</td>
			<td>15630-080</td>
			<td></td>
		</tr>
		<tr>
			<td>Isoton II Diluent</td>
			<td>Beckman Coulter</td>
			<td>8546719</td>
			<td></td>
		</tr>
		<tr>
			<td>L-Glutamine</td>
			<td>Gemini</td>
			<td>400-106</td>
			<td></td>
		</tr>
		<tr>
			<td>Medium 199 (10X)</td>
			<td>Gibco</td>
			<td>11825-015</td>
			<td></td>
		</tr>
		<tr>
			<td>Na pyruvate (100 mM)</td>
			<td>Gibco</td>
			<td>11360-070</td>
			<td></td>
		</tr>
		<tr>
			<td>Paraformaldehyde</td>
			<td>Alfa Aesar</td>
			<td>43368</td>
			<td></td>
		</tr>
		<tr>
			<td>Penicillin/Streptomycin</td>
			<td>Gemini</td>
			<td>400-109</td>
			<td></td>
		</tr>
		<tr>
			<td>Phosphate Buffered Saline (-/-)</td>
			<td>ThermoFisher</td>
			<td>14200166</td>
			<td></td>
		</tr>
		<tr>
			<td>Polypropyline conical Centrifuge Tubes 15 mL</td>
			<td>Cellstar</td>
			<td>188 271</td>
			<td></td>
		</tr>
		<tr>
			<td>Polypropyline conical Centrifuge Tubes 50 mL</td>
			<td>Cellstar</td>
			<td>227 261</td>
			<td></td>
		</tr>
		<tr>
			<td>ProLong Gold antifade reagent</td>
			<td>ThermoFisher</td>
			<td>P36930</td>
			<td></td>
		</tr>
		<tr>
			<td>Rat anti-mouse Lamp-1 antibody</td>
			<td>Developmental Studies Hybridoma Bank</td>
			<td>1D4B</td>
			<td></td>
		</tr>
		<tr>
			<td>Recombinant Human M-CSF</td>
			<td>PeproTech</td>
			<td>300-25</td>
			<td></td>
		</tr>
		<tr>
			<td>Reichert Bright-Line&#160; Hemocytometer&#160;</td>
			<td>Hausser Scientific</td>
			<td>1492</td>
			<td></td>
		</tr>
		<tr>
			<td>RPMI Medium 1640 (1X)</td>
			<td>Gibco</td>
			<td>11875-093</td>
			<td></td>
		</tr>
		<tr>
			<td>Triton X-100 Surfactant</td>
			<td>Millipore Sigma</td>
			<td>TX1568-1</td>
			<td></td>
		</tr>
		<tr>
			<td>Trypan Blue</td>
			<td>Sigma Aldrich</td>
			<td>T8154</td>
			<td></td>
		</tr>
		<tr>
			<td>Delicate Scissors, 4 1/2&#34;</td>
			<td>VWR</td>
			<td>82027-582</td>
			<td></td>
		</tr>
		<tr>
			<td>Dissecting Forceps, Fine Tip</td>
			<td>VWR</td>
			<td>82027-386</td>
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quantification of intracellular growth inside macrophages is a fast and reliable method for assessing the virulence of leishmania parasites

The lifecycle of Leishmania, the causative agent of leishmaniasis, alternates between promastigote and amastigote stages inside the insect and vertebrate hosts, respectively. While pathogenic symptoms of leishmaniasis can vary widely, from benign cutaneous lesions to highly fatal visceral disease forms depending on the infective species, all Leishmania species reside inside host macrophages during the vertebrate stage of their lifecycle. Leishmania infectivity is therefore directly related to its ability to invade, survive and replicate within parasitophorous vacuoles (PVs) inside macrophages. Thus, assessing the parasite&#39;s ability to replicate intracellularly serves as a dependable method for determining virulence. Studying leishmaniasis development using animal models is time-consuming, tedious and often difficult, particularly with the pathogenically important visceral forms. We describe here a methodology to follow the intracellular development of Leishmania in bone marrow-derived macrophages (BMMs). Intracellular parasite numbers are determined at 24 h intervals for 72 - 96 h following infection. This method allows for a reliable determination of the effects of various genetic factors on Leishmania virulence. As an example, we show how a single allele deletion of the Leishmania Mitochondrial Iron Transporter gene (LMIT1) impairs the ability of the Leishmania amazonensis mutant strain LMIT1/&#916;Lmit1 to grow inside BMMs, reflecting a drastic reduction in virulence compared to wild-type. This assay also allows precise control of experimental conditions, which can be individually manipulated to analyze the influence of various factors (nutrients, reactive oxygen species, etc.) on the host-pathogen interaction. Therefore, the appropriate execution and quantification of BMM infection studies provide a non-invasive, rapid, economical, safe and reliable alternative to conventional animal model studies.

Leishmania has two infective forms - metacyclic promastigotes that differentiate from procyclic promastigotes at the stationary phase of culture, and amastigotes, which are the intracellular stages (Figure 1). In some Leishmania species such as L. amazonensis, amastigotes can also be differentiated in axenic culture by shifting the promastigote cells to lower pH (4.5) and elevated temperature (32 &#176;C), conditions similar to thos...

Watch this Scientific Journal Video about Quantification of Intracellular Growth Inside Macrophages is a Fast and Reliable Method for Assessing the Virulence of Leishmania Parasites at JoVE.com

Quantification of Intracellular Growth Inside Macrophages is a Fast and Reliable Method for Assessing the Virulence of Leishmania Parasites

All pathogenic Leishmania species reside and replicate inside macrophages of their vertebrate hosts. Here, we present a protocol to infect murine bone marrow-derived macrophages in culture with Leishmania, followed by precise quantification of intracellular growth kinetics. This method is useful for studying individual factors influencing host-pathogen interaction and Leishmania virulence.

Quantification of Intracellular Growth Inside Macrophages is a Fast and Reliable Method for Assessing the Virulence of Leishmania Parasites

The lifecycle of Leishmania, the causative agent of leishmaniasis, alternates between promastigote and amastigote stages inside the insect and vertebrate ...

quantification-intracellular-growth-inside-macrophages-is-fast

Universidad de Cartagena UDEC

Research

JoVE Journal

Immunology and Infection

10.4K Views.  University of Maryland. All pathogenic Leishmania species reside and replicate inside macrophages of their vertebrate hosts. Here, we present a protocol to infect murine bone marrow-derived macrophages in culture with Leishmania, followed by precise quantification of intracellular growth kinetics. This method is useful for studying individual factors influencing host-pathogen interaction and Leishmania virulence.

Video: Quantification of Intracellular Growth Inside Macrophages is a Fast and Reliable Method for Assessing the Virulence of Leishmania Parasites

In vivo Imaging of Transgenic Leishmania Parasites in a Live Host

Distinct species of Leishmania, a protozoan parasite of the family Trypanosomatidae, typically cause different human disease manifestations. The most common forms of disease are visceral leishmaniasis (VL) and cutaneous leishmaniasis (CL). Mouse models of leishmaniasis are widely used, but quantification of parasite burdens during murine disease requires mice to be euthanized at various times after infection. Parasite loads are then measured either by microscopy, limiting dilution assay, or qPCR amplification of parasite DNA. The in vivo imaging system (IVIS) has an integrated software package that allows the detection of a bioluminescent signal associated with cells in living organisms. Both to minimize animal usage and to follow infection longitudinally in individuals, in vivo models for imaging Leishmania spp. causing VL or CL were established. Parasites were engineered to express luciferase, and these were introduced into mice either intradermally or intravenously. Quantitative measurements of the luciferase driving bioluminescence of the transgenic Leishmania parasites within the mouse were made using IVIS. Individual mice can be imaged multiple times during longitudinal studies, allowing us to assess the inter-animal variation in the initial experimental parasite inocula, and to assess the multiplication of parasites in mouse tissues. Parasites are detected with high sensitivity in cutaneous locations. Although it is very likely that the signal (photons/second/parasite) is lower in deeper visceral organs than the skin, but quantitative comparisons of signals in superficial versus deep sites have not been done. It is possible that parasite numbers between body sites cannot be directly compared, although parasite loads in the same tissues can be compared between mice. Examples of one visceralizing species (L. infantum chagasi) and one species causing cutaneous leishmaniasis (L. mexicana) are shown. The IVIS procedure can be used for monitoring and analyzing small animal models of a wide variety of Leishmania species causing the different forms of human leishmaniasis.

In vivo Imaging of Transgenic Leishmania Parasites in a Live Host

An in vivo imaging system is used to generate quantitative measurements of murine infection with the Trypanosomatid protozoan Leishmania. This is a non-invasive and non-lethal method for detecting parasites expressing luciferase within many tissues throughout the course of chronic Leishmania spp. infection.

Distinct species of Leishmania, a protozoan parasite of the family Trypanosomatidae, typically cause different human disease manifestations. The most ...

Protocol for Production of a Genetic Cross of the Rodent Malaria Parasites

Variation in response to antimalarial drugs and in pathogenicity of malaria parasites is of biologic and medical importance. Linkage mapping has led to successful identification of genes or loci underlying various traits in malaria parasites of rodents1-3 and humans4-6. The malaria parasite Plasmodium yoelii is one of many malaria species isolated from wild African rodents and has been adapted to grow in laboratories. This species reproduces many of the biologic characteristics of the human malaria parasites; genetic markers such as microsatellite and amplified fragment length polymorphism (AFLP) markers have also been developed for the parasite7-9. Thus, genetic studies in rodent malaria parasites can be performed to complement research on Plasmodium falciparum. Here, we demonstrate the techniques for producing a genetic cross in P. yoelii that were first pioneered by Drs. David Walliker, Richard Carter, and colleagues at the University of Edinburgh10.

Genetic crosses in P. yoelii and other rodent malaria parasites are conducted by infecting mice Mus musculus with an inoculum containing gametocytes of two genetically distinct clones that differ in phenotypes of interest and by allowing mosquitoes to feed on the infected mice 4 days after infection. The presence of male and female gametocytes in the mouse blood is microscopically confirmed before feeding. Within 48 hrs after feeding, in the midgut of the mosquito, the haploid gametocytes differentiate into male and female gametes, fertilize, and form a diploid zygote (Fig. 1). During development of a zygote into an ookinete, meiosis appears to occur11. If the zygote is derived through cross-fertilization between gametes of the two genetically distinct parasites, genetic exchanges (chromosomal reassortment and cross-overs between the non-sister chromatids of a pair of homologous chromosomes; Fig. 2) may occur, resulting in recombination of genetic material at homologous loci. Each zygote undergoes two successive nuclear divisions, leading to four haploid nuclei. An ookinete further develops into an oocyst. Once the oocyst matures, thousands of sporozoites (the progeny of the cross) are formed and released into mosquito hemoceal. Sporozoites are harvested from the salivary glands and injected into a new murine host, where pre-erythrocytic and erythrocytic stage development takes place. Erythrocytic forms are cloned and classified with regard to the characters distinguishing the parental lines prior to genetic linkage mapping. Control infections of individual parental clones are performed in the same way as the production of a genetic cross.

Genetic crosses of rodent malaria parasites are performed by feeding two genetically distinct parasites to mosquitoes. Recombinant progeny are cloned from mouse blood after allowing mosquitoes to bite infected mice. This video shows how to produce genetic crosses of Plasmodium yoelii and is applicable to other rodent malaria parasites.

Variation in response to antimalarial drugs and in pathogenicity of malaria parasites is of biologic and medical importance. Linkage mapping has led to ...

A Parasite Rescue and Transformation Assay for Antileishmanial Screening Against Intracellular Leishmania donovani Amastigotes in THP1 Human Acute Monocytic Leukemia Cell Line

Leishmaniasis is one of the world's most neglected diseases, largely affecting the poorest of the poor, mainly in developing countries. Over 350 million people are considered at risk of contracting leishmaniasis, and approximately 2 million new cases occur yearly1. Leishmania donovani is the causative agent for visceral leishmaniasis (VL), the most fatal form of the disease. The choice of drugs available to treat leishmaniasis is limited 2;current treatments provide limited efficacy and many are toxic at therapeutic doses. In addition, most of the first line treatment drugs have already lost their utility due to increasing multiple drug resistance 3. The current pipeline of anti-leishmanial drugs is also severely depleted. Sustained efforts are needed to enrich a new anti-leishmanial drug discovery pipeline, and this endeavor relies on the availability of suitable in vitro screening models.
In vitro promastigotes 4 and axenic amastigotes assays5 are primarily used for anti-leishmanial drug screening however, may not be appropriate due to significant cellular, physiological, biochemical and molecular differences in comparison to intracellular amastigotes. Assays with macrophage-amastigotes models are considered closest to the pathophysiological conditions of leishmaniasis, and are therefore the most appropriate for in vitro screening. Differentiated, non-dividing human acute monocytic leukemia cells (THP1) (make an attractive) alternative to isolated primary macrophages and can be used for assaying anti-leishmanial activity of different compounds against intracellular amastigotes.
Here, we present a parasite-rescue and transformation assay with differentiated THP1 cells infected in vitro with Leishmania donovani for screening pure compounds and natural products extracts and determining the efficacy against the intracellular Leishmania amastigotes. The assay involves the following steps: (1) differentiation of THP1 cells to non-dividing macrophages, (2) infection of macrophages with L. donovani metacyclic promastigotes, (3) treatment of infected cells with test drugs, (4) controlled lysis of infected macrophages, (5) release/rescue of amastigotes and (6) transformation of live amastigotes to promastigotes. The assay was optimized using detergent treatment for controlled lysis of Leishmania-infected THP1 cells to achieve almost complete rescue of viable intracellular amastigotes with minimal effect on their ability to transform to promastigotes. Different macrophage:promastigotes ratios were tested to achieve maximum infection. Quantification of the infection was performed through transformation of live, rescued Leishmania amastigotes to promastigotes and evaluation of their growth by an alamarBlue fluorometric assay in 96-well microplates. This assay is comparable to the currently-used microscopic, transgenic reporter gene and digital-image analysis assays. This assay is robust and measures only the live intracellular amastigotes compared to reporter gene and image analysis assays, which may not differentiate between live and dead amastigotes. Also, the assay has been validated with a current panel of anti-leishmanial drugs and has been successfully applied to large-scale screening of pure compounds and a library of natural products fractions (Tekwani et al. unpublished).

A Parasite Rescue and Transformation Assay for Antileishmanial Screening Against Intracellular Leishmania donovani Amastigotes in THP1 Human Acute Monocytic Leukemia Cell Line

A parasite-rescue and transformation assay with THP1 cells infected in vitro with Leishmania donovani has been optimized for anti-leishmanial drug screening. The assay involves differentiation of THP1 cells, infection with promastigotes, treatment with test drugs, controlled lysis of the infected macrophages, rescue of amastigotes, transformation to promastigotes and monitoring promastigote growth and proliferation with a fluorometric assay.

Leishmaniasis is  one of the world's most neglected diseases, largely affecting the  poorest of the poor, mainly in developing countries. Over 350 million ...

Establishment of an In vitro System to Study Intracellular Behavior of Candida glabrata in Human THP-1 Macrophages

A cell culture model system, if a close mimic of host environmental conditions, can serve as an inexpensive, reproducible and easily manipulatable alternative to animal model systems for the study of a specific step of microbial pathogen infection. A human monocytic cell line THP-1 which, upon phorbol ester treatment, is differentiated into macrophages, has previously been used to study virulence strategies of many intracellular pathogens including Mycobacterium tuberculosis. Here, we discuss a protocol to enact an in vitro cell culture model system using THP-1 macrophages to delineate the interaction of an opportunistic human yeast pathogen Candida glabrata with host phagocytic cells. This model system is simple, fast, amenable to high-throughput mutant screens, and requires no sophisticated equipment. A typical THP-1 macrophage infection experiment takes approximately 24 hr with an additional 24-48 hr to allow recovered intracellular yeast to grow on rich medium for colony forming unit-based viability analysis. Like other in vitro model systems, a possible limitation of this approach is difficulty in extrapolating the results obtained to a highly complex immune cell circuitry existing in the human host. However, despite this, the current protocol is very useful to elucidate the strategies that a fungal pathogen may employ to evade/counteract antimicrobial response and survive, adapt, and proliferate in the nutrient-poor environment of host immune cells.

Establishment of an In vitro System to Study Intracellular Behavior of Candida glabrata in Human THP-1 Macrophages

The current article outlines a protocol to establish an in vitro cell culture model system to study the interaction of a facultative intracellular human fungal pathogen Candida glabrata with human macrophages which will be a useful tool to advance our knowledge of fungal virulence mechanisms.

A cell culture model system, if a close mimic of host environmental conditions, can serve as an inexpensive, reproducible and easily manipulatable ...

A Microscopic Phenotypic Assay for the Quantification of Intracellular Mycobacteria Adapted for High-throughput/High-content Screening

Despite the availability of therapy and vaccine, tuberculosis (TB) remains one of the most deadly and widespread bacterial infections in the world. Since several decades, the sudden burst of multi- and extensively-drug resistant strains is a serious threat for the control of tuberculosis. Therefore, it is essential to identify new targets and pathways critical for the causative agent of the tuberculosis, Mycobacterium tuberculosis (Mtb) and to search for novel chemicals that could become TB drugs. One approach is to set&#160;up methods suitable for the genetic and chemical screens of large scale libraries enabling the search of a needle in a haystack. To this end, we developed a phenotypic assay relying on the detection of fluorescently labeled Mtb within fluorescently labeled host&#160;cells using automated confocal microscopy. This in vitro assay allows an image based quantification of the colonization process of Mtb&#160;into the host and was optimized for the 384-well microplate format, which is proper for screens of siRNA-, chemical compound- or Mtb mutant-libraries. The images are then processed for multiparametric analysis, which provides read&#160;out inferring on the pathogenesis of Mtb&#160;within host cells.

Here, we describe a phenotypic assay applicable to the High-throughput/High-content screens of small-interfering synthetic RNA (siRNA), chemical compound, and Mycobacterium tuberculosis mutant libraries. This method relies on the detection of fluorescently labeled Mycobacterium tuberculosis within fluorescently labeled host&#160;cell using automated confocal microscopy.

Despite the availability of therapy and vaccine, tuberculosis (TB) remains one of the most deadly and widespread bacterial infections in the world. Since ...

Monitoring the Assembly of a Secreted Bacterial Virulence Factor Using Site-specific Crosslinking

This article describes a method to detect and analyze dynamic interactions between a protein of interest and other factors in vivo. Our method is based on the amber suppression technology that was originally developed by Peter Schultz and colleagues1. An amber mutation is first introduced at a specific codon of the gene encoding the protein of interest. The amber mutant is then expressed in E. coli together with genes encoding an amber suppressor tRNA and an amino acyl-tRNA synthetase derived from Methanococcus jannaschii. Using this system, the photo activatable amino acid analog p-benzoylphenylalanine (Bpa) is incorporated at the amber codon. Cells are then irradiated with ultraviolet light to covalently link the Bpa residue to proteins that are located within 3-8 &#197;. Photocrosslinking is performed in combination with pulse-chase labeling and immunoprecipitation of the protein of interest in order to monitor changes in protein-protein interactions that occur over a time scale of seconds to minutes. We optimized the procedure to study the assembly of a bacterial virulence factor that consists of two independent domains, a domain that is integrated into the outer membrane and a domain that is translocated into the extracellular space, but the method can be used to study many different assembly processes and biological pathways in both prokaryotic and eukaryotic cells. In principle interacting factors and even specific residues of interacting factors that bind to a protein of interest can be identified by mass spectrometry.

This article illustrates the use of pulse-chase radio labeling in combination with site-specific photocrosslinking to monitor interactions between a protein of interest and other factors in E. coli. Unlike traditional chemical cross-linking methods, this approach generates high resolution &#8220;snapshots&#8221; of an ordered assembly pathway in a living cell.

This article describes a method to detect and analyze dynamic interactions between a protein of interest and other factors in vivo. Our method is based on ...

ScanLag: High-throughput Quantification of Colony Growth and Lag Time

Growth dynamics are fundamental characteristics of microorganisms. Quantifying growth precisely is an important goal in microbiology. Growth dynamics are affected both by the doubling time of the microorganism and by any delay in growth upon transfer from one condition to another, the lag. The ScanLag method enables the characterization of these two independent properties at the level of colonies originating each from a single cell, generating a two-dimensional distribution of the lag time and of the growth time. In ScanLag, measurement of the time it takes for colonies on conventional nutrient agar plates to be detected is automated on an array of commercial scanners controlled by an in house application. Petri dishes are placed on the scanners, and the application acquires images periodically. Automated analysis of colony growth is then done by an application that returns the appearance time and growth rate of each colony. Other parameters, such as the shape, texture and color of the colony, can be extracted for multidimensional mapping of sub-populations of cells. Finally, the method enables the retrieval of rare variants with specific growth phenotypes for further characterization. The technique could be applied in bacteriology for the identification of long lag that can cause persistence to antibiotics, as well as a general low cost technique for phenotypic screens.

ScanLag is a high-throughput method for measuring the delay in growth, namely lag time, as well as the growth rate of colonies for thousands of cells in parallel. Screening using ScanLag enables the discrimination between long lag-time and slow growth at the level of a single variant.

Growth dynamics are fundamental characteristics of microorganisms. Quantifying growth precisely is an important goal in microbiology. Growth dynamics are ...

Assessing Anti-fungal Activity of Isolated Alveolar Macrophages by Confocal Microscopy

The lung is an interface where host cells are routinely exposed to microbes and microbial products. Alveolar macrophages are the first-line phagocytic cells that encounter inhaled fungi and other microbes. Macrophages and other immune cells recognize Aspergillus motifs by pathogen recognition receptors and initiate downstream inflammatory responses. The phagocyte NADPH oxidase generates reactive oxygen intermediates (ROIs) and is critical for host defense. Although NADPH oxidase is critical for neutrophil-mediated host defense1-3, the importance of NADPH oxidase in macrophages is not well defined. The goal of this study was to delineate the specific role of NADPH oxidase in macrophages in mediating host defense against A. fumigatus. We found that NADPH oxidase in alveolar macrophages controls the growth of phagocytosed A. fumigatus spores4. Here, we describe a method for assessing the ability of mouse alveolar macrophages (AMs) to control the growth of phagocytosed Aspergillus spores (conidia). Alveolar macrophages are stained in vivo and ten days later isolated from mice by bronchoalveolar lavage (BAL). Macrophages are plated onto glass coverslips, then seeded with green fluorescent protein (GFP)-expressing A. fumigatus spores. At specified times, cells are fixed and the number of intact macrophages with phagocytosed spores is assessed by confocal microscopy.

A method to evaluate the ability of isolated mouse alveolar macrophages to control the growth of phagocytosed Aspergillus spores by confocal microscopy.

The lung is an interface where host cells are routinely exposed to microbes and microbial products. Alveolar macrophages are the first-line phagocytic ...

Applying an Inducible Expression System to Study Interference of Bacterial Virulence Factors with Intracellular Signaling

The technique presented here allows one to analyze at which step a target protein, or alternatively a small molecule, interacts with the components of a signaling pathway. The method is based, on the one hand, on the inducible expression of a specific protein to initiate a signaling event at a defined and predetermined step in the selected signaling cascade. Concomitant expression, on the other hand, of the gene of interest then allows the investigator to evaluate if the activity of the expressed target protein is located upstream or downstream of the initiated signaling event, depending on the readout of the signaling pathway that is obtained. Here, the apoptotic cascade was selected as a defined signaling pathway to demonstrate protocol functionality. Pathogenic bacteria, such as Coxiella burnetii, translocate effector proteins that interfere with host cell death induction in the host cell to ensure bacterial survival in the cell and to promote their dissemination in the organism. The C. burnetii effector protein CaeB effectively inhibits host cell death after induction of apoptosis with UV-light or with staurosporine. To narrow down at which step CaeB interferes with the propagation of the apoptotic signal, selected proteins with well-characterized pro-apoptotic activity were expressed transiently in a doxycycline-inducible manner. If CaeB acts upstream of these proteins, apoptosis will proceed unhindered. If CaeB acts downstream, cell death will be inhibited. The test proteins selected were Bax, which acts at the level of the mitochondria, and caspase 3, which is the major executioner protease. CaeB interferes with cell death induced by Bax expression, but not by caspase 3 expression. CaeB, thus, interacts with the apoptotic cascade between these two proteins.

The method described here is used to induce the apoptotic signaling cascade at defined steps in order to dissect the activity of an anti-apoptotic bacterial effector protein. This method can also be used for inducible expression of pro-apoptotic or toxic proteins, or for dissecting interference with other signaling pathways.

The technique presented here allows one to analyze at which step a target protein, or alternatively a small molecule, interacts with the components of a ...

Identification of Virulence Markers of Mycobacterium abscessus for Intracellular Replication in Phagocytes

What differentiates Mycobacterium abscessus from other saprophytic mycobacteria is the ability to resist phagocytosis by human macrophages and the ability to multiply inside such cells. These virulence traits render M. abscessus pathogenic, especially in vulnerable hosts with underlying structural lung disease, such as cystic fibrosis, bronchiectasis or tuberculosis. How patients become infected with M. abscessus remains unclear. Unlike many mycobacteria, M. abscessus is not found in the environment but might reside inside amoebae, environmental phagocytes that represent a potential reservoir for M. abscessus. Indeed, M. abscessus is resistant to amoebal phagocytosis and the intra-amoeba life seems to increase M. abscessus virulence in an experimental model of infection. However, little is known about M. abscessus virulence in itself. To decipher the genes conferring an advantage to M. abscessus intracellular life, a screening of a M. abscessus transposon mutant library was developed. In parallel, a method of RNA extraction from intracellular Mycobacteria after co-culture with amoebae was developed. This method was validated and allowed the sequencing of whole M. abscessus transcriptomes inside the cells; providing, for the first time, a global view on M. abscessus adaptation to intracellular life. Both approaches give us an insight into M. abscessus virulence factors that enable M. abscessus to colonize the airways in humans.

Identification of Virulence Markers of Mycobacterium abscessus for Intracellular Replication in Phagocytes

Here, we present two protocols to study Phagocyte-Mycobacterium abscessus interactions: the screening of a transposon mutant library for bacterial intracellular deficiency and the determination of bacterial intracellular transcriptome from RNA sequencing. Both approaches provide insight into the genomic advantages and transcriptomic adaptations enhancing intracellular bacteria fitness.

What differentiates Mycobacterium abscessus from other saprophytic mycobacteria is the ability to resist phagocytosis by human macrophages and the ability ...

Quantification of Intracellular Growth Inside Macrophages is a Fast and Reliable Method for Assessing the Virulence of Leishmania Parasites

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Chapters in this video

In vivo Imaging of Transgenic Leishmania Parasites in a Live Host

Protocol for Production of a Genetic Cross of the Rodent Malaria Parasites

A Parasite Rescue and Transformation Assay for Antileishmanial Screening Against Intracellular Leishmania donovani Amastigotes in THP1 Human Acute Monocytic Leukemia Cell Line

Establishment of an In vitro System to Study Intracellular Behavior of Candida glabrata in Human THP-1 Macrophages

A Microscopic Phenotypic Assay for the Quantification of Intracellular Mycobacteria Adapted for High-throughput/High-content Screening

Monitoring the Assembly of a Secreted Bacterial Virulence Factor Using Site-specific Crosslinking

ScanLag: High-throughput Quantification of Colony Growth and Lag Time

Assessing Anti-fungal Activity of Isolated Alveolar Macrophages by Confocal Microscopy

Applying an Inducible Expression System to Study Interference of Bacterial Virulence Factors with Intracellular Signaling

Identification of Virulence Markers of Mycobacterium abscessus for Intracellular Replication in Phagocytes