The overall goal of this procedure is to study and compare the virulence of Mycobacterium abscessus strains and to describe the interactions between these bacteria and the host innate immune system in vivo using transparency zebrafish embryos. This is accomplished by first preparing homogenous bacterial inocular. Next, the embryos are intravenously injected with the bacterial suspensions at 30 hours post fertilization.
Additionally, to study more precisely the role of macrophages in the infection control, a lipo choate based injection procedure is used to generate macrophage depleted embryos. Finally, the infected embryos are prepared and imaged using fluorescence and confocal microscopy to monitor the progression of the infection. Ultimately, the results which can be obtained show the specific interactions between the fluorescent phagocytic cells and M abscessus, either as individual basi or as serpentine cords inside a transparent embryo.
The main advantage of using zebra fish over other animal models like mice is that it allows to specifically investigate the infection process in real time and to study an image the specific interactions between microbacterium abscesses and microphages or neutrophils. This step is a critical issue for the subsequent injection pressure in zebrafish. Due to high propensity of rough mycobacterium abscessus to form large aggregates and to produce codes, a specific treatment is therefore necessary to prepare homogenous and quantitatively controlled inocular prior to microinjection.
To prepare m abscessus, inocular harvest exponentially growing cultures from 150 square centimeter tissue culture flasks into 50 milliliter sterile plastic tubes and centrifuge at 4, 000 times G and room temperature for 15 minutes. Use one milliliter of Middlebrook seven H nine, supplemented with the OADC enrichment, plus tween 80 hereafter referred to as seven H nine medium to resuspend the pellet and Eloqua. 200 microliters of the bacterial suspensions into 1.5 milliliter tubes with a 26 gauge needle homogenize each ALI quad of bacterial suspension 15 times.
Then using a water bath sonicate sonicate three times for 10 seconds each with ten second breaks. In between, add one milliliter of seven H nine, medium and briefly vortex, then centrifuge at 100 times G for three minutes. Carefully collect the mycobacteria containing supernatants to avoid clumps and pull the homogeneous suspensions in a 50 milliliter sterile plastic tube.
Centrifuge the suspension at 4, 000 times G for five minutes and use 200 microliters of seven H nine medium to resuspend the pellet assess final bacterial inoculum. According to the text protocol, The visual demonstration of this method, it's critical as the steps are difficult to learn because the precise gentle manipulation of the injection in zebrafish embryos under the microscope as well as the acquisition of the microinjection technique required time To carry out microinjection of M abscessus at about 24 hours. Post fertilization or HPF, use tweezers to coate embryos in a 100 millimeter Petri dish and keep them at 28.5 degrees Celsius.
Transfer 30 48 HPF embryos to a V-shaped positioning chamber filled with 25 milliliters of fish water containing 270 milligrams per liter of trica with a clipped micro loader tip. Lay the embryos properly in the channels for easier manipulation. Use PBST with 0.05%between 80 to dilute the microbacterial inoculum to the desired CFU to be delivered and include 10%phenol red to check proper injection with a micro loader tip.
Load a microinjection needle with five to 10 microliters of the bacterial inoculum. Connect the microinjection needle into the holder of a micro manipulator connected to the injector, and use fine tweezers to break off the top of the needle. To obtain an opening diameter of five to 10 micrometers.
Calibrate the injection volume by adjusting the microinjection pressure and time. Then to obtain the required injection volume, measure the diameter of a droplet expelled into the yolk of one embryo to micro inject into the coddle vein. Position the embryo with the ventral side facing the needle and place the needle tip close to the urogenital opening.
And gently push the tip of the needle into the embryo until it just pierces the coddle vein region. Then deliver the desired volume of bacterial suspension, usually one to three nanoliters containing around 100 CFUs per nanoliter. Incubate and monitor the embryos according to the text protocol to carry out lipo Choate, depletion of macrophages and embryos at 24 HP.Fate, the embryos, and transfer them to an injection dish filled with fish water with trica.
Then orient them dorsal side downwards. After vortexing the liposome encapsulated clodronate solution, load the micro capillary needle and calibrate the injection volume. Pierce the coddle vein region as demonstrated earlier, and inject two to three liters of solution.
Repeat the injection three times. Incubate the embryos at 28.5 degrees Celsius and use fluorescence microscopy to control the proper depletion of macrophages before infecting. As demonstrated earlier in this video to enumerate colony forming units or CFUs at the desired time point.
Collect five embryos per infection condition and transfer each embryo into a 1.5 milliliter micro centrifuge tube. Cryo anesthetize the embryos by incubation on ice for 10 minutes after euthanizing the embryos with 300 to 500 milligrams per liter.Trica. Use sterile water to wash the embryos twice in a new tube.
Next, after removing water at 2%Triton X 100 in one XPBS to each embryo, and use a 26 gauge needle to homogenize the tissue for complete lysis. After centrifuging the suspension, use one X-P-B-S-T to resuspend the pellet. Then plate serial dilution of the homogenous on Middlebrook seven H 10 OADC.
Supplemented with B-B-L-M-G-I-T Panta incubate the plates at 30 degrees Celsius for four days before counting the colonies for live imaging of M abscessus infection, mount trica anesthetized embryos in 1%low melting point aros in a 35 millimeter glass bottom Petri dish for an inverted microscope or a single cavity depression slide for upright confocal microscopy. Orient the embryo to the desired position and cover the solidified aros with fish water containing trica for fixed imaging of M abscessus infection. After euthanizing the embryos, transfer them to 1.5 milliliter micro centrifuge tubes, and fix the embryos in 4%Paraform aldehyde in one X-P-B-S-T for two hours.
At room temperature, remove the paraform aldehyde by washing the embryos twice with one X-P-B-S-T for 10 minutes to preserve the integrity of the tissues and fluorescence successively. Incubate the embryos in increasing concentrations of glycerol solution for 10 minutes per condition. Lay the embryo embedded in 50%glycerol in a viewing chamber.
Finally, use a fluorescence microscope with a 10 x objective or a fluorescence confocal microscope with 40 x or 63 x objectives for sequential fluorescence acquisition and transmission imaging of the embryo. To investigate and compare the virulence of rough and smooth M abscessus variants fluorescent bacteria were injected intravenously in 30 HPFC proficient embryos. In contrast to the smooth variant, the rough one induces a more robust and lethal infection with the development of abscesses within central nervous system.
Differences in virulence were quantified either by enumerating the CFU, or by determining the fluorescent pixel counts which are correlated. This points to higher bacterial loads for the rough variant As seen in this figure, the kinetics of cord formation can be monitored and imaged by video microscopy in a noninvasive manner, demonstrating the propensity of the rough variant to replicate extracellularly as shown here. Infection of macrophage depleted embryos with the rough M abscessus leads to a massive increase in the bacterial loads and cord production and to rapid larval death.
This clearly indicates that macrophages are required to control M abscessus infection to observe in real time the interactions between macrophages and M abscessus. The EG one M cherry transgenic line that produces red fluorescent macrophages can be used. Infections induce a marked recruitment of macrophages with efficient phagocytosis of individual bacteria, whereas mycobacterial cords represent a strategy evolved by am abscesses to avoid being phagocytosed After it's development.
This technique paved the way to explore the important role of coding as a new mechanism of immune evasion. Thanks to the zebrafish mole of infection, it becomes now possible to observe and describe the in vivo contribution of codes in the pathogenesis of mycobacterium abscesses.
