Geração e Multi-fenotípica rastreio de alto teor de<em> Coxiella burnetii</em> mutantes por transposões

The overall goal of this procedure is to identify at a large scale. The COE factors implicated in the key steps of the infectious cycle, including entry into host cells generation of a bacterial replicative niche and persistence. This is accomplished by first generating a GF PT co mutant library by transpose on mutagenesis.

Next, the host cells are infected with every isolated mutant. Then the intracellular replication of every mutant is followed in real time using a microplate reader in order to identify the mutations that are most detrimental to coxiella infection. Finally, the coxiella infected cells are analyzed by automated microscopy.

Ultimately, automated image analysis allows the morphological characterization of every phenotype obtained to associate each mutated gene with a putative function in the coxiella infectious cycle. This method can help address key issues in the field of host pathogen interactions, such as the large scale identification of the essential genes of a given pathogen required to interact with the the whole cell and hijack its molecular machineries to its benefit. Visual demonstration of this method is critical as the plating and isolation of COE colonies are difficult to achieve.

Indeed, COE forms very small colonies that need to be carefully extracted from soft ACCM two aro. How this method can provide insight into coccal lab infection. It can also be applied to other intracellular pathogenic bacteria such as salmonella, burel, mycobacterium, et cetera.

After transforming competent coxiella with transposon and transposase coating plasmids according to the text protocol, to isolate individual mutants, prepare bottom aros by mixing 10 milliliters of melted 0.5%aros with 10 milliliters of two X accm, two in a microbial safety cabinet or MSC and add the appropriate antibiotics immediately pour into a Petri dish and allow it to cool uncovered for 30 minutes and then air dry for 20 minutes. To prepare top bag aros, mix 1.25 milliliters of two x accm two with 0.75 milliliters of water in a 15 milliliter polystyrene tube. Add the appropriate antibiotics and incubate at 37 degrees Celsius.

Then add one to 100 microliters of bacterial culture and vortex for five seconds. Next, add 0.5 milliliters of melted aros mix and immediately pour over the bottom aros. Allow the plate to cool for 20 minutes.

Place the lid on top and incubate at four degrees Celsius for 20 minutes to help solidify. Then remove the lid and air dry the plate for 20 minutes before transferring to a 37 degree Celsius humidified incubator with 5%carbon dioxide and 2.5%oxygen for six to seven days. Once colonies are detectable, collect them by cutting the end of a one milliliter pipette tip and using it to isolate plugs containing single colonies.

Then transferring them into the wells of a 24 well plate containing 1.5 milliliters of accm two. After preparing a standard curve, according to the text protocol, quantitate each bacterial suspension by dispensing five microliters of 10%Triton X 100 per well. In a 96 well microplate with black walls and bottom, add 50 microliters of the bacterial suspensions to each well and incubate on a plate shaker for 10 minutes.

At room temperature, use one XTE buffer to dilute double stranded DNA quantitation reagent one to 200, and add 55 microliters of the diluted reagent to each sample. In the 96 well microplate with a plate shaker mixed well before incubating in the dark at room temperature for two to five minutes, measure the fluorescence of the samples using a fluorescence microplate reader and filters for standard fluorescein wavelengths. To obtain the bacterial DNA concentration plot, the fluorescence readings in the standard curve previously prepared, divide the DNA concentration by the mass of the coxiella genome to obtain bacterial concentration.

Express the results in genome equivalents per milliliter after carrying out single primer colony PCR and DNA sequencing according to the text protocol. Using sequence analysis software, load the complete annotated genome of Coxiella Burnett I 4 93 NM one with the align to reference function, load and align the sequence results using blast N and determine the site of transposition. After preparing a Vero cell suspension of 10 to the fifth cells per milliliter in RPMI medium, according to the text protocol, dispense 100 microliters of the suspension in each well of a black 96 well plate with a flat, transparent bottom, centrifuge the plate at 400 times G and room for five minutes and incubate at 37 degrees Celsius and 5%carbon dioxide overnight.

The following day fall at room temperature 96 well plates containing the coxiella mutants and dilute 150 microliters of bacterial suspensions in 300 microliters of RPMI without phenol red and FBS in a deep well 96 well plate next, remove the medium from the Vero cells and dispense 100 microliters per well of diluted coxiella mutants. Use the A one well as a negative control and wells a two and a three as positive controls using an aerosol tight centrifuge plate holder centrifuge the plate at 400 times G and room temperature for 10 minutes. Then after incubating the plate at 37 degrees Celsius in a humidified atmosphere of 5%carbon dioxide for two hours, replace the bacteria containing medium with 100 microliters per well of fresh complete RPMI medium with a fluorescence microplate reader and filters for fluorescein.

Measure GFP fluorescence every day for seven days On day seven post-infection, remove the medium from the plate and replace it with 50 microliters per well of fresh complete medium containing a one to 1000 dilution of cell permeable fluorescent dye. Incubate the cells for 30 to 60 minutes after the incubation, replace the medium with 50 microliters per well of 4%PFA in PBS incubate at room temperature for 30 minutes. Then remove the PFA containing buffer before using PBS to wash the wells three times After removing the final wash, dispense 50 microliters of blocking solution to each well and incubate at room temperature for 30 minutes.

Then replace the blocking solution with 40 microliters per well, a fresh blocking solution and a one to 500 dilution of anti lamp one antibody. After incubating at room temperature for 30 minutes, remove the solution and use a plate washer to wash the wells five times by applying 100 microliters per well of PBS. Then add 40 microliters per well of blocking solution containing the appropriate fluorescent secondary antibody and hooked 3 3 2 5 8 at five micrograms per milliliter.

Incubate at room temperature for 30 minutes. Wash the samples five times and leave the final PBS wash in the wells to keep them from drying out. To carry out image acquisition, use an epi fluorescence automated microscope equipped with a 20 x objective and 3 4 88 5 55 and six 15 nanometer channels.

Acquire 21 independent fields per well in order to image a minimum of 5, 000 cells per sample. Apply auto focusing using the host cell nuclei channel as a reference. Finally, use automated image analysis to extrapolate a number of relevant features from the acquired images.

This figure illustrates 38 transpose on mutants in 16 point ICM Coxe L at Genes aen growth curves that assess the viability of mutants are shown here.Here. Intracellular growth curves for coxiella mutants incubated with epithelial cells provide quantitative analysis of the phenotypes associated with transpose on insertions in the coxiella genome. In this figure, automated image acquisition was performed to identify and characterize host cell nuclei, cell contours, lysosomes, and coxiella colonies.

Correlating coxiella colonies with cells and lysosomes allows the morphological analysis of coxiella containing vacuoles. Correlating coxiella colonies with host cell contours allows the morphological analysis of infected cells. Finally, the four channels are merged.

The average area of coxiella colonies is plotted against the number of colonies per cell in order to identify mutations that affect intracellular replication of coxiella and or the capacity of bacteria to invade host cells. Mutants were observed in three clusters mutations that affected the intracellular growth of coxiella coxiella internalization in cells and non-significant phenotypes. Here, the average area of coxiella colonies are plotted against the number of host cells surviving infection to identify mutations that confer cytotoxicity to coxiella After its development.

This technique paved the way for researchers in the field of bacterial infections to explore host pathogen interactions at a large scale, and identified the host and bacterial targets for development of new therapies to counter infectious disease.