Identification of Host Pathways Targeted by Bacterial Effector Proteins using Yeast Toxicity and Suppressor Screens

Intracellular bacteria secrete effector proteins into the host that can manipulate biological pathways to promote pathogen survival. Revealing host targets of effectors is essential for understanding intracellular pathogens like Chlamydia trachomatis. Yeast toxicity and suppressor screens can provide key insight regarding the natural biological target of bacterial effector proteins and can be particularly useful when a binding partner is unknown.

We demonstrate the yeast toxicity and suppressor assays here for screening for Chlamydia trachomatis effector proteins, but this technique has also been used for characterizing Legionella pneumophila and Coxiella burnetii effectors. Begin by inoculating five milliliters of single drop-out broth with the single colony of yeast transformed with the effector protein containing plasmid. Use yeast transformed with the vector alone as a negative control and incubate the inoculum overnight at 30 degrees Celsius while shaking.

On the next day, add 180 microliters of sterile water to the A2 through A6 wells of a 96-well plate. Vortex the overnight culture and add 180 microliters of yeast to well A1.Then serially dilute the yeast in the wells with water. Use a multi-channel pipette to spot five microliters of each dilution onto single drop-out glucose and single drop-out galactose agar plates and incubate the plates at 30 degrees Celsius for 48 hours.

After the incubation, visually assess toxicity by comparing the growth of yeast expressing the effector protein grown on the galactose-containing media to the growth of yeast expressing the vector alone. Inoculate 100 milliliters of the single drop-out broth with one milliliter of the previously prepared yeast stock and incubate it for 16 to 24 hours at 30 degrees Celsius with shaking at 150 RPM. On the next day, add the entire 100 milliliters of the overnight culture to 900 milliliters of pre-warmed single drop-out broth and incubate the flask for four to five hours at 30 degrees Celsius while shaking.

After the incubation, pellet the culture at 6, 000 times g for 10 minutes at four degrees Celsius. Discard the supernatant and resuspend the pellet in 250 milliliters of sterile water. Repeat the centrifugation and discard the supernatant.

Then resuspend the pellet in 250 milliliters of one millimolar lithium acetate. Pellet the culture by centrifugation, remove the lithium acetate, and resuspend the pellet in 9.6 milliliters of 50%PEG 3350. Then add transformation reagents as described in the manuscript and adjust the volume to 15 milliliters with sterile water inverting gently to mix.

Incubate the mixture at 30 degrees Celsius for 30 minutes. Then add 750 microliters of DMSO and incubate in a water bath at 42 degrees Celsius for another 30 minutes. Adding the DMSO before heat shock is crucial to achieve a high transformation efficiency.

After the incubation, pellet the yeast by centrifuging at 3, 000 times g for five minutes. Discard the supernatant and wash the pellet with 10 milliliters of sterile water then repeat the centrifugation to pellet the yeast. Resuspend the pellet in eight milliliters of water.

Determine the transformation efficiency by diluting the sample one to 10 and plating 100 microliters of each dilution onto double drop-out agar. Then plate 200 microliters of the sample on double drop-out galactose agar plates and incubate the plates at 30 degrees Celsius for 48 to 96 hours or until colonies appear. Once colonies have appeared, patch them on double drop-out galactose agar and incubate them for another 24 to 48 hours.

Use part of the patch to inoculate five milliliters of double drop-out broth and incubate the inoculum overnight at 26 degrees Celsius with shaking at 150 RPM. On the next day, add 180 microliters of sterile water to five wells of a 96-well plate starting with well A2.Vortex the overnight culture mix. Add 180 microliters of yeast to well A1 and serially dilute it using the water in wells A2 through A6.Spot five microliters of each dilution on single drop-out glucose and single drop-out galactose agar plates making sure to include the toxic effector alone as a control.

Incubate the plates at 30 degrees Celsius for 48 hours then compare the growth of the yeast with toxic effector alone to the growth of yeast with the potential suppressor. To confirm suppressors that have demonstrated diminished toxicity compared to the toxic effector alone, isolate the plasmid according to manuscript directions and re-transform it into the toxic yeast. Inoculate double drop-out glucose broth with a colony from the transformation plate.

Incubate it overnight and spot on double drop-out glucose and galactose agar to confirm suppression of toxicity. Prior to performing the yeast suppressor screen, the effector proteins of interest were tested for toxicity in yeast. The protein of interest was expressed in yeast under the control of a galactose inducible promoter and growth on galactose was compared to growth on glucose.

When CT229 was tested for toxicity, smaller colonies and growth suppression were observed. Ideally, a two to three log decrease should be observed to proceed with yeast suppressor screens. The suppressor screen was conducted by transforming the toxic strain with the yeast genomic library pYAP 13 and plating the transformants on galactose agar.

The obtained suppressor clones were spotted on double drop-out galactose agar to confirm suppression of toxicity. Suppressors pSup1 and pSup2 suppressed the toxicity of the effector protein whereas pSup3 did not. These procedures require careful attention to detail in addition to critical preparatory work including having large amounts of media and plates ready prior to performing the experiment.

Once suppressors are identified, experiments can be performed to verify interactions with the effector of interest including pull downs, immunofluorescence colocalization, effector knockout or knockdown of host factors identified in the screens. Development of this technique for Chlamydia effectors has allowed for expedited preliminary characterization of effector proteins and helped to focus our efforts on elucidating the underlying molecular mechanisms that mediate host pathogen interactions.