Name: identification of host pathways targeted by bacterial effector proteins using yeast toxicity and suppressor screens
Uploaded: 2019-10-25T15:00:00
Description: Watch this Scientific Journal Video about Identification of Host Pathways Targeted by Bacterial Effector Proteins using Yeast Toxicity and Suppressor Screens at JoVE.com

We thank Shelby Andersen, Abby McCullough, and Laurel Woods for their assistance with these techniques. This study was funded by startup funds from the University of Iowa Department of Microbiology and Immunology to Mary M. Weber.

1. Preparation of media and reagents
NOTE: Plates should be prepared before the day of the assay and are good for 1 month. Media and reagents can be made at any point and are good for 1 month.
<ol>
	<li>Prepare 1 L of the glucose solution (10% w/v) by dissolving 100 g of D-(+)-glucose in 800 mL of distilled water in a 1, 000 mL beaker. Adjust the volume to 1 L with distilled water. Filter through a 0.2 &#181;m sterile filter into a sterile 1 L media storage bottle.</li>
	<li>Prepare 1 L of t...

<ol>
	<li>Tan, Y., Luo, Z. Q. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Legionella+pneumophila+SidD+is+a+deAMPylase+that+modifies+Rab1.">Legionella pneumophila SidD is a deAMPylase that modifies Rab1.</a> Nature. 475 (7357), 506-509 (2011).</li><li>Guo, Z., Stephenson, R., Qiu, J., Zheng, S., Luo, Z. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+Legionella+effector+modulates+host+cytoskeletal+structure+by+inhibiting+actin+polymerization.">A Legionella effector modulates host cytoskeletal structure by inhibiting actin polymerization.</a> Microbes and Infection. 16 (3), 225-236 (2014).</li><li>Tan, Y., Arnold, R. J., Luo, Z. -. Q. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Legionella+pneumophila+regulates+the+small+GTPase+Rab1+activity+by+reversible+phosphorylcholination.">Legionella pneumophila regulates the small GTPase Rab1 activity by reversible phosphorylcholination.</a> Proceedings of the National Academy of Sciences of the United States of America. 108 (52), 21212-21217 (2011).</li><li>Weber, M. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+type+IV+secreted+effector+protein+CirA+stimulates+the+GTPase+activity+of+RhoA+and+is+required+for+virulence+in+a+mouse+model+of+Coxiella+burnetii+infection.">The type IV secreted effector protein CirA stimulates the GTPase activity of RhoA and is required for virulence in a mouse model of Coxiella burnetii infection.</a> Infection and Immunity. 84, (2016).</li><li>Faris, R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chlamydia+trachomatis+CT229+subverts+Rab+GTPase-dependent+CCV+trafficking+pathways+to+promote+chlamydial+infection.">Chlamydia trachomatis CT229 subverts Rab GTPase-dependent CCV trafficking pathways to promote chlamydial infection.</a> Cell Reports. 26, 3380-3390 (2019).</li><li>Mumberg, D., M&#252;ller, R., Funk, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Yeast+vectors+for+the+controlled+expression+of+heterologous+proteins+in+different+genetic+backgrounds.">Yeast vectors for the controlled expression of heterologous proteins in different genetic backgrounds.</a> Gene. 156, 119 (1995).</li><li>Hill, J., Donald, K. A. G., Griffiths, D. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=DMSO-enhanced+whole+cell+yeast+transformation.">DMSO-enhanced whole cell yeast transformation.</a> Nucleic Acids Research. 19 (20), 41070 (1991).</li><li>Kramer, R. W., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Yeast+Functional+Genomic+Screens+Lead+to+Identification+of+a+Role+for+a+Bacterial+Effector+in+Innate+Immunity+Regulation.">Yeast Functional Genomic Screens Lead to Identification of a Role for a Bacterial Effector in Innate Immunity Regulation.</a> PLoS Pathogens. 3 (2), 21 (2007).</li><li>H&#228;user, R. T. S., Rajagopala, S. V., Uetz, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Array-Based+Yeast+Two-Hybrid+Screens:+A+Practical+Guide.">Array-Based Yeast Two-Hybrid Screens: A Practical Guide.</a> Two Hybrid Technologies: Methods and Protocols, Methods in Molecular Biology. , 21-38 (2012).</li><li>Mirrashidi, K. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Global+mapping+of+the+inc-human+interactome+reveals+that+retromer+restricts+chlamydia+infection.">Global mapping of the inc-human interactome reveals that retromer restricts chlamydia infection.</a> Cell Host and Microbe. 18 (1), 109-121 (2015).</li><li>Wang, Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Development+of+a+transformation+system+for+chlamydia+trachomatis:+Restoration+of+glycogen+biosynthesis+by+acquisition+of+a+plasmid+shuttle+vector.">Development of a transformation system for chlamydia trachomatis: Restoration of glycogen biosynthesis by acquisition of a plasmid shuttle vector.</a> PLoS Pathogens. 7 (9), 1002258 (2011).</li><li>Mueller, K. E., Wolf, K., Fields, K. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Gene+deletion+by+fluorescence-reported+allelic+exchange+mutagenesis+in+Chlamydia+trachomatis.">Gene deletion by fluorescence-reported allelic exchange mutagenesis in Chlamydia trachomatis.</a> mBio. 7 (1), 1-9 (2016).</li><li>Johnson, C. M., Specific Fisher, D. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Site-Specific,+Insertional+Inactivation+of+incA+in+Chlamydia+trachomatis+Using+a+Group+II+Intron.">Site-Specific, Insertional Inactivation of incA in Chlamydia trachomatis Using a Group II Intron.</a> PLoS ONE. 8 (12), 83989 (2013).</li><li>Weber, M. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Absence+of+specific+Chlamydia+trachomatis+inclusion+membrane+proteins+triggers+premature+inclusion+membrane+lysis+and+host+cell+death.">Absence of specific Chlamydia trachomatis inclusion membrane proteins triggers premature inclusion membrane lysis and host cell death.</a> Cell Reports. 19 (7), 1406-1417 (2017).</li><li>Weber, M. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+functional+core+of+IncA+is+required+for+Chlamydia+trachomatis+inclusion+fusion.">A functional core of IncA is required for Chlamydia trachomatis inclusion fusion.</a> Journal of Bacteriology. 198 (8), 1347-1355 (2016).</li></ol>

This protocol outlines step-by-step procedures for identifying host biological pathways targeted by bacterial effector proteins using a modified yeast toxicity and suppressor screen. The yeast strain used, S. cerevisiae W303, is auxotrophic for both uracil and leucine. Uracil auxotrophy of the strain is used to select yeast carrying the protein of interest on the pYesNTA-Kan vector while leucine auxotrophy is used to select for the yeast genomic library vector pYep13. The yeast genomic library plasmids carry 3&#...

The first report of procedures similar to those presented here characterized the Legionella pneumophila type IV effector SidD, a deAMPylase that modifies Rab11. Comparable techniques were used for the characterization of several L. pneumophila effectors1,2,3. The assay was adapted to characterize a Coxiella burnetii type IV effector protein4, and recently the utility of this technique was expanded for the characterization of Chlamydia trachomatis inclusion membrane proteins5.
This protocol can be broken into two major parts: 1) the yeast toxicity screen, in which the bacterial effector protein of interest is expressed in yeast and clones are screened for a toxic phenotype as evidenced by a growth defect, and 2) the yeast suppressor screen, in which the toxic phenotype is suppressed by expression of a yeast genomic library in the toxic strain. Thus, the toxicity screen is a screen for toxic phenotypes that manifest as growth defects when the bacterial effector of interest is overexpressed. Toxic clones, successfully transformed with and expressing the bacterial effector, are selected and saved for the next step. The second major step involves overexpressing a partially digested yeast genomic library in the toxic yeast clone. Plasmids making up the yeast genomic library suggested for the use in this protocol carry 5&#8722;20 kb inserts, usually corresponding to 3&#8722;13 yeast open reading frames (ORF) of an average gene size of ~1.5 kb across all plasmids, representing the whole yeast genome covered approximately 10x. This part of the assay is called the suppressor screen, as the goal is to suppress the toxicity of the bacterial effector protein. Potential suppressor plasmids are isolated from yeast, sequenced, and the suppressing ORFs identified. The rationale underlying the suppressor screen is that the effector protein binds, interacts with, and/or overwhelms components of the host pathway it targets, and that providing those host proteins back in excess can rescue the toxic effect on the pathway and thus, the growth defect. Thus, identified ORFs that suppress toxicity often represent multiple participants of a host pathway. Orthogonal experiments are then performed to verify that the bacterial effector indeed interacts with the implicated pathway. This is especially necessary if a binding partner such as clathrin or actin has been identified, because these proteins are involved in a multitude of host processes. Further experiments can then elucidate the physiological function of the effector protein during infection. The toxicity and suppressor screens are also powerful tools for deciphering the physiological function of bacterial effector proteins that do not physically bind host proteins with affinities sufficient to detect by immunoprecipitation or that interact with the host in enzymatic hit-and-run interactions that may not be detected by a yeast-two hybrid screen.
Although the suppressor screen can be a powerful method to reveal potential physiological interactions between bacterial effector proteins and host pathways, the bacterial effector protein must induce a growth defect in yeast, otherwise using it in the suppressor screen will be of little use. Furthermore, the toxic phenotype must result in at least a 2&#8722;3 log10 deficit in growth or it will be difficult to identify suppressors. If a laboratory is set up for cell culture, screening effector proteins for toxicity in common cell lines such as HeLa can often give insight as to whether it is worth the effort to proceed with the yeast toxicity screen. Ectopic expression of the effector protein in HeLa cells sometimes results in toxicity that correlates very strongly with toxicity in the yeast strain used for these screens4. Observable hallmarks of stress in HeLa cells include loss of stress fibers, cell detachment from the plate, and nuclear condensation indicating apoptosis. Any visual indication of stress in HeLa cells make the protein of interest a good candidate for inducing a growth defect in yeast, which replicate much more rapidly and are thus more responsive to perturbation of essential pathways.
It should be noted that the suppressor screen does not always identify host binding partners as suppressors, but it can still implicate critical components of the host pathway(s) targeted, yielding a holistic view of the biological processes being hijacked by the bacterial effector protein. On the surface, this seems counterintuitive, because providing the binding partner of the effector protein in excess would be expected to rescue the growth defect. In efforts to identify pathways targeted by the C. trachomatis effector protein CT229 (CpoS), which binds to at least 10 different Rab GTPases during infection5, none of the Rab binding partners suppressed the toxicity of CT229. However, numerous suppressors involved in clathrin-coated vesicle (CCV) trafficking were identified, which led to further work demonstrating that CT229 specifically subverts Rab-dependent CCV trafficking. Similarly, when investigating the C. burnetii effector protein Cbu0041 (CirA) several Rho GTPases that rescued the yeast growth defect were identified, and it was later found that CirA functions as a GTPase activating protein (GAP) for RhoA4.
The usefulness of the yeast suppressor screen for elucidating host pathways targeted by bacterial effector proteins cannot be overstated, and other researchers attempting to characterize intracellular bacterial effector proteins can greatly benefit from these techniques. These assays are of value if immunoprecipitations and/or yeast-two hybrid screens have failed to find a binding partner and can elucidate which pathways are targeted by the bacterial effector protein. Here, detailed protocols for the toxicity and suppressor screens to identify host biological pathways targeted by intracellular bacterial effector proteins are provided, as well as some of the common obstacles experienced when using these assays and their corresponding solutions.

<table border="0" cellpadding="0" cellspacing="0" style="width:871px;" width="870">
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	</colgroup>
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:319px;">Agar</td>
			<td style="width:199px;">Fisher Scientific</td>
			<td style="width:119px;">BP2641500</td>
			<td style="width:235px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:319px;">Galactose</td>
			<td style="width:199px;">MilliporeSigma</td>
			<td>G0750-1KG</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:319px;">GeneJet Gel extraction kit</td>
			<td style="width:199px;">ThermoFisher Scientific</td>
			<td style="width:119px;">K0691</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:319px;">GeneJet PCR purification kit</td>
			<td style="width:199px;">ThermoFisher Scientific</td>
			<td style="width:119px;">K0701</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:319px;">GeneJet plasmid miniprep kit</td>
			<td style="width:199px;">Thermo</td>
			<td style="width:119px;">K0503</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:319px;">Glucose</td>
			<td style="width:199px;">MilliporeSigma</td>
			<td>G8270-1KG</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:319px;">Herring Sperm DNA</td>
			<td style="width:199px;">Promega</td>
			<td>D1811</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:319px;">KpnI-HF</td>
			<td style="width:199px;">New England Biolabs</td>
			<td style="width:119px;">R3142S</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Lithium acetate dihydrate</td>
			<td style="width:199px;">MilliporeSigma</td>
			<td>L6883-250G</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Peptone</td>
			<td style="width:199px;">Fisher Scientific</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:319px;">Phusion High-Fidelity DNA Polymerase</td>
			<td style="width:199px;">New England Biolabs</td>
			<td style="width:119px;">M0530</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Poly(ethylene glycol) 3350</td>
			<td style="width:199px;">MilliporeSigma</td>
			<td>1546547-1G</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:319px;">pYep13</td>
			<td style="width:199px;">ATCC</td>
			<td style="width:119px;">37323</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:319px;">T4 DNA ligase</td>
			<td style="width:199px;">New England Biolabs</td>
			<td style="width:119px;">M0202S</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:319px;">Tryptophan</td>
			<td style="width:199px;">MilliporeSigma</td>
			<td>470031-1G</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:319px;">XhoI-HF</td>
			<td style="width:199px;">New England Biolabs</td>
			<td style="width:119px;">R0146S</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:319px;">Yeast extract</td>
			<td style="width:199px;">Fisher Scientific</td>
			<td>BP1422-500</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:319px;">Yeast miniprep kit</td>
			<td style="width:199px;">Zymo</td>
			<td style="width:119px;">D2001</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:319px;">Yeast nitrogen base without amino acids</td>
			<td style="width:199px;">MilliporeSigma</td>
			<td>Y0626-250G</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Yeast Synthetic Drop-out Medium Supplements</td>
			<td style="width:199px;">MilliporeSigma</td>
			<td>Y1501-20G</td>
			<td>without uracil</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Yeast Synthetic Drop-out Medium Supplements</td>
			<td style="width:199px;">MilliporeSigma</td>
			<td>Y1771-20G</td>
			<td>without uracil, leucine, tryptophan</td>
		</tr>
	</tbody>
</table>

identification of host pathways targeted by bacterial effector proteins using yeast toxicity and suppressor screens

Intracellular bacteria secrete virulence factors called effector proteins into the host cytosol that act to subvert host proteins and/or their associated biological pathways to the benefit of the bacterium. Identification of putative bacterial effector proteins has become more manageable due to advances in bacterial genome sequencing and the advent of algorithms that allow in silico identification of genes encoding secretion candidates and/or eukaryotic-like domains. However, identification of these important virulence factors is only an initial step. Naturally, the goal is to determine the molecular function of effector proteins and elucidate how they interact with the host. In recent years, techniques like the yeast two-hybrid screen and large-scale immunoprecipitations coupled with mass spectrometry have aided in the identification of protein-protein interactions. Although identification of a host binding partner is the crucial first step toward elucidating the molecular function of a bacterial effector protein, sometimes the host protein is found to have multiple biological functions (e.g., actin, clathrin, tubulin), or the bacterial protein may not physically bind host proteins, depriving the researcher of crucial information about the precise host pathway being manipulated. A modified yeast toxicity screen coupled with a suppressor screen has been adapted to identify host pathways impacted by bacterial effector proteins. The toxicity screen relies on a toxic effect in yeast caused by the effector protein interfering with the host biological pathways, which often manifests as a growth defect. Expression of a yeast genomic library is used to identify host factors that suppress the toxicity of the bacterial effector protein and thus identify proteins in the pathway that the effector protein targets. This protocol contains detailed instructions for both the toxicity and suppressor screens. These techniques can be performed in any lab capable of molecular cloning and cultivation of yeast and Escherichia coli.

Before the actual yeast suppressor screen can be performed, the effector protein of interest must be tested for toxicity in yeast. This is accomplished by expressing the protein of interest in yeast under the control of a galactose-inducible promoter. Growth on glucose (noninducing conditions) should first be compared to ensure toxicity is specifically due to the expression of the protein of interest and is not a general defect. As shown in Figure 3, toxicity manifests as smaller colonies an...

Watch this Scientific Journal Video about Identification of Host Pathways Targeted by Bacterial Effector Proteins using Yeast Toxicity and Suppressor Screens at JoVE.com

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

Bacterial pathogens secrete proteins into the host that target crucial biological processes. Identifying the host pathways targeted by bacterial effector proteins is key to addressing molecular pathogenesis. Here, a method using a modified yeast suppressor and toxicity screen to elucidate host pathways targeted by toxic bacterial effector proteins is described.

Intracellular bacteria secrete virulence factors called effector proteins into the host cytosol that act to subvert host proteins and/or their associated ...

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.

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A Deep-sequencing-assisted, Spontaneous Suppressor Screen in the Fission Yeast Schizosaccharomyces pombe

A genetic screen for mutant alleles that suppress phenotypic defects caused by a mutation is a powerful approach to identify genes that belong to closely ...

Enhanced Yeast One-hybrid Screens To Identify Transcription Factor Binding To Human DNA Sequences

Identifying the sets of transcription factors (TFs) that regulate each human gene is a daunting task that requires integrating numerous experimental and ...

Pooled CRISPR-Based Genetic Screens in Mammalian Cells

Genome editing using the CRISPR-Cas system has vastly advanced the ability to precisely edit the genomes of various organisms. In the context of mammalian ...