A Comparative Approach to Characterize the Landscape of Host-Pathogen Protein-Protein Interactions

1. Yeast Two-hybrid Screenings

1. Make the following required solutions
   1. Complete Synthetic Drop Out (SD): dissolve 26.7 g of minimal SD base with glucose in 1 L of water. Add 2 g of amino acid mixture prepared as follows: 2 g Adenine hemisulfate, 2 g Arginine HCl, 2 g Histidine HCl, 2 g Isoleucine, 4 g Leucine, 2 g Lysine HCl, 2 g Methionine, 3 g Phenylalanine, 2 g L-Serine, 2 g L-Threonine, 3 g Tryptophan, 2 g Tyrosine, 1.2 g Uracil, 9 g Valine.
   2. Selective Drop Out SD-L/-W/-H: SD containing every essential amino acids except those specified (-L: -Leucine ; -W: -Tryptophan; -H: -Histidine).
   3. YPGLU: For 1 L, weight 10 g of yeast extract, 10 g of Bacto Peptone and 20 g of glucose. Complete with 25 g Bacto agar for solid media.
   4. YCM: Same as YPGLU, but pH adjusted to 4.5.
2. Mating and spreading
   1. Use frozen aliquots of pre-transformed Y187 yeast strain (mating type α) containing a cDNA library cloned in pACT2. Check cell viability by plating serial dilutions on SD-L plates.
   2. Clone the pathogen ORF into pGBKT7 vector, and transform the recombinant plasmid using a standard procedure into AH109 yeast strain (mating type a).
   3. Spread pGBKT7-transformed AH109 yeast on SD-W plates. Incubate at least two days 30 °C in a humidified incubator. Plates can be stored at 4 °C until the two-hybrid screening.
   4. Use 3-aminotriazole (3-AT), a competitive inhibitor of the HIS3 enzyme, to counteract the potential self-transactivation of the HIS3 reporter gene by the pathogens’ proteins. Perform an auto-activation test by determining the concentration of 3-AT, which prevents growth of pGBKT7-transformed yeast on SD-W/-H plates.
   5. Seed 30 ml of SD-W with few colonies of pGBKT7-transformed AH109. Incubate 30 hr at 30 °C with rotation.
   6. Seed 200 ml of SD-W with the 30 ml of the AH109 cultures. Incubate with rotation around 20 hr at 30 °C.
   7. Incubate thawed cDNA library-transformed Y187 in 20 ml YPGLU, incubate 10 min at 30 °C with rotation.
   8. Centrifuge a volume of pGBKT7-transformed AH109 culture corresponding to an equivalent amount of viable Y187 yeast cells for 5 min at 3,500 rpm at 20 °C. Carefully withdraw the supernatant and resuspend pellet in 10 ml YPGLU.
   9. Mix the resuspended AH109 yeast with Y187 containing library. Transfer in a 50 ml tube. Centrifuge for 5 min at 3,500 rpm at 20 °C, carefully withdraw supernatant (fragile pellet).
   10. Resuspend pellet in YPGLU to get a total of 1.5 ml.
   11. Spread yeast on to 3 YCM-Agar 150 mm plates, 0.5 ml/plate. Incubate 4.5 hr at 30 °C.
   12. Collect mated yeast from YCM plates by scraping with a rake glass in 10 ml of SD-L/-W/-H. Rinse the plates two times with 5 ml SD-L/-W/-H medium and pool.
   13. Centrifuge 5 min at 3,500 rpm, 20 °C. Discard supernatant and resuspend the yeast pellet in 5 ml SD-L/-W/-H medium.
   14. Spread mated yeast on to 10 plates (0.5 ml/plate) of SD-L/-W/-H Agar supplemented with the appropriate concentration of 3-aminotriazole (3-AT) determined in the auto-activation test. Incubate at 30 °C in a humidified atmosphere for 6-10 days.
   15. Determine the diploid (2n) rate in order to evaluate mating efficiency by spreading 250 μl of serial dilutions (10^-4 to 10^-6) of the mated yeast suspension on SD-L/-W plates. Incubate plates at 30 °C for two days. Count colonies grown on SD-L/-W, and deduce the total diploid number present in the initial volume of mated yeast. Report this diploid number to viable library-containing yeast number to calculate the diploid rate. It should range around 10-25%.
   16. 6-10 days after incubation at 30 °C, pick mated yeast colonies in fresh SD-L/-W/-H + 3-AT plates. [Tip: order the yeast in a scheme of 8 lanes of 12 wells reproducing a 96 well plate so that it will be easier afterward for sequencing]. Let yeast colonies grow for 4-5 days at 30 °C in a humidified atmosphere.
3. Screening of his3 positive clones by PCR and sequencing
   The goal is to PCR amplify the ORF contained in the pACT2 vectors of yeast colonies grown on selective SD-L/-H/-W plates.
   1. Put a 96 well PCR plate on ice.
   2. To lyse yeast cells, add 50 μl per well of a Zymolase 20T solution (2.5 mg/ml in 10 mM HEPES Buffer pH 7.7).
   3. Gently resuspend one colony per well.
   4. Incubate 15 min at 37 °C and 15 min at 95 °C.
   5. Put the plate back on ice. Add 50 μl of distilled water per well. Mix well by pipetting up and down.
   6. Centrifuge for 5 min at 3,500 rpm and 4 °C.
   7. PCR amplify 10 μl to detect an insert using primers annealing either sides of the ORF derived from cDNA in pACT2 vectors. The protocol is given for amplification of 96 lysed yeast colonies with the ExTaq polymerase. Prepare the following mix: 525 μl 10X ExTaq buffer, 420 μl dNTPs mix (2.5 mM), 10.5 μl Forward Primer (1 μg/μl), 10.5 μl Reverse Primer (1 μg/μl), 31.5 μl ExTaq (5 U/μl) and 3202.5 μl H₂0. Distribute 40 μl of the mix per well in a 96 well PCR plate. Add 10 μl of lysed yeast per well. Perform amplification as follows: 94 °C for 3 min, then 35 cycles at 94 °C for 30 sec, 60 °C for 1 min, 72 °C for 4 min, and end with 72 °C for 7 min. Run 3 μl of the PCR products on a 1.2% agarose gel to detect PCR-positive clones. Note that using pre-cast 96 well agarose gels is recommended.
      [Tip: plate can be stored at -20 °C for a few months and re-used for new PCR]
   8. Sequence the PCR amplified fragments isolated from HIS3 positive clones. Perform a BLAST analysis to identify cellular ORF. Low-confidence alignments (E value >10^-10, identity <80%, peptide length <20 amino acids) are discarded as well as frameshifted and premature STOP codon containing sequences.

2. Matrix building for HT-GPCA

HT-GPCA is a mammalian cell-based assay where both pathogen and host proteins are transiently expressed fused to complementary fragments of the split Gaussia princeps luciferase. Upon interaction of the partners, this enzyme is reconstituted allowing the measure of its enzymatic activity. The interaction intensity is thus deduced from a Normalized Luminescence Ratio (see below and Figure 1)

4. Selection of host cell partners
   1. Select proteins that are identified more than three times in the Y2H screenings for further validation in mammalian cells to increase the dataset confidence ⁹.
   2. Add known interacting partners of the pathogen proteins as positive controls.
5. Transfer into HT-GPCA compatible vectors
   1. Clone each pathogen protein ORF in a pSPICA-N2 vector to generate pathogen proteins with amino acids 110 to 185 of the humanized Gaussia princeps luciferase fused at their N-terminus (GL2-pathogen protein fusions).
   2. PCR amplify cellular protein ORF directly from the Y2H positive clones or from other ORF resources (Human ORFeome http://horfdb.dfci.harvard.edu/) and transfer them into pSPICA-N1 vectors to generate proteins fused at the N-terminus with the amino acids 18 to 109 of humanized Gaussia princeps luciferase (GL1-host protein fusions).
      [Tip: For large-scale cloning, the Gateway cloning system is recommended. In that case, for PCR amplification, use primers designed to contain Gateway compatible sites].
   3. Sequence all expression vectors. Sequences of the pSPICA-N1 and pSPICA-N2 can be found in reference 8.

Note that plasmid constructs can be inversed, i.e. pathogens proteins in pSPICA-N1 and host protein into pSPICA-N2.

3. High-Throughput Gaussia princeps Luciferase-Based Protein Complementation Assay (HT-GPCA)

6. Cell Transfection
   1. Seed 293T cells at 350,000 cells/ml of DMEM with 10% Fetal Bovine Serum (FBS) without antibiotic. Distribute 100 μl/well in sterile white culture plates. Do not exceed 10 plates in a single experiment. Let cells grow for 24 hr at 37 °C with 5% CO₂.
      [Tip: Since the pSPICA plasmids contain an SV40 origin, the use of 293T cells allows their replication in transfected cells and thus leads to overexpression of the two luciferase fragments.]
   2. The following day, transfect cells using any suitable transfection protocol. Note that for certain transfection reagents, the number of cells seeded may have to be adapted. Use 100 ng per well of both the pathogen and host ORF plasmid constructs. Co-transfect 10 ng per well of a of CMV-Firefly luciferase plasmid to normalize for transfection efficiency. Each point is tested in triplicate.
      [Tip: DNA mixes can be performed the day before transfection and stored at -20 °C with adhesive seals.]
   3. Transfer transfection mix to the plates of cultured 293T cells. Be careful to gently deposit the DNA mix onto the cells, as 293T are not strongly adherent and easily detach from the bottom of the well. Incubate in a cell culture incubator for 24-30 hr at 37 °C with 5% CO₂.

7. Cell lysis and Gaussia luciferase dosage
   1. Wash cells with PBS (100 μl/well). Add 40 μl 1X Renilla lysis buffer. Incubate for 30 min at room temperature under agitation.
   2. Save 10 μl of cell lysates in another white plate for subsequent dosage of the Firefly luciferase activity. Store at 4 °C or alternatively at -20 °C with sealing.
   3. Measure the Gaussia activity on the remaining 30μl of cell lysates by adding 100 μl of Renilla luciferase assay reagent and counting luminescence for 10 sec with a luminometer. Dosage of a 96 well plate takes about half an hour. Perform measurement with fresh cell extracts since freezing or long-term storage may affect interaction-mediated Gaussia activity.

8. Measurement of Firefly activity
   1. Measure the Firefly activity on the saved 10 μl of cell lysates by adding 50 μl of Firefly luciferase assay reagent and counting luminescence for 10 sec. Dosage of a 96 well plate takes about half an hour. [Tip: The dosage of Firefly luciferase can be performed the day after on frozen lysates.]

9. NLR Calculations
   1. For each interaction, calculate the ratio between Gaussia luciferase activity and Firefly luciferase activity to obtain a normalized Gaussia luminescence value taking into account transfection efficiency and cell viability. Calculate the mean of the triplicates.
   2. To monitor the interaction, estimate a Normalized Luminescence Ratio (NLR) for each pathogen-host pair. To do so, divide the normalized Gaussia luminescence activity detected in presence of both pathogen and host proteins by the sum of the activities measured in control wells (Figure 1 adapted from reference 8). The NLR cut-off value for positive interactions has been estimated to be around 3.5 ⁸.

10. Data analysis
    1. Perform hierarchical clustering using the R statistic package. First, group the NLR data into categories, then calculate an interaction dendrogram using the complete linkage method. Visualize this dendrogram with JavaTreeView ¹⁰. Calculate the distance between interaction dendrogram and phylogenetic tree using a pearson correlation coefficient.
    2. To generate interaction networks, select the positive interactions from the HT-GPCA dataset and use the Cytoscape software ¹. Extract the degrees of the host proteins and plot their distribution to access the network local dynamics. Host-pathogens networks can be superimposed to the host interactome to provide an appraisal of the global impact of pathogen proteins in the host cellular network.
    3. Extract the GO (Gene Ontology) terms associated with the targeted cellular factors with the DAVID bioinformatics database ¹² to assess the functional enrichment of the dataset.