The overall goal of the following experiment is to perform ectomic studies to compare interaction profiles of proteins from different pathogen variants against a common set of cellular factors. This is achieved by mating based yeast, two hybrid screenings of CDNA to identify potential interacting partners of proteins from different pathogen variants. As a second step, the interacting partners open reading frames are sequenced and transferred into vectors compatible for further validation.
Next, a set of individual partners emerging from all two hybrid screenings is selected and retested for their interaction with the complete range of strains studied. This is done using an orthogonal gia, luciferase based protein fragment complementation assay performed in mammalian cells in order to provide robust comparative interaction data sets. Results show different interaction intensities between the pathogen proteins and the cellular proteins based on the luciferase measures obtained, and thus provide a comparative overview of pathogen host protein protein interactions.
Processing of the interaction profiles by hierarchical clustering correlates specific host pathogen interactions with pathological traits and gives insights on the involvement of pathogen proteins in pathogenesis. The main advantage of this approach over other methods mapping pathogen host protein protein interactions is that it provides stringent comparative interaction dataset between multiple strain variants. This techniques provide insight into the interaction network of a pathogen protein, but it can also be applied to other systems as an infectious molecule using complete viruses and rivers genetics, for example, Begin this protocol with mating and spreading the yeast cells as described in the written protocol seed.
30 milliliters of selective dropout medium lacking tryptophan with few colonies of AH 1 0 9 yeast strain transformed with the P-G-B-K-T seven plasmid expressing the pathogen protein. Incubate 30 hours at 30 degrees Celsius with rotation. Next seed, 200 milliliters of selective dropout medium minus tryptophan with the 30 milliliters of the AH 1 0 9.
Cultures incubate with rotation for 20 hours at 30 degrees Celsius. Also, incubate thought CDNA library transformed Y 187 in 20 milliliters of YP glue for 10 minutes at 30 degrees Celsius with rotation. Then centrifuge a volume of the PG BK T seven transformed AH 109 culture corresponding to an equivalent amount of viable Y 187 yeast cells for five minutes at 3, 500 RPM and 20 degrees Celsius.
Carefully withdraw the supernatant and Resus suspend the palate in 10 milliliters of YP glue. Mix the resuspended AH 109 yeast with the Y 187 containing library. Transfer to a 50 milliliter tube before centrifuging for five minutes at 3, 500 RPM and 20 degrees Celsius.
After carefully withdrawing the super dat Resus, suspend the pellet in YP glue to get a total of 1.5 milliliters. Next, spread the yeast onto three YCM agar, 150 millimeter plates at 0.5 milliliters per plate and incubate 4.5 hours at 30 degrees Celsius. Collect the mated yeast from YCM plates by scraping with a rake glass in 10 milliliters of selective dropout medium minus leucine tryptophan and histidine.
Rinse the plate two times with five milliliters of the same medium and pool following centrifugation for five minutes at 3, 500 RPM and 20 degrees Celsius. Discard supernatant and reus. Suspend the yeast pellet in five milliliters of selective dropout medium minus leucine tryptophan and histidine.
Then spread the mated yeast onto 10 plates of selective dropout agar minus leucine, tryptophan and histamine supplemented with the appropriate concentration of three amino triazole or three at as determined in the auto activation test. Calculate the diploid rate as described in the written protocol after incubating at 30 degrees Celsius in a humidified atmosphere for six to 10 days. Pick yeast colonies and transfer to plates prepared fresh from selective dropout agar minus leucine tryptophan and histidine plus three a t.
Order the yeast in a scheme of eight lanes of 12 wells reproducing a 96 well plate so that later it will be easier for sequencing. Let the yeast colonies grow for four to five days at 30 degrees Celsius in a humidified atmosphere. The goal of this section is to PCR, amplify the CDNA contained in the PA ct two vectors of lysed yeast colonies grown on selective dropout agar minus leucine, trytophan and histidine plates.
Begin by placing a 96 well PCR plate on ice to lyce yeast cells at 50 microliters per well of xmal 20 T solution. Gently resuspend one colony per well. Incubate the plate 15 minutes at 37 degrees Celsius and 15 minutes at 95 degrees Celsius.
After placing the plate back on ice, add 50 microliters of distilled water per well mix by pipetting up and down, and proceed to centrifuge for five minutes at 3, 500 RPM and four degrees Celsius. After preparing the X tack mix as described in the text protocol, distribute 40 microliters of the mix per well in the 96 well PCR plate. Add 10 microliters of the lysed yeast per well and perform amplification as listed in the text protocol.
Run three microliters of the PCR products on a 1.2%agros gel to detect PCR positive clones. Sequence the PCR amplified fragments isolated from his three positive clones. Then perform a blast analysis to identify cellular open reading frames, discarding low confidence alignments as well as frameshift and premature stop code on containing sequences prior to cell transfection.
Transfer the pathogen protein and cellular protein open reading frames into their respective vectors to generate proteins fused to fragments of the Gaia luciferase as detailed in the text protocol seed 2 9 3 T cells at 350, 000 cells per milliliter of DMEM with 10%fetal bovine serum without antibiotic. Distribute 100 microliters per well in sterile white culture plates before growing for 24 hours at 37 degrees Celsius with 5%carbon dioxide the following day. Transfect cells using any suitable transfection protocol use 100 nanograms per well of both pathogen and host open reading frame plasmid constructs transfect 10 nanograms per well of CMV firefly luciferase plasmid to normalize for transfection efficiency.
Each point is tested in triplicate transfer transfection mix to the plates of cultured 2 9 3 T cells. Be careful to gently deposit the DNA mix onto the cells as 2 9 3 T cells are not strongly adherent and are easily detached from the bottom of the well incubate in a cell culture incubator for 24 to 30 hours at 37 degrees Celsius with 5%carbon dioxide wash cells with PBS at 100 microliters per well. Add 40 microliters of one x vanilla lysis, buffer, and incubate for 30 minutes at room temperature.
Under agitation, save 10 microliters of cell lysates in another white plate for subsequent dosage of the firefly luciferase stored four degrees Celsius or alternatively at negative 20 degrees Celsius with ceiling to to measure Gaia activity. Place the plate into a luminometer and inject 100 microliters of vanilla luciferase assay reagent onto cell lysates. Then count luminescence for 10 seconds.
Dosage of a 96 well plate takes about half an hour. Always use fresh extracts since freezing or long-term storage may affect interaction mediated Gaia activity on the remaining 10 microliter cell extracts. Inject 50 microliters of firefly luciferase assay reagent onto cell lysates using a luminometer and counting luminescence for 10 seconds.
Dosage of a 96 well plate takes about half an hour for each interaction. Calculate the ratio between Gaia luciferase activity and firefly luciferase activity to obtain a normalized GAIA luminescence value. Taking into account transfection efficiency and cell viability.
Calculate the mean of the triplicates to monitor the interaction. Estimate a normalized luminescence ratio or NLR for each pathogen host pair. To do so, divide the normalized GAIA luminescence activity detected in presence of both pathogen and host proteins by the sum of the activities measured in control wells.
The NLR cutoff value for positive interactions has been estimated in a previous study to be around 3.5. A major strength of the high throughput GIA precepts, protein COMPLEMENTATION assay, or H-T-G-P-C-A lies in its high sensitivity as illustrated by the assessment of false positive and false negative rates for the HPVE two protein to determine the false negative rate. Known interactions of E two from HPV 16 were assessed by H-T-G-P-C-A four out of 18 interactions were not recovered corresponding to a 22%false negative rate.
The false positive interactions were measured to be 5.8%using 12 HPVE two proteins against a random set of cellular proteins. The strong specificity of the method is highlighted in this figure showing that a single point mutation known to interfere with E two binding to its cell partner BRD four annihilates, the NLR ratio. This large scale comparative ectomic approach has been recently successfully applied to three early proteins of human papilloma viruses, E two, E six and E seven, originating from different genotypes, representative of their natural diversity in tropisms and pathologies for each of the early proteins.
Hierarchical clustering of the interaction profiles, mostly recapitulates HPV Phylogeny proving the robustness of the approach and the pance of interaction data sets. It can be used to correlate specific virus host interaction profiles with pathological traits, thereby giving clues on the involvement of viral proteins. In pathogenesis, genotype specific interactions can be extracted, which potentially correspond to tropism or pathogenic biomarkers.
As illustrated here for E six interacting partners, cellular targets were sorted based on the interaction intensity and grouped according to interacting HPV type. This representation allows the identification of specific biomarkers such as fad, which interacts only with the E six proteins of oncogenic HPV. Such targeting must be crucial for the carcinogenic trait of all oncogenic HPV and thus constitutes a good candidate to be used either as a surrogate marker for HPV infection or as a therapeutic target.
Following this procedure. The H-T-G-P-C-A can be adapted to be used with a third protein expressed as a fusion with a biotin tag. This allows the capture of three partner complexes on strep diving color plates, and this can answer the question of the formation of large protein complexes.
After watching this video, you should have a good understanding of how to characterize pathogen, host pot pot interactions. Third, by probing interaction partners of multiple strain variants. Second, by comparing their interaction with a whole set of pathogen types using a protein protein duration, say in mammalian cells.
