Pasteurella multocida toxin (PMT) is an intracellular acting bacterial protein known for its potent mitogenic properties in vitro and in vivo and its ability to induce strong anchorage-independent growth for certain type of cells. These properties suggest that PMT may have the potential to act as a tumor promotter especially in the case of chronic infections. The detailed mechanism behind mitogenic properties of PMT is unknown. We previously revealed that rPMT hijacks cellular signal transduction pathways via deamidation of heterotrimeric G-proteins and leads to a sustained activation of mTOR signaling via a Gαq/11/PLCβ/PKC mediated pathway, which in part, leads to cell proliferation and migration. In addition, showed that rPMT treated cells secrete an autocrine/pracrine factor(s) that is responsible of mTOR and MAPK signaling but not the RTK pathway activation in a manner independent of Gαq/11 deamidation. This observation is in agreement with the report showing that experimental injection of rPMT in animal causes cell proliferation at distal sites, including the epithelium of the bladder and ureter. In an effort to find other diffusible factors, we used the mouse cytokine array. Each membrane array contains 40 different anti-cytokine antibodies, three positive controls, and one negative control printed in duplicate. Conditioned media from control nontreated cells and cells treated with rPMT for 24 h were diluted and mixed with a cocktail of biotinylated detection antibodies. The samples/antibody mixture was then incubated with the membranes. Any cytokine/detection antibody complex present was bound by its cognate immobilized capture antibody on the membrane. The membranes were revealed using streptavidin-HRP and chemiluminescent detection reagent. We found that three cytokines including interleukin IL-6, keratinocyte-derived chemokine (KC) and monocyte chemotactic protein-1 (MCP1) are significantly upregulated in rPMT treated cells. This result was validated using ELISA technique. Importantly, IL-6 was able to activate MAPK in serum starved fibroblast cells. Since rPMT is known to activate other signaling pathways independently of Gαq. To determine the specific role of Gαq deamidation induced by rPMT treatment, we choose to establish a cell line that harbor Gαq mutation of the key catalytic residue of the inherent GTPase activity leading to the synthesis of the active form of Gαq protein using clustered regularly interspaced short palindromic repeats (CRISPR) technology. The Cas9 nuclease from Streptococcus pyogenes can be directed by a chimeric single-guided RNA (sgRNA) to any genomic locus followed by a 5-NGG (where N can be any nucleotide.) protospacer adjacent motif (PAM). A 20 nucleotide guide sequence within the sgRNA directs Cas9 to genomic DNA target via Watson-crick base pairing. The resulting complex leads to site-specific double strand break (DSB) three base pairs upstream of PAM sequence by Cas9. The DSBs are repaired by the cell either by homologous recombination (HR) in the presence of a DNA donor template or by nonhomologous end joining (NHEJ) repair mechanisms. Imprecise NHEJ-mediated repair can produce insertion and/or deletion mutations of variable length at the site of the DSB. HR-mediated repair, however, can introduce precise point mutations or insertions from a single-stranded or double-stranded DNA donor template. Five 20 bp-long Gαq sgRNAs, designed using several online bioinformatics resources, were cloned in CRISPR/cas9 plasmid downstream of U6 promoter. To test the cleavage efficiency of these gRNAs, we used an in vitro assay. We prepare the in vitro cleavage assay by mixing recombinant Cas9 protein derived from Streptococcus pyogenes with in vitro transcribed Gαq sgRNAs that are complementary to the genomic DNA sequence of Gαq. We found that 4 out of 5 Gαq sgRNAs are efficient at cuttting Gαq genomic DNA. To test the specificity of Gαq sgRNA-mediated DSBs, we introduced a mutation in the PAM sequences. These mutations prevent all Gαq sgRNAs to cut DNA. This result shows that Gαq sgRNAs-induced DNA cleavage is very specific for each sgRNA. We next used these 4 Gαq sgRNAs in subsequent experiment to test their ability to induce genomic DNA editing in the cell leading to a mutation in Gαq gene. Cells were transfected with constructs that express both Gαq sgRNA and Cas9 protein along with a DNA donor template that have the desired mutation. Forty eight hours post transfection, genomic DNA samples isolated from mock and Gαq sgRNAs transfected cells were used for PCR amplifications. The PCR products were subjected to Restriction fragment length polymorphism (RFLP), CRISPR/Cas-derived RNA-guided engineered nucleases (RGENs), and the surveyor assays to detect Cas9-induced mutations and the cuting efficiency of each sgRNA. Using all these assays, we found that only one Gαq sgRNA out of four is efficient to induce a modification in cellular Gαq genomic DNA. This difference in Gαq sgRNAs efficiency is not due to a difference in sgRNAs or Cas9 protein expression levels. Currently, we are in the process of isolating the clones either by cell sorting or puromycin selection. In addition, we used targeted integration system that uses ϕC31 integrase that can integrate a donor plasmid of any size into an intergenic region with high transcription activity, as a single copy, and requires no cofactors. The integrated transgenes with a desired mutation are stably expressed and heritable. This level of targeting control allows for the study of phenotypic effects free from context and positional variations, which results in more accurate genotype to phenotype correlations.
| Oubrahim, Hammou; Wong, Allison; Wilson, Brenda A et al. (2013) Pasteurella multocida toxin (PMT) upregulates CTGF which leads to mTORC1 activation in Swiss 3T3 cells. Cell Signal 25:1136-48 |
| Oubrahim, Hammou; Wong, Allison; Wilson, Brenda A et al. (2013) Mammalian target of rapamycin complex 1 (mTORC1) plays a role in Pasteurella multocida toxin (PMT)-induced protein synthesis and proliferation in Swiss 3T3 cells. J Biol Chem 288:2805-15 |
