Mechanism of PMT-Induced Anchorage-Independent Growth and mTOR Signaling

多杀性巴氏杆菌毒素(PMT)是一种细胞内作用的细菌蛋白，在体内和体外都有很强的促有丝分裂能力，并能诱导某些类型细胞的强烈非贴壁生长。这些特性表明PMT可能具有作为肿瘤促进剂的潜力，特别是在慢性感染的情况下。PMT有丝分裂特性背后的详细机制尚不清楚。我们先前发现，rPMT通过异源三聚体G蛋白的脱酰胺化劫持细胞信号转导通路，并通过G&11/PLC/PKC介导的途径导致mTOR信号的持续激活，这在一定程度上导致细胞的增殖和迁移。 此外，RPMT处理的细胞分泌一种自分泌/实践因子(S)，该因子负责mTOR和MAPK信号转导，但不以一种独立于G&Q/11脱酰胺的方式激活RTK通路。这一观察结果与报告一致，该报告显示，在动物体内实验性注射rPMT会导致远端部位的细胞增殖，包括膀胱和输尿管上皮。为了寻找其他扩散因子，我们使用了小鼠细胞因子阵列。每个膜阵列包含40种不同的抗细胞因子抗体，3个阳性对照，1个阴性对照，一式两份。将对照组、未处理细胞和rPMT处理24 h的细胞的条件培养液稀释后与生物素标记的检测抗体混合。然后将样品/抗体混合物与膜孵育。存在的任何细胞因子/检测抗体复合体都与其在膜上的同源固定化捕获抗体结合。用链霉亲和素-辣根过氧化物酶(Streptavidin-HRP)和化学发光检测试剂揭示膜。我们发现，在rPMT处理的细胞中，IL-6、角质形成细胞衍生的趋化因子(KC)和单核细胞趋化蛋白-1(MCP1)这三种细胞因子显著上调。用ELISA法对结果进行了验证。重要的是，IL-6能够激活血清饥饿成纤维细胞中的MAPK。 由于已知rPMT可以独立于G&Q激活其他信号通路。为了确定RPMT诱导的G&Q脱酰胺化的具体作用，我们选择建立一个细胞系，该细胞系含有固有GTP酶活性的关键催化残基的G&Q突变，导致利用簇状规则间隔短回文重复序列(CRISPR)技术合成G&Q蛋白的活性形式。来自化脓性链球菌的Cas9核酸酶可以被嵌合的单引导RNA(SgRNA)定向到任何基因组位置，后面跟着5-NGG(其中N可以是任何核苷酸)。保护邻近基序(PAM)。SgRNA中的20个核苷酸引导序列通过Watson-Crick碱基配对将Cas9定向到基因组DNA靶点。得到的复合体导致CAS9在PAM序列上游三个碱基对的位点特异性双链断裂(DSB)。DSB由细胞在DNA供体模板存在下通过同源重组(HR)或通过非同源末端连接(NHEJ)修复机制修复。不精确的NHEJ介导的修复可在DSB位点产生长度可变的插入和/或缺失突变。然而，HR介导的修复可以从单链或双链DNA供体模板引入精确点突变或插入。 利用多个在线生物信息学资源设计了5个20bp长的G&q sgRNAs，克隆到U6启动子下游的CRISPR/Cas9载体中。为了测试这些gRNAs的切割效率，我们使用了体外实验。我们将来自化脓性链球菌的重组Cas9蛋白与与G&Q基因组DNA序列互补的体外转录的G&Q sgRNAs混合，制备了体外切割实验。我们发现，5个G&Q sgRNAs中有4个能有效切割G&Q基因组DNA。为了测试G&Q sgRNA介导的DSB的特异性，我们在PAM序列中引入了一个突变。这些突变阻止所有G&Q sgRNAs切割DNA。这一结果表明G&Q sgRNAs诱导的DNA切割对每个sgRNA都是非常特异的。 接下来，我们在随后的实验中使用这4个G&Q sgRNAs来测试它们在细胞中诱导基因组DNA编辑的能力，从而导致G&Q基因的突变。将同时表达G&Q sgRNA和Cas9蛋白的构建体与具有所需突变的DNA供体模板一起导入细胞。48h后，分别从模拟细胞和G&#q sgRNAs细胞中提取基因组DNA，进行聚合酶链式反应。对扩增产物进行限制性片段长度多态性(RFLP)、CRISPR/Cas来源的RNA引导的工程核酸酶(RANS)分析，并用测量员检测Cas9诱导的突变和每个sgRNA的切割效率。使用所有这些分析，我们发现四个G&Q sgRNA中只有一个能有效地诱导细胞G&Q基因组DNA的修饰。G&Q sgRNAs效率的这种差异并不是由于sgRNAs或Cas9蛋白表达水平的差异。目前，我们正在通过细胞分选或嘌呤霉素选择来分离克隆。 此外，我们使用了靶向整合系统，该系统使用&#981；C31整合酶，可以将任何大小的供体质粒整合到具有高转录活性的基因间隔区，作为单拷贝，并且不需要辅助因子。具有所需突变的整合转基因稳定表达并可遗传。这种水平的靶向控制使表型效应的研究不受背景和位置变化的影响，从而导致更准确的基因型与表型相关性。

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.