Numerous bacterial pathogens use type III secretion systems (T3SSs) or T4SSs to inject or translocate virulence proteins into eukaryotic cells. Several different reporter systems have been developed to measure the translocation of these proteins. In this study, a peptide tag-based reporter system was developed and used to monitor the injection of T3S and T4S substrates. The glycogen synthase kinase (GSK) tag is a 13-residue phosphorylatable peptide tag derived from the human GSK-3␤ kinase. Translocation of a GSK-tagged protein into a eukaryotic cell results in host cell protein kinase-dependent phosphorylation of the tag, which can be detected with phosphospecific GSK-3␤ antibodies. A series of expression plasmids encoding Yop-GSK fusion proteins were constructed to evaluate the ability of the GSK tag to measure the injection of Yops by the Yersinia pestis T3SS. GSK-tagged YopE, YopH, LcrQ, YopK, YopN, and YopJ were efficiently phosphorylated when translocated into HeLa cells. Similarly, the injection of GSK-CagA by the Helicobacter pylori T4SS into different cell types was measured via phosphorylation of the GSK tag. The GSK tag provides a simple method to monitor the translocation of T3S and T4S substrates.Many bacterial pathogens use type III secretion systems (T3SSs) or T4SSs to inject or translocate effector proteins into eukaryotic cells (12,23,34). Injected effector proteins function to disrupt host cell signaling pathways that normally function to limit bacterial growth. The T3S apparatus is a complex supramolecular structure that spans the bacterial inner and outer membranes and is topped by a needle-like structure. T4SSs also utilize a multicomponent membrane-bound secretion apparatus to translocate effector proteins or protein-DNA complexes into targeted eukaryotic cells. The mechanism by which T3SSs and T4SSs recognize substrates and transport these substrates across both bacterial and host membranes is not well understood.The injection process can be divided into two distinct steps, (i) secretion of effector proteins across the bacterial membranes and (ii) translocation of effector proteins across a eukaryotic membrane. Recognition of cytosolic T3S substrates requires N-terminal signals, whereas recent analyses suggest that recognition of T4S substrates relies upon C-terminal signals (31,55,60,61). A role for specific chaperone-like proteins has also been confirmed for some, but not all, T3S and T4S substrates (16,17). Secretion chaperones may assist in the targeting of secretion substrates and/or maintain secretion substrates in a secretion-competent state.The identity of the T3S or T4S apparatus components that specifically recognize T3S or T4S signals is not known. Recent studies suggest that a conserved T3S ATPase plays an important role early in the T3S process. The T3S ATPase has been shown to directly interact with both secretion substrates and T3S chaperones (1, 22). ATPase activity is required for unfolding of T3S substrates and for the release of chaperones from their substrates (1). Not s...

The needle structures of type III secretion (T3S) systems are formed by the secretion and polymerization of a needle subunit protein, YscF in Yersinia pestis. A subset of T3S systems employ unique heterodimeric chaperones, YscE and YscG in Y. pestis, to prevent the polymerization of needle subunits within the bacterial cell. We demonstrate that the YscE/YscG chaperone is also required for stable YscF expression and for secretion of YscF. Overexpression of a functional maltose-binding protein (MBP)-YscG hybrid protein stabilized cytoplasmic YscF but YscF was not secreted in the absence of YscE. Furthermore, a YscE mutant protein was identified that functioned with YscG to stabilize cytosolic YscF; however, YscF was not secreted. These findings confirm a role for the YscE/YscG chaperone in YscF secretion and suggest that YscE may have a specific role in this process. Recent studies have shown that YscF deleted of its N-terminal 15 residues is still secreted and functional, suggesting that YscF may not require an N-terminal secretion signal. However, we demonstrate that YscF contains an N-terminal secretion signal and that a functional N-terminal signal is required for YscF secretion.

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Disk diffusion assays have traditionally been used as a simple and effective way to screen compounds for antibacterial activity and to determine the susceptibility of pathogens to antibiotics; however, they are limited to detecting growth inhibition only. Consequently, antimicrobial agents that inhibit virulence factors, but not growth, would not be detected.

Platinum resistance remains a major challenge in the chemotherapeutic management of ovarian cancer. The anti-diabetic drug metformin has been previously shown to induce cytotoxicity in platinum resistant ovarian cancer cells and overexpression and increased phosphorylation of a tyrosine kinase called Focal Adhesion Kinase (FAK) has been implicated in the development of this platinum resistance. Therefore, in the present study we evaluated the combined cytotoxic efficacy of Metformin and the focal adhesion kinase inhibitor 1,2,4,5-Benzenetetraamine tetrahydrochloride (Y15) in platinum resistant OVCAR3 ovarian cancer cells. Cells were initially treated with concentrations of Y15 ranging from 10-100 μM, and metformin from 10-100mM to determine 1C50 values. Subsequently, cells were treated with Y15 (80 μM) and metformin (26mM) alone and in combination. All treatments were triplicated with duration of 24hrs and control cells exposed to media only. The cytotoxic profile of each treatment was assessed using the automated trypan blue assay. DNA fragmentation and poly ADP ribose polymerase (PARP) cleavage assays were performed to evaluate the mechanism of cell death and we further evaluated the expression of phosphorylated FAK, p53 and p21 in response to treatments using western blot. Y15 alone produced 48% cell death. In combination, Y15 significantly increased the cytotoxic efficacy of metformin by 22%, when compared to the metformin only treatment. Cell death by apoptosis was confirmed by PARP cleavage and the presence of DNA fragments in Y15, metformin, and metformin +Y15 treatment groups. The Metformin +Y15 combination significantly downregulated the expression of phosphorylated FAK when compared to the individual treatments and control and this confirmed reduced FAK activity. Reduced FAK auto phosphorylation also correlated with increased expression of p53 AND p21 in metformin and Y15 treatment groups. Our findings show that Y15 significantly enhances the cytotoxic profile of metformin in platinum resistant OVCAR-3 cells. Furthermore, a FAK dependent apoptotic mechanism appears to underlie the cytotoxic effect of metformin as well as Y15 as both drugs significantly reduced the phosphorylation of FAK alone, and in combination. Reduced FAK activity also correlated with increased p53 and p21 expression. This study is the first to report a FAK dependent cytotoxic mechanism of metformin in ovarian cancer and in further work we will evaluate the mechanisms why which metformin cooperates with Y15 to inhibit FAK activity in platinum resistant ovarian cancer. Citation Format: Arkene S. Levy, Zara Khan, Samuel Batko, Keerthi Thallapureddy, Robert Smith, Thanigaivelan Kanagasabai, Julie Torruellas Garcia, Appu Rathinavelu. Evaluation of the cytotoxic profile of Metformin and Y15 in platinum resistant ovarian cancer cells. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 2090.