Genetically Programmable Pathogen Sense and Destroy

<i>Pseudomonas aeruginosa</i> (<i>P. aeruginosa</i>) is a major cause of urinary tract and nosocomial infections. Here, we propose and demonstrate proof-of-principle for a potential cell therapy approach against <i>P. aeruginosa</i>. Using principles of synthetic biology, we genetically modified <i>E. coli</i> to specifically detect wild type <i>P. aeruginosa</i> (PAO1) via its quorum sensing (QS) molecule, 3OC₁₂HSL. Engineered <i>E. coli</i> sentinels respond to the presence of 3OC₁₂HSL by secreting CoPy, a novel pathogen-specific engineered chimeric bacteriocin, into the extracellular medium using the flagellar secretion tag FlgM. Extracellular FlgM-CoPy is designed to kill PAO1 specifically. CoPy was constructed by replacing the receptor and translocase domain of Colicin E3 with that of Pyocin S3. We show that CoPy toxicity is PAO1 specific, not affecting sentinel <i>E. coli</i> or the other bacterial strains tested. In order to define the system’s basic requirements and PAO1-killing capabilities, we further determined the growth rates of PAO1 under different conditions and concentrations of purified and secreted FlgM-CoPy. The integrated system was tested by co-culturing PAO1 cells, on semisolid agar plates, together with engineered sentinel <i>E. coli</i>, capable of secreting FlgM-CoPy when induced by 3OC₁₂HSL. Optical microscopy results show that the engineered <i>E. coli</i> sentinels successfully inhibit PAO1 growth

Chemotherapeutic Drug-Induced ABCG2 Promoter Demethylation as a Novel Mechanism of Acquired Multidrug Resistance12

ABCG2 is an efflux transporter conferring multidrug resistance (MDR) on cancer cells. However, the initial molecular events leading to its up-regulation in MDR tumor cells are poorly understood. Herein, we explored the impact of drug treatment on the methylation status of the ABCG2 promoter and consequent reactivation of ABCG2 gene expression in parental tumor cell lines and their MDR sublines. We demonstrate that ABCG2 promoter methylation is common in T-cell acute lymphoblastic leukemia (T-ALL) lines, also present in primary T-ALL lymphoblast specimens. Furthermore, drug selection with sulfasalazine and topotecan induced a complete demethylation of the ABCG2 promoter in the T-ALL and ovarian carcinoma model cell lines CCRF-CEM and IGROV1, respectively. This resulted in a dramatic induction of ABCG2 messenger RNA levels (235- and 743-fold, respectively) and consequent acquisition of an ABCG2-dependent MDR phenotype. Quantitative genomic polymerase chain reaction and ABCG2 promoter-luciferase reporter assay did not reveal ABCG2 gene amplification or differential transcriptional trans-activation, which could account for ABCG2 up-regulation in these MDR cells. Remarkably, mimicking cytotoxic bolus drug treatment through 12- to 24-hour pulse exposure of ABCG2-silenced leukemia cells, to clinically relevant concentrations of the chemotherapeutic agents daunorubicin and mitoxantrone, resulted in a marked transcriptional up-regulation of ABCG2. Our findings establish that antitumor drug-induced epigenetic reactivation of ABCG2 gene expression in cancer cells is an early molecular event leading to MDR. These findings have important implications for the emergence, clonal selection, and expansion of malignant cells with the MDR phenotype during chemotherapy

Mutant Gly482 and Thr482 ABCG2 mediate high-level resistance to lipophilic antifolates

Cellular uptake of hydrophilic antifolates proceeds via the reduced folate carrier whereas lipophilic antifolates enter cells by diffusion. Recently we have shown that transfectant cells overexpressing the mutant G482 ABCG2 displayed 120-6,250-fold resistance to hydrophilic antifolates than untransfected cells upon 4 h drug exposure, but lost almost all their antifolate resistance upon 72 h drug exposure (Shafran et al. in Cancer Res 65:8414-8422, 2005). Here we explored the ability of the wild type (WT) R482-as well as the mutant G482-and T482 ABCG2 to confer resistance to lipophilic antifolate inhibitors of dihydrofolate reductase (trimetrexate, piritrexim, metoprine and pyrimethamine) and thymidylate synthase (AG337, AG377 and AG331). Lipophilic antifolate resistance was determined using growth inhibition assays upon 72 h drug exposure. Cells overexpressing these mutant efflux transporters displayed up to 106-fold resistance to lipophilic antifolates relative to untransfected cells; this resistance was reversed by the specific and potent ABCG2 efflux inhibitor Ko143. In contrast, cells overexpressing the WT R482 ABCG2 exhibited either no or only a low-level of lipophilic antifolate resistance. These results provide the first evidence that overexpression of the mutant G482- and T482 but not the WT R482 ABCG2 confers a high-level of resistance to lipophilic antifolates. The high membrane partitioning of lipophilic antifolates along with the large confinement of ABCG2 to the plasma membrane suggest that these mutant ABCG2 transporters may possibly recognize and extrude lipophilic antifolates from the lipid bilayer. The potential implications to cancer chemotherapy as well as the mechanism of anticancer drug extrusion by these mutant exporters are discussed

Schematic model for the effect of different chemical chaperones groups on CA stability and assembly.

<p>The presence of polyols or sugars (dark grey circles) induces compactization of CA structure and inhibits the formation of high-order CA structures. In contrast, the presence of the methylamines TMAO, betaine or sarcosine (light grey circles) destabilizes CA structure and thus promoting CA-CA interactions, resulting in the formation of CA cylinders. Structure of HIV-1 CA (151–231) created using PDB 1A8O <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0060867#pone.0060867-Gamble1" target="_blank">[52]</a> represents full-length CA protein. The scale bars are 100 nm (left image) and 200 nm (right image).</p

Values of kinetic parameters of CA assembly either in the absence or presence of different chemical chaperones.

a<p>t₅₀ is the time at which the optical density (OD) is equal to one-half the optical density extrapolated at infinite time. The values given were obtained by fitting the data to <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0060867#pone.0060867.e001" target="_blank">equation 1</a>. The fitting errors are indicated. For linear-fitted curves, the t₅₀ values cannot be calculated but rather estimated to be >40 min, since the maximal t value is 80 min.</p>b<p>The linear polymerization rate is the average increase in optical density per minute for the approximate linear part of the polymerization curve. Two values of assembly rate are indicated for curves with two phases of linear increase.</p

Dynamic light scattering measurements of CA assemblies.

<p>Assembly of CA was carried out using the same conditions as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0060867#pone-0060867-g001" target="_blank">Fig. 1</a>. The hydrodynamic radius (nm) of CA particles was measured after 30 min incubation in the absence (black solid line) or presence of 0.5 M of (<b>A</b>) polyols: sorbitol (grey solid line); adonitol (black dashed line); meso-erythritol (grey dashed line); glycerol (dotted line) or ethylene glycol (dashed-dotted line), 5 min incubation in the absence (black solid line) or presence of (<b>B</b>) sugars: maltose (grey solid line); arabinose (black dashed line); trehalose (grey dashed line) or mannose (dotted line), and 10 min incubation in the absence (black solid line) or presence of (<b>C</b>) methylamines: TMAO (grey solid line); betaine (black dashed line); sarcosine (grey dashed line) or choline chloride (dotted line).</p