TFEB Regulates ATP7B Expression to Promote Platinum Chemoresistance in Human Ovarian Cancer Cells

ATP7B is a hepato-specific Golgi-located ATPase, which plays a key role in the regulation of copper (Cu) homeostasis and signaling. In response to elevated Cu levels, ATP7B traffics from the Golgi to endo-lysosomal structures, where it sequesters excess copper and further promotes its excretion to the bile at the apical surface of hepatocytes. In addition to liver, high ATP7B expression has been reported in tumors with elevated resistance to platinum (Pt)-based chemotherapy. Chemoresistance to Pt drugs represents the current major obstacle for the treatment of large cohorts of cancer patients. Although the mechanisms underlying Pt-tolerance are still ambiguous, accumulating evidence suggests that lysosomal sequestration of Pt drugs by ion transporters (including ATP7B) might significantly contribute to drug resistance development. In this context, signaling mechanisms regulating the expression of transporters such as ATP7B are of great importance. Considering this notion, we investigated whether ATP7B expression in Pt-resistant cells might be driven by transcription factor EB (TFEB), a master regulator of lysosomal gene transcription. Using resistant ovarian cancer IGROV-CP20 cells, we found that TFEB directly binds to the predicted coordinated lysosomal expression and regulation (CLEAR) sites in the proximal promoter and first intron region of ATP7B upon Pt exposure. This binding accelerates transcription of luciferase reporters containing ATP7B CLEAR regions, while suppression of TFEB inhibits ATP7B expression and stimulates cisplatin toxicity in resistant cells. Thus, these data have uncovered a Pt-dependent transcriptional mechanism that contributes to cancer chemoresistance and might be further explored for therapeutic purposes.

Toll-like receptor 5 as a novel receptor for fungal zymosan

Microbial pathogens carry specific structural patterns which were termed pathogenassociated molecular patterns (PAMPs). Toll-like receptors (TLRs) as key elements for the recognition of microbial pathogens are necessary for the activation of innate immune pathways. TLRs are activated by binding PAMPs of bacteria, viruses and fungi and initiate a signaling pathway resulting in the activation of transcription factors which modulate the production of various proinflammatory cytokines. It is not fully clear in detail which microbial pattern is recognized by which TLR. Here we show for the first time that TLR5 is a strong receptor for the yeast particle zymosan. We have generated stable human cell lines with combinations of TLR2 and TLR5 knock in/knock out together with stable nuclear factor kappaB (NF-κB) luciferase reporters. We found that both receptors TLR5 and TLR2 lead to an independent activation of the NF-κB pathway when simulated with zymosan. Our results demonstrate that TLR5 is a receptor for the fungal particle zymosan in addition to bacterial fragments like flagellin. Distinct cytokine patterns might suggest that TLR5 is potentially important for the differentiation in the recognition of the specific type of the foreign microorganisms and in the specific host defense response.

Multiplexed bioluminescence microscopy via phasor analysis

Microscopic bioluminescence imaging has been historically challenging due to a lack of detection methods and easily resolved probes. Here we combine bioluminescence with phasor analysis, an optical method commonly used to distinguish spectrally similar fluorophores. Bioluminescent phasor enabled rapid differentiation of multiple luciferase reporters and resonance energy transfer processes. The merger of bioluminescence and phasor analysis provides a platform for routine, time-lapse imaging of collections of cellular features.

#99: Streptococcus pyogenes Utilizes the Peptide-Based Rgg 2/3 Quorum Sensing System During Oropharyngeal Colonization

Background Streptococcus pyogenes is a human-restricted pathogen most often found in the human nasopharynx. Multiple bacterial factors have been found to contribute to persistent colonization of this niche, and many of these factors are important in mucosal immunity and vaccine development. In this work, we infected mice intranasally with transcriptional regulator mutants of the Rgg2/3 quorum sensing (QS) system—a peptide-based signaling system conserved in all sequenced isolates of S. pyogenes. Methods Three-week-old CD1 mice were intranasally infected with ~107 CFU of S. pyogenes strain MGAS315. Calcium alginate throat swabs were used to monitor nasopharyngeal colonization by the bacteria over time. Luciferase reporters used alongside an IVIS camera were able to show quorum sensing activity levels after inoculation into the mouse nose. Bacterial RNA was isolated from the throat of the mice and quantitative RT–PCR was performed on the samples to corroborate the luciferase reporter data. The nasal-associated lymphoid tissue (NALT) was excised and its supernatants were subjected to 32-plex murine cytokine and chemokine analysis (Millipore). Results Deletion of the QS system’s transcriptional activator (Δrgg2) dramatically diminished the percentage of colonized mice. Deletion of the transcriptional repressor (Δrgg3) increased the percentage of colonized mice compared with wild type. Stimulation of the QS system using synthetic pheromones prior to inoculation did not significantly increase the percentage of animals colonized, indicating that activity of the QS system is responsive to conditions of the host nasopharynx. Mice inoculated with QS-dependent luciferase reporters were subjected to in vivo imaging and showed activation within 1 hour. Bacterial RNA extracted directly from oropharyngeal swabs and evaluated by quantitative RT–PCR subsequently confirmed QS upregulation within 1 hour of inoculation. In the nasal-associated lymphoid tissue (NALT), a muted inflammatory response to the Δrgg2 bacteria suggests that their rapid elimination fails to elicit the previously characterized response to intranasal inoculation of GAS. Conclusions Deletion of the Rgg2 transcriptional activator of the Rgg 2/3 quorum sensing system eliminates colonization of the murine nasopharynx and changes the transcriptional profile of the bacteria in this niche. An existing small-molecule inhibitor of the Rgg2/3 system was unable to inhibit QS activation in vivo, likely due to the suboptimal achievable doses; however, results of our study indicate inhibition of QS may diminish the oropharyngeal colonization of S. pyogenes and argue for further development.

APPLICATION OF BIOLUMINESCENT METHODS TO STUDY STRESS EFFECTS ON LIVING SYSTEMS

Bioluminescent methods based on firefly luciferase for detecting the effect of membrane-active agents (antibiotics and saponins) on living prokaryotic and eukaryotic cells have been developed. New data on the kinetics and mechanism of action of colistin on E. coli cells and digitonin cells on NEK293 cells were obtained. Examples of the use of constitutive and inducible luciferase reporters in cell biology are given.

OsPP2C09 Is a Bifunctional Regulator in Both ABA-Dependent and Independent Abiotic Stress Signaling Pathways

Clade A Type 2C protein phosphatases (PP2CAs) negatively regulate abscisic acid (ABA) signaling and have diverse functions in plant development and in response to various stresses. In this study, we showed that overexpression of the rice ABA receptor OsPYL/RCAR3 reduces the growth retardation observed in plants exposed to osmotic stress. By contrast, overexpression of the OsPYL/RCAR3-interacting protein OsPP2C09 rendered plant growth more sensitive to osmotic stress. We tested whether OsPP2CAs activate an ABA-independent signaling cascade by transfecting rice protoplasts with luciferase reporters containing the drought-responsive element (DRE) or ABA-responsive element (ABRE). We observed that OsPP2CAs activated gene expression via the cis-acting drought-responsive element. In agreement with this observation, transcriptome analysis of plants overexpressing OsPP2C09 indicated that OsPP2C09 induces the expression of genes whose promoters contain DREs. Further analysis showed that OsPP2C09 interacts with DRE-binding (DREB) transcription factors and activates reporters containing DRE. We conclude that, through activating DRE-containing promoters, OsPP2C09 positively regulates the drought response regulon and activates an ABA-independent signaling pathway.

Accurate and sensitive detection of Salmonella in foods by engineered bacteriophages

Salmonella is a major causative agent of foodborne illness and rapid identification of this pathogen is essential to prevent disease. Currently most assays require high bacterial burdens or prolonged enrichment to achieve acceptable performance. A reduction in testing time without loss of sensitivity is critical to allow food processors to safely decrease product holding time. To meet this need, a method was developed to detect Salmonella using luciferase reporter bacteriophages. Bacteriophages were engineered to express NanoLuc, a novel optimized luciferase originating from the deep-sea shrimp Oplophorus gracilirostris. NanoLuc-expressing bacteriophages had a limit of detection of 10–100 CFU per mL in culture without enrichment. Luciferase reporters demonstrated a broad host range covering all Salmonella species with one reporter detecting 99.3% of 269 inclusivity strains. Cross-reactivity was limited and only observed with other members of the Enterobacteriaceae family. In food matrix studies, a cocktail of engineered bacteriophages accurately detected 1 CFU in either 25 g of ground turkey with a 7 h enrichment or 100 g of powdered infant formula with a 16 h enrichment. Use of the NanoLuc reporter assay described herein resulted in a considerable reduction in enrichment time without a loss of sensitivity.

Metabolic Activation of CsgD in the Regulation of Salmonella Biofilms

Among human food-borne pathogens, gastroenteritis-causing Salmonella strains have the most real-world impact. Like all pathogens, their success relies on efficient transmission. Biofilm formation, a specialized physiology characterized by multicellular aggregation and persistence, is proposed to play an important role in the Salmonella transmission cycle. In this manuscript, we used luciferase reporters to examine the expression of csgD, which encodes the master biofilm regulator. We observed that the CsgD-regulated biofilm system responds differently to regulatory inputs once it is activated. Notably, the CsgD system became unresponsive to repression by Cpx and H-NS in high osmolarity conditions and less responsive to the addition of amino acids. Temperature-mediated regulation of csgD on agar was altered by intracellular levels of RpoS and cyclic-di-GMP. In contrast, the addition of glucose repressed CsgD biofilms seemingly independent of other signals. Understanding the fine-tuned regulation of csgD can help us to piece together how regulation occurs in natural environments, knowing that all Salmonella strains face strong selection pressures both within and outside their hosts. Ultimately, we can use this information to better control Salmonella and develop strategies to break the transmission cycle.

Custom-designed, degradation-resistant messenger RNAs in yeast

ABSTRACTStructured RNA elements that protect RNA transcripts from 5’→3’ degradation are becoming useful research tools. Here we show that exonuclease-resistant RNA structures (xrRNAs) from Flaviviruses can be used to protect heterologous messenger RNAs (mRNAs) from 5’→3’ degradation in budding yeast. Installation of xrRNAs ahead of a downstream internal ribosome entry site (IRES) leads to the accumulation of partially-degraded mRNAs that are substrates for cap-independent translation of a LacZ reporter, yielding a 30-fold increase in measured β-galactosidase activity. Additionally, by monitoring the translation of dual-luciferase reporters we show that xrRNA sequences do not interfere with the progression of an elongating ribosome. Combined these data indicate that xrRNA elements can be used in creative ways to stabilize RNAs with potentially useful applications.