Kelly Flentie

Advances in bioluminescence imaging of live animal models.

Many of the obligate steps of physiology and disease are dynamic in time and space, and thus, end-point assays do not always provide a full understanding of these processes. Comprehensive understanding of the functional complexity of protein interactions and cell trafficking requires mapping of cellular and molecular function within complex systems over biologically relevant time scales. New approaches to bioluminescence imaging of cell migration, signaling pathways, drug action, and interacting protein partners in vivo allow the study of biology and disease within the context of living animals.

Mycobacterium tuberculosis Transcription Machinery: Ready To Respond to Host Attacks

Regulating responses to stress is critical for all bacteria, whether they are environmental, commensal, or pathogenic species. For pathogenic bacteria, successful colonization and survival in the host are dependent on adaptation to diverse conditions imposed by the host tissue architecture and the immune response. Once the bacterium senses a hostile environment, it must enact a change in physiology that contributes to the organisms survival strategy. Inappropriate responses have consequences; hence, the execution of the appropriate response is essential for survival of the bacterium in its niche. Stress responses are most often regulated at the level of gene expression and, more specifically, transcription. This minireview focuses on mechanisms of regulating transcription initiation that are required by Mycobacterium tuberculosis to respond to the arsenal of defenses imposed by the host during infection. In particular, we highlight how certain features of M. tuberculosis physiology allow this pathogen to respond swiftly and effectively to host defenses. By enacting highly integrated and coordinated gene expression changes in response to stress,M. tuberculosis is prepared for battle against the host defense and able to persist within the human population.

Stably integrated luxCDABE for assessment of salmonella invasion kinetics

Salmonella Typhimurium is a common cause of gastroenteritis in humans and also localizes to neoplastic tumors in animals. Invasion of specific eukaryotic cells is a key mechanism of Salmonella interactions with host tissues. Early stages of gastrointestinal cell invasion are mediated by a Salmonella type III secretion system, powered by the adenosine triphosphatase invC. The aim of this work was to characterize the invC dependence of invasion kinetics into disparate eukaryotic cells traditionally used as models of gut epithelium or neoplasms. Thus, a nondestructive real-time assay was developed to report eukaryotic cell invasion kinetics using lux+ Salmonella that contain chromosomally integrated luxCDABE genes. Bioluminescence-based invasion assays using lux+ Salmonella exhibited inoculum dose-response correlation, distinguished invasion-competent from invasion-incompetent Salmonella, and discriminated relative Salmonella invasiveness in accordance with environmental conditions that induce invasion gene expression. In standard gentamicin protection assays, bioluminescence from lux+ Salmonella correlated with recovery of colony-forming units of internalized bacteria and could be visualized by bioluminescence microscopy. Furthermore, this assay distinguished invasion-competent from invasion-incompetent bacteria independent of gentamicin treatment in real time. Bioluminescence reported Salmonella invasion of disparate eukaryotic cell lines, including neoplastic melanoma, colon adenocarcinoma, and glioma cell lines used in animal models of malignancy. In each case, Salmonella invasion of eukaryotic cells was invC dependent.

A Bioluminescent Transposon Reporter-Trap Identifies Tumor-Specific Microenvironment-Induced Promoters in Salmonella for Conditional Bacterial-Based Tumor Therapy

UNLABELLED Salmonella specifically localize to malignant tumors in vivo, a trait potentially exploitable as a delivery system for cancer therapeutics. To characterize mechanisms and genetic responses of Salmonella during interaction with living neoplastic cells, we custom-designed a promoterless transposon reporter containing bacterial luciferase. Analysis of a library containing 7,400 independent Salmonella transposon insertion mutants in coculture with melanoma or colon carcinoma cells identified five bacterial genes specifically activated by cancer cells: adiY, yohJ, STM1787, STM1791, and STM1793. Experiments linked acidic pH, a common characteristic of the tumor microenvironment, to a strong, specific, and reversible stimulus for activation of these Salmonella genes in vitro and in vivo. Indeed, a Salmonella reporter strain encoding a luciferase transgene regulated by the STM1787 promoter, which contains a tusp motif, showed tumor-induced bioluminescence in vivo. Furthermore, Salmonella expressing Shiga toxin from the STM1787 promoter provided potent and selective antitumor activity in vitro and in vivo, showing the potential for a conditional bacterial-based tumor-specific therapeutic. SIGNIFICANCE Salmonella, which often encounter acidic environments during classical host infection, may co-opt evolutionarily conserved pathways for tumor colonization in response to the acidic tumor microenvironment. We identified specific promoter sequences that provide a platform for targeted Salmonella-based tumor therapy in vivo.

Synthetic (p)ppGpp analogue is an inhibitor of stringent response in mycobacteria

ABSTRACT Bacteria elicit an adaptive response against hostile conditions such as starvation and other kinds of stresses. Their ability to survive such conditions depends, in part, on stringent response pathways. (p)ppGpp, considered to be the master regulator of the stringent response, is a novel target for inhibiting the survival of bacteria. In mycobacteria, the (p)ppGpp synthetase activity of bifunctional Rel is critical for stress response and persistence inside a host. Our aim was to design an inhibitor of (p)ppGpp synthesis, monitor its efficiency using enzyme kinetics, and assess its phenotypic effects in mycobacteria. As such, new sets of inhibitors targeting (p)ppGpp synthesis were synthesized and characterized by mass spectrometry and nuclear magnetic resonance spectroscopy. We observed significant inhibition of (p)ppGpp synthesis by RelMsm in the presence of designed inhibitors in a dose-dependent manner, which we further confirmed by monitoring the enzyme kinetics. The Rel enzyme inhibitor binding kinetics were investigated by isothermal titration calorimetry. Subsequently, the effects of the compounds on long-term persistence, biofilm formation, and biofilm disruption were assayed in Mycobacterium smegmatis, where inhibition in each case was observed. In vivo, (p)ppGpp levels were found to be downregulated in M. smegmatis treated with the synthetic inhibitors. The compounds reported here also inhibited biofilm formation by the pathogen Mycobacterium tuberculosis. The compounds were tested for toxicity by using an MTT assay with H460 cells and a hemolysis assay with human red blood cells, for which they were found to be nontoxic. The permeability of compounds across the cell membrane of human lung epithelial cells was also confirmed by mass spectrometry.

Characterization of phthiocerol and phthiodiolone dimycocerosate esters of M. tuberculosis by multiple-stage linear ion-trap MS.

Both phthiocerol/phthiodiolone dimycocerosate (PDIM) and phenolic glycolipids are abundant virulent lipids in the cell wall of various pathogenic mycobacteria, which can synthesize a wide range of complex high-molecular-mass lipids. In this article, we describe linear ion-trap MSn mass spectrometric approach for structural study of PDIMs, which were desorbed as the [M + Li]+ and [M + NH4]+ ions by ESI. We also applied charge-switch strategy to convert the mycocerosic acid substituents to their N-(4-aminomethylphenyl) pyridinium (AMPP) derivatives and analyzed them as M + ions, following alkaline hydrolysis of the PDIM to release mycocerosic acids. The structural information from MSn on the [M + Li]+ and [M + NH4]+ molecular species and on the M + ions of the mycocerosic acid-AMPP derivative affords realization of the complex structures of PDIMs in Mycobacterium tuberculosis biofilm, differentiation of phthiocerol and phthiodiolone lipid families and complete structure identification, including the phthiocerol and phthiodiolone backbones, and the mycocerosic acid substituents, including the locations of their multiple methyl side chains, can be achieved.

Analysis of the contribution of MTP and the predicted Flp pilus genes to Mycobacterium tuberculosis pathogenesis

Mycobacterium tuberculosis (Mtb) is one of the worlds most successful pathogens. Millions of new cases of tuberculosis occur each year, emphasizing the need for better methods of treatment. The design of novel therapeutics is dependent on our understanding of factors that are essential for pathogenesis. Many bacterial pathogens use pili and other adhesins to mediate pathogenesis. The recently identified Mycobacterium tuberculosis pilus (MTP) and the hypothetical, widely conserved Flp pilus have been speculated to be important for Mtb virulence based on in vitro studies and homology to other pili, respectively. However, the roles for these pili during infection have yet to be tested. We addressed this gap in knowledge and found that neither MTP nor the hypothetical Flp pilus is required for Mtb survival in mouse models of infection, although MTP can contribute to biofilm formation and subsequent isoniazid tolerance. However, differences in mtp expression did affect lesion architecture in infected lungs. Deletion of mtp did not correlate with loss of cell-associated extracellular structures as visualized by transmission electron microscopy in Mtb Erdman and HN878 strains, suggesting that the phenotypes of the mtp mutants were not due to defects in production of extracellular structures. These findings highlight the importance of testing the virulence of adhesion mutants in animal models to assess the contribution of the adhesin to infection. This study also underscores the need for further investigation into additional strategies that Mtb may use to adhere to its host so that we may understand how this pathogen invades, colonizes and disseminates.

Nucleoside Diphosphate Kinase-3 (NME3) Enhances TLR5-Induced NFκB Activation

Bacterial flagellin is a potent activator of NFκB signaling, inflammation, and host innate immunity, and recent data indicate that flagellin represents a novel antitumor ligand acting through toll-like receptor 5 (TLR5) and the NFκB pathway to induce host immunity and aid in the clearance of tumor xenografts. To identify innate signaling components of TLR5 responsible for these antitumor effects, a loss-of-function high-throughput screen was employed utilizing carcinoma cells expressing a dynamic NFκB bioluminescent reporter stimulated by Salmonella typhimurium expressing flagellin. A live cell screen of a siRNA library targeting 691 known and predicted human kinases to identify novel tumor cell modulators of TLR5-induced NFκB activation uncovered several interesting positive and negative candidate regulators not previously recognized, including nucleoside diphosphate kinase 3 (NME3), characterized as an enhancer of signaling responses to flagellin. Targeted knockdown and overexpression assays confirmed the regulatory contribution of NME3 to TLR5-mediated NFκB signaling, mechanistically downstream of MyD88. Furthermore, Kaplan–Meier survival analysis showed that NME3 expression correlated highly with TLR5 expression in breast, lung, ovarian, and gastric cancers, and furthermore, high-level expression of NME3 increased overall survival for patients with breast, lung, and ovarian cancer, but the opposite in gastric cancer. Together, these data identify a previously unrecognized proinflammatory role for NME3 in signaling downstream of TLR5 that may potentiate cancer immunotherapies. Implications: Proinflammatory signaling mediated by innate immunity engagement of flagellin-activated TLR5 in tumor cells results in antitumor effects through NME3 kinase, a positive downstream regulator of flagellin-mediated NFκB signaling, enhancing survival for several human cancers. Mol Cancer Res; 16(6); 986–99. ©2018 AACR.

Abstract 4880: A high-throughput siRNA screen identifies Nucleoside Diphosphate Kinase (NME3) as a novel host regulator of NF-êB signaling in response to Salmonella-induced activation of TLR-5

Bacterial flagellin is a potent activator of NF-eB signaling, inflammation and host innate immunity. In comparison to TNFa, perhaps the most studied NF-eB-inducing ligand, flagellin elicits an overlapping, yet unique downstream transcriptional response by host cells. In contrast to the pro-oncogenic functions of TNFa, recent data indicate that flagellin may represent a novel anti-tumor ligand that acts through TLR5 and the NF-eB pathway to induce host immunity and to aid in the clearance of tumor xenografts. To begin to characterize the molecular basis for the anti-tumor effect of flagellin and to identify downstream innate signaling components of NF-eB that are responsible for the pro-inflammatory program necessary for these anti-tumor effects, we employed a high throughput screen. Utilizing HCT116 colon carcinoma cells expressing a dynamic NF-eB bioluminescent reporter and Salmonella typhimurium expressing flagellin, we performed a live cell screen of a siRNA library targeting 691 known and predicted human kinases to identify novel modulators of flagellin-induced NF-eB activation. This screen uncovered nucleoside diphosphate kinase (NME3) as a previously unrecognized, positive regulator of NF-eB signaling in response to flagellin. Targeted knockdown and overexpression assays confirmed the regulatory contribution of NME3. Furthermore, Kaplan-Meier survival analysis for breast, lung and ovarian cancer patients showed that high level expression of NME3 correlated with increased overall survival. Together, these data identify a novel pro-inflammatory role for NME3 in NF-eB signaling that may potentially be exploited to optimize cancer immunotherapy. Citation Format: Caleb Gonzalez, Kelly Flentie, Brandon Kocher, Jayne Jayne Marasa, David Piwnica-Worms. A high-throughput siRNA screen identifies Nucleoside Diphosphate Kinase (NME3) as a novel host regulator of NF-eB signaling in response to Salmonella -induced activation of TLR-5. [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 4880.

Abstract LB-367: Bioluminescent transposon screen identifies cancer cell specific promoters in Salmonella typhimurium

Proceedings: AACR 103rd Annual Meeting 2012‐‐ Mar 31‐Apr 4, 2012; Chicago, IL The ability of Salmonella Typhimurium to specifically localize to malignant tumors in vivo has been well documented and offers a powerful avenue for targeted anti-cancer therapies. S. Typhimurium, a common gastrointestinal pathogen, utilizes a complex array of type III secretion protein complexes, effector molecules and transcription factors during its normal pathophysiology. The expression of these genes is highly regulated in a spatiotemporal manner in response to the host environment. Akin to other host environments, such as the gastrointestinal tract and phagosomes, tumors have specific extracellular properties such as low pH and hypoxia. Identifying S. Typhimurium genes and their promoters that respond specifically to these tumor environmental properties has served as the basis for several lines of research with the hope of utilizing S. Typhimurium for anti-cancer based therapies. To identify such genes that are regulated in a tumor specific manner, we engineered a bioluminescent transposon reporter-trap to screen a S. Typhimurium library for genes specifically regulated by co-culture with malignant cells in vitro. Five previously undiscovered genes were identified (adiY, yohJ, STM1787, STM1791, and STM1793) and their promoter sequences were found to be specifically activated by the acidic microenvironment associated with cancer cells in vitro and tumors in vivo. Three of the genes (STM1787, STM1791, and STM1793) are controlled by a single promoter, which contains a recently discovered conserved tusp motif. We have uncovered that transcriptional regulation of this motif is more complex as it is sensitive to low pH in addition to hypoxia as was originally ascribed. The genes identified herein are highly expressed in an acidic pH tumor environment, but are not required for bacterial tumor targeting. Therefore, the promoters regulating these genes may be ideal candidates for utilization in therapeutic gene, pro-drug or toxin delivery studies. To address this inquiry, we utilized the cancer cell-activated STM1787 promoter sequence to regulate the expression of a toxic tumor transgene in a wild type strain of S. Typhimurium. Shiga-like toxin 2 (Stx2) is a holoenzyme produced by some strains of E.coli. It has been extensively studied for the potent translational inhibition characteristics of the A subunit in addition to the high tumor cell binding-specificity of the B subunit. Conditionally regulating the tumor-specific expression of Stx2 by STM1787s promoter resulted in striking cancer cell death in vitro, and shows promise in mouse xenografts in vivo. In conclusion, we have identified novel tumor-specific genes expressed by S. Typhimurium, identified the acidic pH-responsiveness of the tusp motif, and demonstrated that these promoters can be successfully engineered for targeted anti-cancer therapy using live S. Typhimurium. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr LB-367. doi:1538-7445.AM2012-LB-367