Real-Time Bioluminescence Imaging Of Salmonella-Neoplastic Cell Interactions

Salmonella Typhimurium is a Gram-negative bacterial pathogen and a common cause of gastroenteritis in humans. The organism utilizes a multitude of well-studied virulence factors to invade and replicate in host intestinal epithelial cells and macrophages. Interestingly, Salmonella is also capable of localizing to tumors in in vivo model systems, and while the typical route of Salmonella infection and pathogenesis has been thoroughly investigated, the behavior of Salmonella in the tumor microenvironment has not. Therefore, to investigate Salmonella and host behavior during bacterial-neoplastic cell interactions, I utilized two high-throughput screens. In the first, I designed a bioluminescent transposon-reporter trap to identify specific Salmonella genes activated in the context of cancer cell co-culture conditions. Through this work, I identified five Salmonella genes reproducibly activated by co-culture with cancer cells, and further isolated the activating stimulus to low pH. Because low pH is a common characteristic of the tumor microenvironment, I also demonstrated the pH inducibility and reversibility of Salmonellagene activation in tumors ex vivo and in vivo. In a separate study, to better understand how host neoplastic cells respond to Salmonella, I investigated the ability of Salmonellato induce pro-inflammatory responses in HCT116 colon carcinoma cells, specifically, NF-κB activation. Then, I performed a high-throughput siRNA screen to identify novel host kinases and phosphatases involved in detection of Salmonellaand activation of NF-κB signaling. For this work, I used a reporter construct consisting of an IκBα-firefly luciferase fusion protein transcriptionally activated by NF-κB. The reporter permitted imaging of both degradation of the NF-κB negative inhibitor IκBα and its resynthesis, which is dependent on NF-κB activation, following stimulus with Salmonella. The host kinase, NME3, was identified in the screen as a specific modulator of NF-κB. Knockdown of NME3 prevents proper activation of NF-κB signaling pathways in HCT116 cells exposed to Salmonella, demonstrating the role of this kinase as a positive regulator of NF-κB pro-inflammatory signaling in colon carcinoma cells

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

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 Rel(Msm) 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

An experimental study of Taylor-Goertler vortices in a curved rectangular channel

A rectangular cross section channel, designed and built by R. McKee to create the laminar secondary Taylor-Goertler vortex flow, was modified to expand the experimental investigation of the vortex flow. The modification allowed the hot wire anemometer, used to obtain velocity profiles, to be moved in both the radial and transverse direction. this additional freedom permitted a rectangular cross section of the flow to be investigated. It was concluded that the onset of Taylor-Goertler vortices was in agreement with existing information, that the intensity of the vortices increased with increasing flow rate, and turbulent fluctuations grew within the vortex structure. It was also concluded that flow is a curved channel resulted in distortion of the normally parabolic velocity profile shifting the maximum mean velocity toward the concave wall.http://archive.org/details/experimentalstud00flenLieutenant Commander, United States NavyApproved for public release; distribution is unlimited

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 organism's 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

Identification of 4-amino-thieno[2,3-d]pyrimidines as QcrB inhibitors in Mycobacterium tuberculosis

Antibiotic resistance is a global crisis that threatens our ability to treat bacterial infections, such as tuberculosis, caused b

In Vivo Bioluminescent Imaging (BLI): Noninvasive Visualization and Interrogation of Biological Processes in Living Animals

In vivo bioluminescent imaging (BLI) is increasingly being utilized as a method for modern biological research. This process, which involves the noninvasive interrogation of living animals using light emitted from luciferase-expressing bioreporter cells, has been applied to study a wide range of biomolecular functions such as gene function, drug discovery and development, cellular trafficking, protein-protein interactions, and especially tumorigenesis, cancer treatment, and disease progression. This article will review the various bioreporter/biosensor integrations of BLI and discuss how BLI is being applied towards a new visual understanding of biological processes within the living organism