Salmonella enterica serovar Typhimurium adhesion and cytotoxicity during epithelial cell stress is reduced by Lactobacillus rhamnosus GG

<p>Abstract</p> <p>Background</p> <p>Physiological stressors may alter susceptibility of the host intestinal epithelium to infection by enteric pathogens. In the current study, cytotoxic effect, adhesion and invasion of <it>Salmonella enterica </it>serovar Typhimurium (<it>S</it>. Typhimurium) to Caco-2 cells exposed to thermal stress (41°C, 1 h) was investigated. Probiotic bacteria have been shown to reduce interaction of pathogens with the epithelium under non-stress conditions and may have a significant effect on epithelial viability during infection; however, probiotic effect on pathogen interaction with epithelial cells under physiological stress is not known. Therefore, we investigated the influence of <it>Lactobacillus rhamnosus </it>GG and <it>Lactobacillus gasseri </it>on <it>Salmonella </it>adhesion and <it>Salmonella</it>-induced cytotoxicity of Caco-2 cells subjected to thermal stress.</p> <p>Results</p> <p>Thermal stress increased the cytotoxic effect of both <it>S</it>. Typhimurium (P = 0.0001) and nonpathogenic <it>E. coli </it>K12 (P = 0.004) to Caco-2 cells, and resulted in greater susceptibility of cell monolayers to <it>S</it>. Typhimurium adhesion (P = 0.001). Thermal stress had no significant impact on inflammatory cytokines released by Caco-2 cells, although exposure to <it>S</it>. Typhimurium resulted in greater than 80% increase in production of IL-6 and IL-8. Blocking <it>S</it>. Typhimurium with anti-ShdA antibody prior to exposure of <it>Salmonella </it>decreased adhesion (P = 0.01) to non-stressed and thermal-stressed Caco-2 cells. Pre-exposure of Caco-2 cells to <it>L. rhamnosus </it>GG significantly reduced <it>Salmonella</it>-induced cytotoxicity (P = 0.001) and <it>Salmonella </it>adhesion (P = 0.001) to Caco-2 cells during thermal stress, while <it>L. gasseri </it>had no effect.</p> <p>Conclusion</p> <p>Results suggest that thermal stress increases susceptibility of intestinal epithelial Caco-2 cells to <it>Salmonella </it>adhesion, and increases the cytotoxic effect of <it>Salmonella </it>during infection. Use of <it>L. rhamnosus </it>GG as a probiotic may reduce the severity of infection during epithelial cell stress. Mechanisms by which thermal stress increases susceptibility to <it>S</it>. Typhimurium colonization and by which <it>L. rhamnosus </it>GG limits the severity of infection remain to be elucidated.</p

The role of Listeria adhesion protein (LAP) during the intestinal phase of Listeria monocytogenes pathogenesis

Listeria monocytogenes is an intracellular, foodborne pathogen which causes systemic listeriosis in physiologically stressed or immunocompromised individuals. Early interaction with the intestinal epithelium is essential for Listeria infection, and involves the concerted action of multiple virulence factors. We identified Listeria adhesion protein (LAP) and demonstrated that LAP facilitated adhesion to intestinal epithelial cells and promoted full virulence during oral infection of mice. Our early work indicated that LAP expression was influenced by stressors common to the host gastrointestinal (GI) tract, including anaerobiosis. However, the mechanism by which LAP was exported to the bacterial cell surface was unclear, as was the influence of GI-related stressors on LAP-mediated infectivity. The epithelial receptor for LAP was identified as the human stress response chaperone, heat shock protein 60 (Hsp60). Hsp60 was shown to be necessary for LAP-mediated binding to intestinal epithelial cells, but the precise role for the LAP-Hsp60 interaction during Listeria infection was unknown. Others have shown that stressors, including infection, can alter host Hsp60 expression, but it was unclear how such a stress response might impact LAP-mediated infection. Therefore, the goals of the following studies were to characterize the role of both LAP and Hsp60 during Listeria infection. Here we examined a potential mechanism for LAP secretion and surface localization, as well as the influence of anaerobiosis on LAP cellular localization and LAP-mediated infectivity. We also evaluated the role of the LAP-Hsp60 interaction in bacterial adhesion to, invasion of and transepithelial translocation through Caco-2 intestinal epithelial monolayers, and determined the influence of Listeria infection on host Hsp60 expression and LAP-mediated infection. Data indicate that LAP is secreted by the SecA2 system, and that anaerobiosis increases LAP secretion in L. monocytogenes. Exposure of L. monocytogenes to an anaerobic environment also increased LAP-mediated adhesion to Caco-2 cells and enhanced bacterial translocation to the liver and spleen of mice in a LAP-specific manner. By altering Hsp60 expression in Caco-2 cells, we confirmed that the LAP-Hsp60 interaction promotes Listeria adhesion, and also demonstrated its role in mediating bacterial transepithelial translocation. Further, L. monocytogenes infection increased Caco-2 expression of Hsp60, which rendered host cells more susceptible to LAP-mediated adhesion and translocation by L. monocytogenes. Data offer new insight into the role of LAP as a virulence factor during the intestinal phase of Listeria pathogenesis, and pose new questions regarding the dynamics between the host stress response and pathogen infection

Subtropical to Subpolar Lagrangian Pathways in the North Atlantic and Their Impact on High Latitude Property Fields

<p>In response to the differential heating of the earth, atmospheric and oceanic flows constantly act to carry surplus energy from low to high latitudes. In the ocean, this poleward energy flux occurs as part of the large scale meridional overturning circulation: warm, shallow waters are transported to high latitudes where they cool and sink, then follow subsurface pathways equatorward until they are once again upwelled to the surface and reheated. In the North Atlantic, the upper limb of this circulation has always been explained in simplistic terms: the Gulf Stream/North Atlantic Current system carries surface waters directly to high latitudes, resulting in elevated sea surface temperatures in the eastern subpolar gyre, and, because the prevailing winds sweeping across the Atlantic are warmed by these waters, anomalously warm temperatures in Western Europe. This view has long been supported by Eulerian measurements of North Atlantic sea surface temperature and surface velocities, which imply a direct and continuous transport of surface waters between the two gyres. However, though the importance of this redistribution of heat from low to high latitudes has been broadly recognized, few studies have focused on this transport within the Lagrangian frame. </p><p>The three studies included in this dissertation use data from the observational record and from a high resolution model of ocean circulation to re-examine our understanding of upper limb transport between the subtropical and subpolar gyres. Specifically, each chapter explores intergyre Lagrangian pathways and investigates the impact of those pathways on subpolar property fields. The findings from the studies suggest that intergyre transport pathways are primarily located beneath the surface and that subtropical surface waters are largely absent from the intergyre exchange process, a very different image of intergyre transport than that compiled from Eulerian data alone. As such, these studies also highlight the importance of including 3d Lagrangian information in examinations of transport pathways.</p>Dissertatio

Longitudinal Study of the Impacts of a Climate Change Curriculum on Undergraduate Student Learning: Initial Results

The present study assesses the efficacy of a semester-long undergraduate sustainability curriculum designed from a systems approach. The three-course curriculum, which incorporated environmental science and ethics courses along with an integrative course using a community-based learning pedagogy, was intended to provide students with experience using knowledge and skills from distinct disciplines in a holistic way in order to address the complex problems of the human acceptance of and response to anthropogenic climate change. In the fall of 2013, 23 of the 24 sophomore general education students enrolled in the three courses were surveyed at the beginning and end of the semester; 17 of those same students completed the survey again in the spring of 2016, their senior year. Results, which focus on the 17 students who continued to participate through their senior year, were analyzed with quantitative and qualitative methodologies. The pre/post data from the surveys demonstrated significant improvement in climate literacy, certainty, concern and urgency over the course of the semester; the senior data indicated that those improvements were largely retained. The study also suggests that the nine-credit curriculum improved transferable skills such as interdisciplinary thinking, self-confidence and public speaking. A qualitative analysis of three student cases, informed by a focus group (n = 7) of seniors along with other sources of information, suggested retention of such transferable skills, and, in some cases, deeper involvement in climate and sustainability action

Bacteria-mediated delivery of nanoparticles and cargo into cells

Nanoparticles and bacteria can be used, independently, to deliver genes and proteins into mammalian cells for monitoring or altering gene expression and protein production. Here, we show the simultaneous use of nanoparticles and bacteria to deliver DNA-based model drug molecules in vivo and in vitro. In our approach, cargo (in this case, a fluorescent or a bioluminescent gene) is loaded onto the nanoparticles, which are carried on the bacteria surface. When incubated with cells, the cargo-carrying bacteria (‘microbots’) were internalized by the cells, and the genes released from the nanoparticles were expressed in the cells. Mice injected with microbots also successfully expressed the genes as seen by the luminescence in different organs. This new approach may be used to deliver different types of cargo into live animals and a variety of cells in culture without the need for complicated genetic manipulations