Carbon-Starvation Induces Cross-Resistance to Thermal, Acid, and Oxidative Stress in Serratia marcescens.

The broad host-range pathogen Serratia marcescens survives in diverse host and non-host environments, often enduring conditions in which the concentration of essential nutrients is growth-limiting. In such environments, carbon and energy source starvation (carbon-starvation) is one of the most common forms of stress encountered by S. marcescens. Related members of the family Enterobacteriaceae are known to undergo substantial changes in gene expression and physiology in response to the specific stress of carbon-starvation, enabling non-spore-forming cells to survive periods of prolonged starvation and exposure to other forms of stress (i.e., starvation-induced cross-resistance). To determine if carbon-starvation also results in elevated levels of cross-resistance in S. marcescens, both log-phase and carbon-starved cultures, depleted of glucose before the onset of high cell-density stationary-phase, were grown in minimal media at either 30 °C or 37 °C and were then challenged for resistance to high temperature (50 °C), low pH (pH 2.8), and oxidative stress (15 mM H2O2). In general, carbon-starved cells exhibited a higher level of resistance to thermal stress, acid stress, and oxidative stress compared to log-phase cells. The extent of carbon-starvation-induced cross-resistance was dependent on incubation temperature and on the particular strain of S. marcescens. In addition, strain- and temperature-dependent variations in long-term starvation survival were also observed. The enhanced stress-resistance of starved S. marcescens cells could be an important factor in their survival and persistence in many non-host environments and within certain host microenvironments where the availability of carbon sources is suboptimal for growth.

Proteomic analysis of cross protection provided between cold and osmotic stress in Listeria monocytogenes.

Listeria monocytogenes is a Gram-positive, foodborne pathogen responsible for approximately 28% of all food-related deaths each year in the United States. L. monocytogenes infections are linked to the consumption of minimally processed ready-to-eat (RTE) products such as cheese, deli meats, and cold-smoked finfish products. L. monocytogenes is resistant to stresses commonly encountered in the food-processing environment, including low pH, high salinity, oxygen content, and various temperatures. The purpose of this study was to determine if cells habituated at low temperatures would result in cross-protective effects against osmotic stress. We found that cells exposed to refrigerated temperatures prior to a mild salt stress treatment had increased survival in NaCl concentrations of 3%. Additionally, the longer the cells were pre-exposed to cold temperatures, the greater the increase in survival in 3% NaCl. A proteomics analysis was performed in triplicate in order to elucidate mechanisms involved in cold-stress induced cross protection against osmotic stress. Proteins involved in maintenance of the cell wall and cellular processes, such as penicillin binding proteins and osmolyte transporters, and processes involving amino acid metabolism, such as osmolyte synthesis, transport, and lipid biosynthesis, had the greatest increase in expression when cells were exposed to cold temperatures prior to salt. By gaining a better understanding of how this pathogen adapts physiologically to various environmental conditions, improvements can be made in detection and mitigation strategies.

Proteomic analysis of the response of Listeria monocytogenes to bile salts under anaerobic conditions.

Listeria monocytogenes is a food-borne pathogen responsible for the disease listeriosis. The infectious process depends on survival in the high bile-salt conditions encountered throughout the gastrointestinal tract, including the gallbladder. However, it is not clear how bile-salt resistance mechanisms are induced, especially under physiologically relevant conditions. This study sought to determine how the L. monocytogenes strains EGDe (serovar 1/2a), F2365 (serovar 4a) and HCC23 (serovar 4b) respond to bile salts under anaerobic conditions. Changes in the expressed proteome were analysed using multidimensional protein identification technology coupled with electrospray ionization tandem mass spectrometry. In general, the response to bile salts among the strains tested involved significant alterations in the presence of cell-wall-associated proteins, DNA repair proteins, protein folding chaperones and oxidative stress-response proteins. Strain viability correlated with an initial osmotic stress response, yet continued survival for EGDe and F2365 involved different mechanisms. Specifically, proteins associated with biofilm formation in EGDe and transmembrane efflux pumps in F2365 were expressed, suggesting that variations exist in how virulent strains respond and adapt to high bile-salt environments. These results indicate that the bile-salt response varies among these serovars and that further research is needed to elucidate how the response to bile salts correlates with colonization potential in vivo.

Proteomic expression profiles of virulent and avirulent strains of Listeria monocytogenes isolated from macrophages.

Listeria monocytogenes is able to survive and proliferate within macrophages. In the current study, the ability of three L. monocytogenes strains (serovar 1/2a strain EGDe, serovar 4b strain F2365, and serovar 4a strain HCC23) to proliferate in the murine macrophage cell line J774.1 was analyzed. We found that the avirulent strain HCC23 was able to initiate an infection but could not establish prolonged infection within the macrophages. By contrast, strains EGDe and F2365 proliferated within macrophages for at least 7 h. We further analyzed these strains by comparing their protein expression profiles at 0 h, 3 h, and 5 h post-infection using multidimensional protein identification technology coupled with electrospray ionization tandem mass spectrometry. Our results indicated that similar metabolic and cell wall associated proteins were expressed by all three strains at 3 h post-infection. However, increased expression of stress response and DNA repair proteins was associated with the ability to proliferate in macrophages at 5 h post-infection. By comparing the protein expression patterns of these three L. monocytogenes strains during intracellular growth in macrophages, we were able to detect biological differences that may determine the ability of L. monocytogenes to survive in macrophages.