Abstract
The pathogen Salmonella enterica is known to cause both food poisoning and typhoid fever. Because of the emergence of antibiotic-resistant isolates and the threat of bioterrorism (e.g. contamination of the food supply), there is a growing need to study this bacterium. In this investigation, comparative peptidomics was used to study S. enterica serovar Typhimurium cultured in either a rich medium or in an acidic, low magnesium, and minimal nutrient medium designed to roughly mimic the macrophage phagosomal environment (within which Salmonella are known to survive). Native peptides from cleared cell lysates were enriched by using isopropanol extraction and analyzed by using both LC-MS/MS and LC-FTICR-MS. We identified and quantified 5,163 peptides originating from 682 proteins, and the data clearly indicated that compared with Salmonella cultured in the rich medium, cells cultured in the phagosome-mimicking medium had dramatically higher abundances of a wide variety of protein degradation products, especially from ribosomal proteins. Salmonella from the same cultures were also analyzed using traditional, bottom-up proteomic methods, and when the peptidomics and proteomics data were analyzed together, two clusters of proteins targeted for proteolysis were tentatively identified. Possible roles of targeted proteolysis by phagocytosed Salmonella are discussed.
Highlights
The pathogen Salmonella enterica is known to cause both food poisoning and typhoid fever
In this article we describe the use of high throughput LC-MS and LC-MS/MS to identify and quantify thousands of peptides generated by S. typhimurium grown in host-free culture conditions designed to mimic the environment of the phagosome
High throughput LC-MS and LCMS/MS were used to identify and quantify thousands of native S. typhimurium peptides generated during conditions designed to mimic the environment of the phagosome
Summary
Reagents—Water was purified using a NANOpure® system (Ն18 M⍀ϫcm, Barnstead International, Dubuque, IA). Cell Culture—Wild type S. enterica serovar typhimurium strains 14028 and LT2 were grown to logarithmic (Log) phase, stationary (Stat) phase, magnesium minimal medium- (MgM-) shocked stationary phase, and linear in N-Salts medium phase and harvested following standard batch culture techniques [11]. Cultures were initiated by inoculating 5 ml of either LB (Luria-Bertani) broth (Log, Stat, and MgM samples) or N-Salts medium (N-Salts samples) with cells from a single colony and grown for 16 h at 37 °C. A 2-liter flask containing 300 ml of either LB broth (Log, Stat, and MgM samples) or N-Salts medium (N-Salts samples) was inoculated with 3 ml of the starter culture and shaken at 200 rpm at 37 °C. The Log, Stat, and N-Salts cultures were harvested at room temperature at an OD600 nm of 0.6, 2.0, and 0.2, respectively. Harvested cell cultures were centrifuged at 5000 ϫ g, cell pellets were washed twice with Dulbecco’s phosphate buffered saline (Mediatech, Inc., Herndon, VA), and cell pellets with an approximate wet weight of 0.1 g/tube were stored at Ϫ80 °C
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