Abstract
We performed proteolytic surface-shaving with trypsin on three strains/sevovars of Salmonella enterica enterica (SEE): Newport, Kentucky, and Thompson. Surfaced-exposed proteins of live bacterial cells were digested for 15 min. A separate 20 h re-digestion was also performed on the supernatant of each shaving experiment to more completely digest protein fragments into detectable peptides for proteomic analysis by nano-liquid chromatography-electrospray ionization-Orbitrap mass spectrometry. Control samples (i.e., no trypsin during surface-shaving step) were also performed in parallel. We detected peptides of flagella proteins: FliC (filament), FliD (cap), and FlgL (hook-filament junction) as well as peptides of FlgM (anti-σ28 factor), i.e., the negative regulator of flagella synthesis. For SEE Newport and Thompson, we detected Salmonella pathogenicity island 1 (SPI-1) secreted effector/invasion proteins: SipA, SipB, SipC, and SipD, whereas no Sip proteins were detected in control samples. No Sip proteins were detected for SEE Kentucky (or its control) although sip genes were confirmed to be present. Our results may suggest a biological response (<15 min) to proteolysis of live cells for these SEE strains and, in the case of Newport and Thompson, a possible invasion response.
Highlights
Bacterial surface-shaving is a technique by which surface-exposed biomolecules are cleaved from the surface of live cells with proteolytic enzymes, e.g., trypsin, followed by detection by liquid chromatography tandem mass spectrometry (LC/MS/MS) [1,2,3]
We identified a number of effector proteins associated with pathogen virulence whose genes are located on pathogenicity island 1 (SPI-1): SipA, SipB, SipC, and SipD
Sample contamination by cytoplasmic proteins is a common problem associated with proteolytic surface-shaving experiments, the amount of cell lysis observed for each strain in our study varied significantly and appeared to be strain dependent
Summary
Bacterial surface-shaving is a technique by which surface-exposed biomolecules (usually proteins) are cleaved from the surface of live cells with proteolytic enzymes, e.g., trypsin, followed by detection by liquid chromatography tandem mass spectrometry (LC/MS/MS) [1,2,3]. Analyses were performed on control samples, i.e., no trypsin during the 15 min surface-shaving step (Table 2 and Supplementary Materials Newport). As with the SEE Newport strain, the number of peptides detected/identified is significantly increased for the 20 h re-digest compared to the 15 min shaving
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