Proteolytic Surface-Shaving and Serotype-Dependent Expression of SPI-1 Invasion Proteins in Subspecies

We performed proteolytic surface-shaving with trypsin on three strains/sevovars of (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-σ factor), i.e., the negative regulator of flagella synthesis. For SEE Newport and Thompson, we detected 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 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.