The terminal O-polysaccharide LY294002 in vivo structures vary greatly among Shigella, thereby giving rise to the immunological specificity that has resulted in distinct serotypes. Although attenuated Shigella strains expressing genetically engineered O-antigens have been tested as vaccine candidates, an effective vaccine against Shigella remains elusive [2], possibly due to the diversity of the O-antigens. Comprehensive proteomic profiling has the potential to identify novel virulence factors in Shigella that could form potential vaccine or therapeutic targets. Proteomic surveys of Shigella have mainly focused on S. flexneri, which causes

endemic shigellosis. Extensive 2D-LC-MS/MS-based profiling of the S. flexneri membrane proteome by Wei et al. resulted in the identification of more than 600 S. flexneri proteins including ca. 200 integral and outer membrane proteins [11]. Immunoproteome buy CUDC-907 analyses of S. flexneri identified several membrane proteins as being antigenic including OmpA, IpaD, Spa33, TolC and YaeT [12, 13]. The S. dysenteriae strain Sd197 was the first S. dysenteriae genome to be sequenced [14], and included sequences of the chromosome, a large virulence-associated CP690550 plasmid (pSD1_197) and a small plasmid (pSD197_Spa). This annotated SD1 genome was exploited

in a comprehensive proteomic survey of S. dysenteriae strain Sd1617 via 2D gel electrophoresis which resulted in the identification of 1061 distinct gene products [15]. Immunoproteome analysis of SD1 Sd1617 identified seven proteins including type III secretion system effectors OspC2 and IpaB as antigens. In this report, a quantitative global proteomic analysis of SD1 cells grown to stationary phase in culture (in vitro) vs. SD1 cells isolated from mammalian

host environment (in vivo) was performed using 2D-LC-MS/MS and APEX, a label-free computationally modified spectral counting method [16]. Data from the SD1 in vitro and in vivo proteomes was analyzed for differential protein expression in the context of virulence and survival Nintedanib (BIBF 1120) in the host. Methods Materials and reagents All reagents for protein extraction from cell lysates and protein analysis by mass spectrometry (MS) were used as previously described [15, 17]. RNase, DNase I (bovine pancreas), triethyl ammonium bicarbonate (TAB) buffer used for tryptic digestion, TCEP (Tris(2-carboxyethyl)phosphine) used as a reducing agent and the bicinchoninic acid (BCA) protein assay kit to estimate protein concentrations were purchased from Sigma-Aldrich (St. Louis, MO). The alkylating agent MMTS (methyl methanethiosulfonate) was purchased from Pierce (Rockford, IL). Sequencing grade porcine trypsin was obtained from Promega (Madison, WI). Triton X-100 was purchased from Calbiochem (LaJolla, CA). SDS-PAGE was performed according to instructions from Invitrogen.

No related posts.