The Proteome of Shigella flexneri 2a 2457T Grown at 30 and 37 °C

Abstract

To upgrade the proteome reference map of Shigella flexneri 2a 2457T, the protein expression profiles of log phase and stationary phase cells grown at 30 and 37°C were thoroughly analyzed using multiple overlapping narrow pH range (between pH 4.0 and 11.0) two-dimensional gel electrophoresis. A total of 723 spots representing 574 protein entries were identified by MALDI-TOF/TOF MS, including the majority of known key virulence factors. 64 hypothetical proteins and six misannotated proteins were also experimentally identified. A comparison between the four proteome maps showed that most of the virulencerelated proteins were up-regulated at 37°C, and the differences were more notable in stationary phase cells, suggesting that the expressions of these virulence factors were not only controlled by temperature but also controlled by the nutrients available in the environment. The expression patterns of some virulence-related genes under the four different conditions suggested that they might also be regulated at the post-transcriptional level. A further significant finding was that the expression of the protein ArgT was dramatically up-regulated at 30°C. The results of semiquantitative RT-PCR analysis showed that expression of argT was not regulated at the transcriptional level. Therefore, we carried out a series of experiments to uncover the mechanism regulating ArgT levels and found that the differential expression of ArgT was due to its degradation by a periplasmic protease, HtrA, whose activity, but not its synthesis, was affected by temperature. The cleavage site in ArgT was between position 160 (Val) and position 161 (Ala). These results may provide useful insights for understanding the physiology and pathogenesis of S. flexneri. Molecular & Cellular Proteomics 9:1209 -1220, 2010.Shigella flexneri is a Gram-negative facultative pathogen that causes the majority of communicable bacterial dysenteries in developing countries. Seven years ago, the whole genome sequence of S. flexneri 2a strain 2457T was determined (1), and a proteome reference map was also constructed at the same time (2). Predicting the pI of all proteins encoded by the bacterial genome revealed that about 45.54% (1972 proteins) were basic. However, all of these basic proteins and the vast majority of virulence-related proteins were missed in the old proteome reference map because of the limitations of the experimental conditions used at that time. Now, the proteomics platform of two-dimensional polyacrylamide gel electrophoresis (2-DE) 1 has been optimized and further developed. The resolution and sensitivity of today's 2-DE technique has been greatly improved. As a result, the expression profiles obtained today are difficult to compare with old profiles. An upgrade of the reference proteome map is urgently required for further proteomics research of S. flexneri, particularly for comparative studies.The virulence genes of Shigella flexneri are located on a large (ϳ230-kb) plasmid acquired by horizontal transfer and encode a type III secretion syst...

Analysis of the Proteome of Intracellular Shigella flexneri Reveals Pathways Important for Intracellular Growth

“…Our 2D gel analysis revealed a C-terminal 28-kDa posttranslational truncation product of the protease SepA (Fig. 1), which was also observed by Zhu et al (89). SepA was quantitatively increased intracellularly.…”

“…Consistent with this, we noted high levels of Ipa, Mxi, and Ics proteins, among other virulence factors, in intracellular bacteria. In an in vitro proteomic analysis of S. flexneri grown at different temperatures, Zhu et al (89) observed that IpaA, IpaC, IpaD, IpgC, and SepA were increased at 37°C versus 30°C. Our 2D gel analysis revealed a C-terminal 28-kDa posttranslational truncation product of the protease SepA (Fig.…”

Analysis of Soluble Protein Complexes in Shigella flexneri Reveals the Influence of Temperature on the Amount of Lipopolysaccharide

“…The gel was electrophoresed at 100 V for 1 h at 4°C. The cathode electrophoresis buffer was then exchanged for cathode buffer without Coomassie Brilliant Blue G-250 and run for a further 16 h at 300 V. For the second dimensional SDS-PAGE, a lane was cut out of the first dimensional gel and dipped into equilibrating buffer (8). SDS-PAGE was performed using a 12.5% separating gel according to standard protocols.…”

Omics in Aid of Host-pathogen Interaction Studies: A Bacterial Perspective

“…Nonetheless, if the focus is on the pathogen's response, its molecular content becomes limiting, making it highly desirable to implement strategies to enrich the molecular content of the pathogen, strategies which are however, not always available or easy to implement. As such, a frequently used alternative approach to study pathogenic responses is to grow the pathogen in conditions mimicking the host environment [8][9][10][11].…”