The proteome of Shigella flexneri 2a 2457T grown at 30 and 37 degrees 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 degrees 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 virulence-related proteins were up-regulated at 37 degrees 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 degrees 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.

Fig. 1.

Two-dimensional gel electrophoresis and identified spots of whole-cell proteins at 37 °C in stationary phase S. flexneri. The identified spots are labeled on the integrated 2-DE map. The whole set of four maps is shown in supplemental Fig. 1 and can be found at the Proteome 2D-PAGE Database.

Fig. 2.

Distribution of proteins according to CAI (A), GRAVY value (B), cellular role categories (C), and localization (D). The whole set of genes encoding proteins (open bars) and identified proteins (black bars) was compared. The single letter codes in C represent a particular functional category as follows: J, translation; A, RNA processing and modification; K, transcription; L, replication, recombination, and repair; D, cell cycle control, mitosis, and meiosis; V, defense mechanisms; T, signal transduction mechanisms; M, cell wall/membrane biogenesis; N, cell motility; U, intracellular trafficking and secretion; O, post-translational modification, protein turnover, chaperones; C, energy production and conversion; G, carbohydrate transport and metabolism; E, amino acid transport and metabolism; F, nucleotide transport and metabolism; H, coenzyme transport and metabolism; I, lipid transport and metabolism; P, inorganic ion transport and metabolism; Q, secondary metabolite biosynthesis, transport, and catabolism; R, general function prediction only; S, function unknown; _, not in COG. The meanings of abbreviations in D are as follows: C, cytoplasmic; CM, cytoplasmic membrane; E, extracellular; OM, outer membrane; P, periplasmic; U, unknown; ML, multiple localization sites.

Fig. 3.

Enlarged images of 2-DE gels highlighting selected differentially expressed proteins. 30lg indicates grown to log phase at 30 °C, 37lg indicates grown to log phase at 37 °C, 30st indicates grown to stationary phase at 30 °C, and 37st indicates grown into stationary phase at 37 °C. The arrows indicated the spots of corresponding proteins listed on the left.

Fig. 4.

Differential expression of ArgT is due to proteolysis. A, semiquantitative RT-PCR analysis of argT. B, overexpression of ArgT at 37 and 30 °C. 2457T/pProEX-argT (2457T carrying pProEX-HTb-argT) was induced by IPTG at 37 and 30 °C, respectively. M, protein marker (Fermentas).

Fig. 5.

ArgT undergoes proteolysis in periplasmic space of S. flexneri. A, the periplasmic proteins (periplasm) and the remaining whole-cell proteins (cytoplasm + membrane) of induced 2457T/pProEX-argT cells were extracted from cultures grown at 37 and 30 °C. ArgT expression products were only detected in the periplasm of 2457T grown at 30 °C. B, 2457T/pGEX-argT (with signal peptide) and 2457T/pGEX-argT2 (without signal peptide) were induced by IPTG at 30 °C and then incubated at 37 and 30 °C for 4 h. Due to the cleavage of the signal peptide, the ArgT detected in 2457T/pGEX-argT is smaller than that in 2457T/pGEX-argT2. M, protein marker (Fermentas).

Fig. 6.

ArgT, overexpressed at 30 °C, was degraded at 37 °C without new protein synthesis. The cultures of 2457T/pProEX-argT were induced to express ArgT at 30 °C for 4 h. Then, chloramphenicol was added to prevent new protein synthesis. After that, the cells were incubated at 30 (left) and 37 °C (right) for another 6 h. CK, the cultures of 2457T/pProEX-argT induced to express at 37 °C. M, protein marker (Fermentas).

Fig. 7.

ArgT was degraded by HtrA protease in vivo and in vitro. A, ArgT could not be degraded in the htrA deletion mutant. Plasmid pProEX-HTb-argT was transformed into 2457T and 2457TΔhtrA, and then strains carrying the recombinant plasmids were induced by IPTG at 37 and 30 °C, respectively. B, 50 μg of GST-ArgT and 0.5 μg of GST-HtrA/HtrAS210A fusion proteins were incubated in a final volume of 100 μl of 50 mm Tris-Cl, pH 8.0 buffer at 37 and 30 °C. 15 μl of the incubation was removed every hour and loaded for SDS-PAGE. M, protein marker (Fermentas).

Fig. 8.

Identification of cleavage site in ArgT. A, GST-ArgT and GST-HtrA were incubated at 37 °C for 2 h, and then the degradation products were separated by 15% SDS-PAGE. The two cleavage fragments (frag1 and frag2) were cut out and digested by trypsin and Glu-C. The resolved digestions were analyzed by MALDI-TOF/TOF. B, the PMF result of frag2 digested by trypsin and the MS/MS result of the target peptide (theoretical m/z 1725.82; sequence, AYANQDLVYSDLAAGR). C, the PMF result of frag2 digested by Glu-C and the MS/MS result of the target peptide (theoretical m/z 2581.22; sequence, AYANQDLVYSDLAAGRLDAALQDE). These two peptides suggest the same cleavage site between position 160 and position 161 (VV↓AY) of ArgT. D, part of the sequence of ArgT. The amino acid sequences of the above two peptides are italicized and underlined, respectively. M, protein marker (Fermentas).