Analysis of Soluble protein complexes in Shigella flexneri reveals the influence of temperature on the amount of lipopolysaccharide

Abstract

Shigella flexneri, which is closely related to Escherichia coli, is the most common cause of the endemic form of shigellosis. In this study, 53 homomultimeric protein complexes and nine heteromultimeric protein complexes from S. flexneri 2a strain 2457T were separated and identified. Among these, three potential homomultimeric protein complexes had not been previously described. One complex, thought to be composed of 12 PhoN1 subunits, is a periplasmic protein with an unknown physiological role encoded on the virulence plasmid of S. flexneri. The abundance of the protein complexes was compared following growth at 37 or 30°C, and the abundance of three protein complexes (PyrB-PyrI, GlmS, and MglB) related to the synthesis of lipopolysaccharides (LPS) appeared to be temperature-dependent. Many studies have shown that LPS is essential to the virulence of S. flexneri. Here, we report the influence of temperature on the amount of LPS.

Fig. 1.

Whole cell soluble protein complexes separated by BN-SDS-PAGE. Two different acrylamide gradients (6–11% (A) and 10–16% (B)) were used in the first dimension.

Fig. 2.

Detection of SodB in protein complex samples using Western blot. Protein complex samples were separated by 10–16% native SDS-PAGE (upper panel) and then immunoblotted with anti-Myc antibody (lower panel).

Fig. 3.

Detection of IpaB in protein complex samples using Western blot. A, protein complex samples were separated by 12% denatured SDS-PAGE and immunoblotted with anti-IpaB antibody. B, protein complex samples were separated by 4–15% native SDS-PAGE and immunoblotted with anti-IpaB antibody.

Fig. 4.

Analysis of soluble protein complexes from S. flexneri grown at 37°C (A) and 30°C (B) by BN-SDS-PAGE. An acrylamide gradient of 4–15% was used in the first dimension. Enlarged images of differential protein spots are shown in the lower panel of C. Transcript levels of pyrB, pyrI, gcvP, mglB, glmS, ipaB, and mxiH were determined by qRT-PCR normalized against the levels of the 16 S rRNA gene in each sample (D).

Fig. 5.

Three of the identified differentially expressed protein complexes were related to LPS biosynthesis. UTP and GlcNAc-1-phosphate are the products of pathways involving aspartate transcarbamylase complex (ATCase) (composed of two catalytic trimers, PyrB, and three regulatory dimers, PyrI) and GFAT (composed of two GlmS subunits), respectively, and can be catalyzed to produce UDP-GlcNAc, which participates in LPS biosynthesis. The substrate transported by the MglABC complex can be directly used as a sugar moiety in LPS biosynthesis. rha, rhamnose; gln, N-acetylglucosamine; glu, glucose; gal, galactose.

Fig. 6.

LPS from S. flexneri grown at 37 and 30°C. A, LPS samples from equal numbers of bacterial cells were loaded into each lane and were analyzed by SDS-PAGE on a 15% (w/v) acrylamide gel, followed by silver staining. B, surface topology of strains detected by transmission electron microscopy after thin sectioning. Scale bar, 100 nm.