Identification of substrates and chaperone from the Yersinia enterocolitica 1B Ysa type III secretion system

Abstract

All pathogenic Yersinia enterocolitica strains carry the pYV plasmid encoding the Ysc-Yop type III secretion (TTS) system, which operates at 37 degrees C. In addition, biovar 1B Y. enterocolitica strains possess a second, chromosomally encoded, TTS system called Ysa, which operates, at least in vitro, under low-temperature and high-salt (LTHS) conditions. Six open reading frames, sycB, yspB, yspC, yspD, yspA, and acpY, neighbor the ysa genes encoding the Ysa TTS apparatus. Here we show that YspA, YspB, YspC, and YspD are secreted by the Ysa TTS system under LTHS conditions. SycB is a chaperone for YspB and YspC and stabilizes YspB. YspB, YspC, and SycB share some similarity with TTS substrates and the chaperone encoded by the Mxi-Spa locus of Shigella flexneri and SPI-1 of Salmonella enterica. In addition, Ysa also secretes the pYV-encoded YopE under LTHS conditions, indicating that YopE is a potential effector of both Y. enterocolitica TTS systems. YspC could also be secreted by S. flexneri, but no functional complementation of ipaC was observed, which indicates that despite their similarity the Ysa and the Mxi-Spa systems are not interchangeable. When expressed from the yopE promoter, YspB and YspC could also be secreted via the Ysc injectisome. However, they could not form detectable pores in eukaryotic target cells and could not substitute for YopB and YopD for translocation of Yop effectors.

FIG. 1.

Identification of proteins secreted by the Ysa TTSS. (A) Detail of the organization of the sycB-yspB-yspC-yspD-yspA operon located 98 bp downstream of ysaU (white arrow). (B) Culture supernatants of 1.5 × 10⁸ Y. enterocolitica 8081 bacteria grown at 26°C for 18 h in LB containing 490 mM NaCl were precipitated with TCA (10%), loaded on an SDS-12% polyacrylamide gel, and stained with Coomassie blue. WT, wild-type strain 8081; ysaN, 8081FM2; yspD, 8081PT15; yspB, 8081BF19; yspA, 8081PT16. Proteins secreted by the Ysa TTS apparatus are labeled by arrows, and their apparent molecular weights (MW; in thousands) are indicated. NI, nonidentified proteins. (C) Western blot of proteins from culture supernatants and bacterial extracts with rabbit polyclonal antibodies directed against YspC (48 kDa), YspD (38 kDa), YopE (23 kDa), and SycE (15 kDa); the same strains as in panel B are shown, with (+) or without (−) their pYV plasmid. (D) Western blot of proteins from the culture supernatant of Y. enterocolitica 8081 carrying (+) or not (−) pMM100 (lacI⁺) and pBF23 [yspB(His)₆⁺] grown at 26°C for 18 h in LB containing 490 mM NaCl. When necessary, IPTG at a 1 mM final concentration was added prior to the 18-h incubation. Numbers at right are molecular weights (MW) in thousands.

FIG. 2.

SycB binds YspB. (A) Detail of the bicistronic constructs of pBF23 and pBF24. (B) Western blot analysis of proteins eluted from glutathione-Sepharose CL-4B or protein A-anti-GST-Sepharose CL-4B loaded with clear lysates of E. coli expressing GST-SycB and YspB-(His)₆ (lanes 1) or GST-SycB_1-21 and YspB-(His)₆ (lanes 2). MW, molecular weights in thousands; Glutathi., glutathione.

FIG. 3.

SycB binds YspC. (A) Coomassie blue-stained SDS-polyacrylamide gel. (B) Western blot analysis of proteins eluted from a glutathione-Sepharose CL-4B column preincubated with extracts of E. coli producing GST alone (lane 2) or GST-SycB (lane 1) and loaded with 20 μg of purified YspC. The Western blot was revealed with an anti-YspC polyclonal antibody (upper part) or anti-GST polyclonal antibody (lower part). MW, molecular weights in thousands.

FIG. 4.

SycB stabilizes YspB. Shown are results of Western blot analysis of crude extracts of E. coli XL-1 Blue(pBF23) (A) and XL-1 Blue(pBF24) (B) showing the steady-state level of YspB-(His)₆ and GST-SycB or GST-SycB_1-21 after inhibition of protein synthesis by addition of chloramphenicol (Chlor.) after 80 min of inoculation under LTHS conditions. YspB-(His)₆ was detected by an anti-His (C-terminal) monoclonal antibody, and GST-SycB or GST-SycB_1-21 was detected with an anti-GST polyclonal antibody. MW, molecular weights in thousands.

FIG. 5.

Lack of activity of YspB-(His)₆ or YspC secreted by the Ysc injectisome of Y. enterocolitica ΔHOPEM. (A) Bacteria (2.5 × 10⁷; C) or the culture supernatant of 5 × 10⁹ bacteria (S) was loaded on an SDS-polyacrylamide gel and analyzed by Western blotting with an anti-His (C-terminal) monoclonal antibody or an anti-YspC polyclonal antibody. The detection was performed with Supersignal chemiluminescent substrate (Pierce). (B) BCECF release from preloaded macrophages upon infection with Y. enterocolitica ΔHOPEM, ΔHOPEMBD, ΔHOPEMBD(pMRS74), and ΔHOPEMBD(pBF15). Results are expressed as the percentages of Triton-lysed macrophages after subtraction of the value measured in the supernatants of uninfected cells. Data show the means and standard deviations of assays performed in triplicate. (C) SDS-polyacrylamide gel stained with Coomassie blue loaded with the culture supernatant of 5 × 10⁹ Y. enterocolitica ΔHOPEM, ΔHOPEMBD, and ΔHOPEMBD(pBF15) bacteria expressing sycB, yspB, and yspC from the yopE promoter under in vitro conditions. MW, molecular weights in thousands.

FIG. 6.

Lack of complementation of S. flexneri mutations. (A) Protein contents of cultures of S. flexneri wild-type (M90T), ipgC (RM97), ipgC carrying plac sycB (pBF15), and ipaB (RM221) strains grown for 4 h at 37°C were analyzed by Western blotting with monoclonal anti-IpaB antibody. (B) Protein contents of cultures of the S. flexneri ipaB strain, wild-type strain, ipaB ipgC (SF1068) strain, and ipaB ipgC sycB⁺ strain carrying pBF25 grown for 4 h at 37°C were analyzed by Western blotting with polyclonal anti-IpaH antibody. (C) Protein contents of culture supernatants of the S. flexneri wild-type strain, ipaB strain, and ipaB sycB⁺ yspB⁺ strain carrying pBF26 grown for 4 h at 37°C were determined by staining with Coomassie blue. (D) Protein contents of supernatants of the S. flexneri ipaC (RM81) strain and ipaC sycB⁺ yspC⁺ strain carrying pBF29 after Congo red induction were analyzed by Western blotting with polyclonal anti-IpaB antibody. WT, wild type; MW, molecular weights in thousands.