Regulated secretion of YopN by the type III machinery of Yersinia enterocolitica

Abstract

During infection, Yersinia enterocolitica exports Yop proteins via a type III secretion pathway. Secretion is activated when the environmental concentration of calcium ions is below 100 microM (low-calcium response). Yersiniae lacking yopN (lcrE), yscB, sycN, or tyeA do not inactivate the type III pathway even when the concentration of calcium is above 100 microM (calcium-blind phenotype). Purified YscB and SycN proteins form cytoplasmic complexes that bind a region including amino acids 16 to 100 of YopN, whereas TyeA binds YopN residues 101 to 294. Translational fusion of yopN gene sequences to the 5' end of the npt reporter generates hybrid proteins that are transported by the type III pathway. The signal necessary and sufficient for the type III secretion of hybrid proteins is located within the first 15 codons of yopN. Expression of plasmid-borne yopN, but not of yopN(1-294)-npt, complements the calcium-blind phenotype of yopN mutants. Surprisingly, yopN mutants respond to environmental changes in calcium concentration and secrete YopN(1-294)-Npt in the absence but not in the presence of calcium. tyeA is required for the low-calcium regulation of YopN(1-294)-Npt secretion, whereas sycN and yscB mutants fail to secrete YopN(1-294)-Npt in the presence of calcium. Experiments with yopN-npt fusions identified two other signals that regulate the secretion of YopN. yopN codons 16 to 100 prevent the entry of YopN into the type III pathway, a negative regulatory effect that is overcome by expression of yscB and sycN. The portion of YopN encoded by codons 101 to 294 prevents transport of the polypeptide across the bacterial double membrane envelope in the presence of functional tyeA. These data support a model whereby YopN transport may serve as a regulatory mechanism for the activity of the type III pathway. YscB/SycN binding facilitates the initiation of YopN into the type III pathway, whereas TyeA binding prevents transport of the polypeptide across the bacterial envelope. Changes in the environmental calcium concentration relieve the TyeA-mediated regulation, triggering YopN transport and activating the type III pathway.

FIG. 1

tyeA, sycN, and yscB mutant Y. enterocolitica strains display a calcium-blind phenotype as well as defects in the regulation of YopN secretion. (A) Y. enterocolitica strain W22703 (wild type [WT]) was grown in TSB in the presence or absence of 5 mM calcium chloride and induced for type III secretion by a temperature shift to 37°C. Cultures were centrifuged, and the extracellular medium was separated with the supernatant (S) from the bacterial pellet (P). Proteins were precipitated with TCA, suspended in sample buffer, separated on SDS-PAGE, and analyzed by immunoblotting with specific antibody (anti-YopE and anti-YopN). Immunoreactive signals were quantified by densitometry scanning, and the amount of secreted polypeptide was calculated. (B) Y. enterocolitica LC4 (tyeA), OK2 (sycN), and OK5 (yscB) carried either no plasmid or plasmids expressing wild-type tyeA, sycN, or yscB under the control of the IPTG-inducible tac promoter.

FIG. 2

tyeA, sycN, and yscB mutant Y. enterocolitica strains display a Los phenotype. HeLa cells were infected with Y. enterocolitica strains W22703 (wild type [WT]), LC4 (tyeA), OK2 (sycN), and OK5 (yscB). After incubation for 3 h at 37°C, the tissue culture medium (M) was decanted and centrifuged to separate secreted proteins from those present within nonadherent bacteria. HeLa cells as well as adherent yersiniae were extracted with digitonin (D), a detergent that solubilizes the eukaryotic plasma membrane but not the bacterial envelope. Extracts were centrifuged to separate proteins solubilized from the HeLa cytoplasm from those that sediment with the bacteria. Proteins were precipitated with chloroform-methanol and analyzed by immunoblotting with anti-YopE and anti-YopN. Immunoreactive signals were quantified by densitometry scanning, and the amount of targeted polypeptide (protein present in the digitonin supernatant) was calculated.

FIG. 3

The phenotype of yscB knockout mutations on the regulation of YopN secretion is epistatic over that of tyeA knockout mutations. (A) Y. enterocolitica strain LC8 (yscB tyeA) was grown in TSB in the presence or absence of 5 mM calcium chloride and induced for type III secretion by a temperature shift to 37°C. Cultures were centrifuged, and the extracellular medium was separated with the supernatant (S) from the bacterial pellet (P). Proteins were precipitated with TCA, suspended in sample buffer, separated on SDS-PAGE, and analyzed by immunoblotting with specific antibody (anti-YopE and anti-YopN). (B) HeLa cells were infected with Y. enterocolitica strain LC8, and samples were subjected to digitonin fractionation. Proteins were precipitated with chloroform-methanol and analyzed by immunoblotting with anti-YopE and anti-YopN. Abbreviations are as in Fig. 2.

FIG. 4

The role of SycN, YscB, and TyeA in the regulated secretion of YopN. (A) Secretion of YopN-Npt hybrids was measured by expressing plasmid-borne yopN-npt translational fusions in Y. enterocolitica W22703 (wild type [WT]) and growing bacteria in TSB with (+) or without (−) 5 mM calcium chloride (Ca²⁺). Bacterial cultures were centrifuged, culture medium and bacterial sediment were separated, and protein secretion was measured by immunoblotting with anti-Npt. Numbers indicate the average amount of secreted polypeptide (standard deviations in parentheses) obtained from three independent experiments. The drawing depicts the primary structure of hybrid polypeptides: (1) YopN-Npt, full-length yopN; (2) YopN_1–100-Npt, codons 1 to 100 of yopN; (3) YopN_1–15-Npt, codons 1 to 15 of yopN; (4) YopN_15–294-Npt; yopN codons 2 to 15 deleted; (5) YopE_1–15-YopN-Npt, replacement of yopN codons 1 to 15 with yopE codons 1 to 15; (6) YopQ_1–15-YopN-Npt, replacement of yopN codons 1 to 15 with yopQ codons 1 to 15. (B) Secretion of YopN-Npt hybrids was measured by expressing plasmid-borne yopN-npt translational fusions in Y. enterocolitica VTL1 (yopN), OK2 (sycN), OK5 (yscB), LC7 (tyeA), or LC8 (tyeA yscB) and growing bacteria in TSB with 5 mM calcium chloride (Ca²⁺).

FIG. 5

SycN/YscB and TyeA copurify with YopN_His6. (A) Y. enterocolitica wild-type (WT) strain W22703 and Y. enterocolitica VTL1(pSM20) (yopN), expressing yopN_His6, were grown in TSB without calcium chloride. Bacteria were harvested and broken in a French press, and extracts were subjected to ultracentrifugation at 100,000 × g. The supernatant (lysate [L]) was applied to chromatography on Ni-NTA Sepharose. Bound proteins were eluted with buffer containing 250 mM imidazole (E1 and E2) and, together with a lysate sample, analyzed by immunoblotting with specific antibody. (B) Cleared lysates obtained from Y. enterocolitica strains W22703, OK2 (sycN), OK5 (yscB), LC4 (tyeA), or LC8 (tyeA yscB) were subjected to chromatography on Ni-NTA Sepharose (Ni) or Ni-NTA Sepharose precharged with purified SycN_His6 (S), YscB_His6 (Y), SycN_His6/YscB_His6 (S/Y), or TyeA_His6 (T). Proteins were eluted and analyzed by immunoblotting with anti-YopN.

FIG. 6

Binding sites of SycN/YscB and TyeA on YopN polypeptide. Cleared lysates were obtained by recovery of the supernatant of ultracentrifuged French press extracts from Y. enterocolitica W22703 harboring pDA82 (YopN-Npt) (I); pDA83 (YopN_1–100-Npt) (II); pDA85 (YopN_1–15-Npt) (III); pDA111 (YopN_15–290-Npt) (IV); and pDA159 (YopN_50–290-Npt) (V). Samples were applied to affinity chromatography on Ni-NTA Sepharose containing immobilized SycN_His6/YscB_His6 (S), TyeA_His6 (T), or SrtA_His6 (Srt [staphylococcal sortase as a control]). Proteins were eluted and analyzed by immunoblotting with anti-Npt. The top panel shows a Coomassie-stained SDS-PAGE gel of eluted SycN_His6/YscB_His6, TyeA_His6, and SrtA_His6. Numbers on the right correspond to molecular weight markers (in thousands).

FIG. 7

Hypothesis for the regulated transport of YopN by the type III machinery of Y. enterocolitica. (A) In the presence of extracellular calcium ions (≥100 μM, ↑Ca²⁺), the secretion signal (yopN codons 1 to 15) and the binding of YscB/SycN to YopN amino acid residues 15 to 100 initiate YopN into the type III pathway. Further progression of YopN along the secretion pathway is stalled by the binding of TyeA to YopN amino acid residues 101 to 294. In the arrested state, the YopN-TyeA complex may act as a repressor for the type III targeting of YopEHMOPT by the Yersinia type III machinery. (B) Insertion of the YscF type III needle into the cytosol of eukaryotic cells may transmit a low-calcium signal (<100 μM calcium, ↓Ca²⁺), resulting in the dissociation of TyeA from YopN polypeptide and in the type III targeting of YopN across bacterial inner membranes (IM) and outer membranes (OM) as well as the plasma membrane (PM) of eukaryotic cells.