Autoproteolysis and intramolecular dissociation of Yersinia YscU precedes secretion of its C-terminal polypeptide YscU(CC)

Abstract

Type III secretion system mediated secretion and translocation of Yop-effector proteins across the eukaryotic target cell membrane by pathogenic Yersinia is highly organized and is dependent on a switching event from secretion of early structural substrates to late effector substrates (Yops). Substrate switching can be mimicked in vitro by modulating the calcium levels in the growth medium. YscU that is essential for regulation of this switch undergoes autoproteolysis at a conserved N↑PTH motif, resulting in a 10 kDa C-terminal polypeptide fragment denoted YscU(CC). Here we show that depletion of calcium induces intramolecular dissociation of YscU(CC) from YscU followed by secretion of the YscU(CC) polypeptide. Thus, YscU(CC) behaved in vivo as a Yop protein with respect to secretion properties. Further, destabilized yscU mutants displayed increased rates of dissociation of YscU(CC)in vitro resulting in enhanced Yop secretion in vivo at 30°C relative to the wild-type strain.These findings provide strong support to the relevance of YscU(CC) dissociation for Yop secretion. We propose that YscU(CC) orchestrates a block in the secretion channel that is eliminated by calcium depletion. Further, the striking homology between different members of the YscU/FlhB family suggests that this protein family possess regulatory functions also in other bacteria using comparable mechanisms.

Figure 1. Domain structure of YscU and crystallographic structure of YscU_C.

(A) Schematic domain structure of the integral membrane protein, YscU of Y. pseudotuberculosis. The full-length protein contains 354 amino acid residues. The N-terminal 210 residues constitute four transmembrane helices (TM). The cytosolic domain of YscU (YscU_C) undergoes autoproteolytic cleavage at the N↑PTH-motif (amino acids 263–266), which leaves an N-terminal cytoplasmic polypeptide, denoted YscU_CN, and a C-terminal polypeptide, denoted YscU_CC. (B) Ribbon drawing of the cleaved cytosolic domain YscU_C (2JLI.PDB) from Y. pestis . YscU_CN and YscU_CC resulting from cleavage at the N↑PTH motif are colored in orange and grey, respectively.

Figure 2. In vitro dissociation of YscU_C.

(A) ¹H-¹⁵N HSQC spectra of YscU_C at 20°C, pH 7.4, before (blue contours) and after (red contours) incubation at 60°C for 10 min. Only resonances that corresponded to YscU_CN were visible after the thermal treatment. (B) Thermal up- and down-scans of YscU_C at pH 7.4 monitored with CD spectroscopy at 220 nm in the absence of calcium. (C) Thermal signatures of P264A, a non-cleavable mutant, at pH 7.4 in the absence of calcium. Up- and down scans of YscU_C and P264A are shown in red and blue circles, respectively.

Figure 3. Calcium effects on YscU_C stability and Yop secretion.

(A) Thermal up- and down-scans of YscU_C at pH 7.4 monitored with CD spectroscopy at 220 nm in the presence of 2.5 mM calcium. Up- and down scans of YscU_C are shown in red and blue circles, respectively. (B) Titration of calcium to YscU_C monitored with CD spectroscopy at 220 nm. The calcium binding isotherm to YscU_C was fit to a one-site binding model (red line). The resulting K_d was 800 µM. (C) Western Blot analysis of YopB, YopD, and YopE in wild-type Y. pseudotuberculosis. The bacteria were grown for 2 h at 26°C and shifted to 37°C for 3 h (temperature shift for induction of Yop secretion) with varying concentrations of free calcium. “Pellet” indicates intracellular proteins; “supernatant” denotes secreted proteins. The LB growth medium was initially supplemented with 1 mM EGTA to complex residual calcium content (approximately 500 µM); thereafter, calcium was added to set the indicated concentrations of free calcium.

Figure 4. In vivo dissociation and secretion of YscU_CC in different Yersinia strains.

Calcium dependent regulation of Yop and YscU_CC secretion in wild-type Y. pseudotuberculosis, in a ΔyscC mutant and ΔyscN mutant strain without and with in trans complementation of YscU_CC. Bacteria transformed with empty pBADmycHis B (pBAD), or pBAD with one additional yscU_CC copy (pBAD(YscU_CC)), were grown for 2 h at 26°C and 3 h at 37°C in calcium depleted (−) or calcium supplemented (+) medium. The expression of yscU_CC was induced by addition of arabinose. Yop secretion is coupled to the secretion of YscU_CC in all analysed Yersinia strains and required a functional T3SS. Secreted Yops visualized on Coomassie stained PAGE gels; YscU_CC visualized on immunoblots with anti-YscU_CC peptide antibodies. “pellet” indicates intracellular proteins; “supernatant” denotes secreted proteins. The YopJ protein (black box) was subjected to densitometric analysis for quantification of secretion levels (see Table 2).

Figure 5. pH-dependencies of YscU_C dissociation in vitro and Yop/YscU_CC secretion in vivo.

(A) Thermal up- and down-scans of YscU_C at pH 6.0, monitored with CD spectroscopy at 220 nm in the absence of calcium. The thermal signature of YscU_C displayed one large-amplitude transition at 55°C. Up- and down scans of YscU_C are shown in red and blue circles, respectively. (B) The pH-dependency of the YscU_C monitored with CD spectroscopy at 220 nm. (C) Chemical shift perturbations of YscU_C quantified from ¹H-¹⁵N HSQC spectra, in response to a pH-shift from 7.4 to 6.0, displayed against the primary sequence. The blue line indicates the threshold value (0.05 ppm) used in Figure 5D. The chemical shift perturbation of the two histidines at positions 266 and 324 are shown in red. (D) Structural distributions of residues that show significant chemical shift perturbations in response to a pH-shift from 7.4 to 6.0 are shown in red on the YscU_C structure (2JLI.PDB). The YscU_CN and YscU_CC fragments are colored orange and gray, respectively. The two histidine residues (266 and 324) in the folded part of YscU_C are indicated. (E) Coomassie stained gels show Yop secretion under different pH conditions. (top panel) “pellet” indicates intracellular proteins; (middle panel) “supernatant” denotes secreted proteins. The YopJ protein (black box) was subjected to densitometric analysis for quantification of secretion levels (see Table 2). (bottom panel) The pH-dependency of YscU_CC secretion was visualized on immunoblots with anti-YscU_CC peptide antibodies.

Figure 6. YscU_C suppressor mutations are buried in the structure.

The spatial locations of single mutations in YscU_C that suppressed the non-secreting ΔyscP phenotype in vivo are shown on the YscU_C structure of Y. pestis (2JLI.PDB). All positions are either fully or partially buried in the protein structure. YscU_CN and YscU_CC polypeptides are colored orange and gray, respectively.

Figure 7. YscU_C suppressor mutant stabilities and dissociation kinetics.

(A) Thermal induced unfolding of YscU_C and suppressor mutants. Dissociation temperatures (T _diss) of single suppressor mutants at pH 7.4 were quantified with the CD signal at 220 nm and a scan rate of 1°C/min; YscU_C (black), A268F (blue), Y287G (green), and V292T (red). All suppressor mutants are destabilized compared to wild-type YscU_C. T _diss values are summarized in Table 3. (B), (C) Dissociation kinetics quantified as dissociation life-times (τ_diss) of YscU_C (black) and V292T (red) at pH 7.4 followed with NMR-spectroscopy at (B) 37°C and (C) 30°C, respectively. Primary NMR data for (B) is shown in Figure S6. Solid lines correspond to fits of the experimental data to single exponential decays. (D) Time dependent GST-pulldown experiments show the dissociation of wild-type YscU_C and the suppressor mutant A268F at 30°C and 37°C after varying incubation times. The suppressor mutant A268F displayed pronounced dissociation of YscU_CC-His₆ at 37°C and moderate dissociation at 30°C; wild-type YscU_C displayed no dissociation of YscU_CC-His₆ at 37°C or at 30°C over the observed time period. Note! Dissociation of YscU_CC is manifested as disappearance of YscU_CC-His₆ over time since the dissociation is irreversible and YscU_CC-His₆ cannot bind itself to the used resin.

Figure 8. Yersinia strains with destabilized yscU suppressor mutants secrete YscU_CC and Yops at lower temperatures (30°C) than wild-type.

Coomassie stained analysis of Yop secretion in Y. pseudotuberculosis incubated at 30°C. Bacteria expressing either wild-type yscU or one of the suppressor mutants, A268F or V292T. “pellet” indicates intracellular proteins; “supernatant” denotes secreted proteins. Secretion of YscU_CC was analyzed on immunoblots with anti-YscU_CC peptide antibodies. Yersinia harboring yscU suppressor mutants showed strongly elevated secretion of Yops after cultivation at 30°C.