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Type III secretion system effector proteins are mechanically labile

Citation

LeBlanc, Marc-Andre; Fink, Morgan; Perkins, Thomas; Sousa, Marcelo (2021), Type III secretion system effector proteins are mechanically labile, Dryad, Dataset, https://doi.org/10.5061/dryad.0rxwdbrzv

Abstract

Multiple Gram-negative bacteria encode Type III secretion systems (T3SS) that allow them to inject effector proteins directly into host cells to facilitate colonization. To be secreted, effector proteins must be at least partially unfolded to pass through the narrow needle-like channel (diameter < 2 nm) of the T3SS. Fusion of effector proteins to tightly packed proteins—such as GFP, ubiquitin, or dihydrofolate reductase (DHFR)—impairs secretion and results in obstruction of the T3SS. Prior observation that unfolding can become rate limiting for secretion has led to the model that T3SS effector proteins have low thermodynamic stability, facilitating their secretion. Here, we first show that the unfolding free energy (ΔG0unfold) of two Salmonella effector proteins, SptP and SopE2, are 6.9 and 6.0 kcal/mol respectively, typical for globular proteins and similar to published ΔG0unfold for GFP, ubiquitin, and DHFR. Next, we mechanically unfolded individual SptP and SopE2 molecules by AFM-based force spectroscopy. SptP and SopE2 unfolded at low force (Funfold ≤ 17 pN @ 100 nm/s), making them among the most mechanically labile proteins studied to date by AFM. Moreover, their mechanical compliance is large, as measured by the distance to the transition state (Δx = 1.6 and 1.5 nm for SptP and SopE2, respectively). In contrast, prior measurements of GFP, ubiquitin, and DHFR show them to be mechanically robust (Funfold > 80 pN) and brittle (Δx < 0.4 nm). These results suggest that effector protein unfolding by T3SS is a mechanical process and that mechanical lability facilitates efficient effector protein secretion.

Methods

Circular dichroism data was collected and analyzed for Fig. 1 as follows:

Protein was removed from the -80 ºC freezer, thawed, and then centrifuged at 21,000 rcf for 5 min. A 10 M urea solution was deionized using BioRad AG 501-X8 resin (50 g beads/L urea) for 1 h and vacuum filtered through a 0.22-μm membrane to remove the resin. Urea concentration was measured using an Abbe refractometer. For equilibrium unfolding measurements, serial dilutions of urea with a fixed protein concentration of 0.05 mg/ml  as well as corresponding no-protein blanks were prepared  by mixing 10X SptP buffer (100 mM Tris base, pH 8.0, 1.5 M sodium sulfate, and 5 mM TCEP) or 10X SopE2 buffer (250 mM HEPES, pH 7.2, 1.5 M NaCl, and 5 mM TCEP) with ultrapure water, 10 M urea stock, and protein to 1X buffer concentration. Samples were incubated in a 25 ºC water bath for 1–3 days to reach equilibrium before CD spectra were collected.

Measurements were performed using a quartz cuvette (Hellma) with a 1-mm path length on an Applied PhotoPhysics ChiraScan Plus spectrophotometer. Measurement parameters: λ = 212.5–260 nm; step size = 0.5 nm; bandwidth = 1.0 nm; time per point = 0.5 s; and 3 repeats. The instrument was thoroughly purged with nitrogen to prevent ozone formation. Temperature was held at 25 °C with a Peltier sample holder and the temperature recorded using the temperature probe. Prior to loading, samples were spun at 18,000 rcf for 5 min. We measured a control sample as “blank” before every protein sample. Following this pair of measurements, the cuvette was serially rinsed with several mL each of 10 M urea, urea free buffer, 1% cleaning solution (Hellmanex), and ultrapure water. The cuvette was then filled with ultrapure water and a CD spectra taken to ensure no protein adhered to the cuvette. The cuvette was then rinsed with absolute ethanol and dried using filtered house air. This was repeated for every concentration of urea.

We analyzed the CD data using Applied Photophysics software. First, the three independent measurements were averaged. The subsequent spectrum was smoothed using the Savitzky–Golay algorithm with a window size of 12 points. This smoothing was done on both the protein-containing sample and the blank. We then subtracted the smoothed blank spectrum from the smoothed protein-containing spectrum to give the final, baseline corrected spectrum. After this analysis was done for all urea concentrations, the ellipicity at λ = 222 nm was plotted as a function of urea concentration. This plot was fit with an equation to determine the free-energy of unfolding assuming a two state system which accounts for a sloping baseline.

AFM data was collected and analyzed as follows for Fig. 2:

AFM experiments were performed on a Cypher ES (Asylum Research) in a temperature-controlled closed fluidic cell (T = 25 ºC). The stiffness (k) of the FIB-modified cantilevers was calibrated using the thermal method (81) far from the surface while sensitivity was measured by pressing the cantilever into hard contact with the surface. The cantilevers had an average k ≈ 6.5 pN/nm. Force-extension curve acquisition was initiated by pressing the cantilever into the surface at 100 pN for 0–200 ms depending on the surface polyprotein concentration. This comparatively low indentation force was enabled by our site-specific, cohesin-dockerin-based coupling between the tip and the polyprotein. To minimize the compliance of the polyprotein construct, we used only a single marker domain and short PEG linkers (MW = 600 D), which facilitated detecting proteins that unfold at low force and low extension [Fig. 2B,C (inset)]. We retracted the cantilever at 100–3,200 nm/s while digitizing at 50 kHz. We acquired multiple traces per sample by probing the surface in a raster scan, moving the AFM tip in a grid pattern with each location separated by 150 nm. Each spot was probed 10 times unless a molecule was detected, in which case the spot was continually sampled until ~20 consecutive attempts failed to yield a connection. This meant that an individual protein could be repeatedly probed. We found that both SopE2CD and SptPCD refolded well, and repeated cycles of unfolding and refolding did not affect the observed unfolding forces (SI Appendix, Fig. S3). The high-bandwidth records were boxcar averaged to the indicated bandwidths for analysis and presentation (1–5 kHz). Force was determined by cantilever deflection accounting for the sensitivity and stiffness of each cantilever. Extension was calculated from the movement of the sample stage minus the deflection of the cantilever. The loading rate (pN/s) for each unfolding event in a force−extension curve was calculated by fitting a line to the force-versus-time curve immediately preceding effector protein unfolding. For the effector protein unfolding-force analysis, only the first unfolding event was used when an unfolding intermediate was observed. A small percentage for the force-extension curves showed atypically high unfolding forces for the initial unfolding of SptPCD SopE2CD (8 and 2% respectively). These records were excluded from analysis as they most likely represented rare tip-sample surface adhesion and/or unfolding of a misfolded protein.

 

The data for Fig. S1 was collected as follows:

To demonstrate reversibility of urea denaturation, we refolded urea denatured SopE2CD and SptPCD by dilution. SopE2CD was buffered with 25 mM HEPES, pH 7.2, 150 mM NaCl, and 0.5 mM TCEP while SptPCD was buffered with 10 mM Tris, pH 8.0, 150 mM sodium sulfate, and 0.5 mM TCEP. Three sample conditions were used, a high urea control sample [4.7 M (SopE2CD); 5.6 M (SptPCD)], low urea control sample [0.38 M (SopE2CD); 0.94 M (SptPCD)], and refolded sample (diluted from 4.7 M to 0.38 M for SopE2CD and from 5.6 M to 0.94 M for SptPCD). Urea buffer without protein was also prepared as a blank. The purpose of the high and low urea controls was to show that the protein denatured under the experimental conditions and to give a signal for native protein for comparison. Samples were prepared and allowed to equilibrate for one day in a 25 ºC water bath. For refolding, the equilibrated samples were added dropwise to a glass vial with a stir bar containing either buffer containing the same amount of urea as the sample (for controls) or buffer with no urea (for the refolding sample). CD spectra were obtained as described in Circular dichroism measurement and analysis in the main text. Two independent high and low urea concentration samples were measured and three independent refolded samples were measured (Fig. S1).

 

The data for Fig. S3 were collected as follows:

Comparisons of the mean Funfold of the first unfolding event of SptP or SopE2 to the mean Funfold of the refolded protein. Measurements were collected with a pulling velocity of 1600 nm/s [Nfirst = 41; Nrefold = 12 (SptP); Nfirst = 18; Nrefold = 27 (SopE2)]. Error bars represent the SEM. In the case of an unfolding intermediate, only the first unfolding event of the effector protein was analyzed.

Usage Notes

Unless noted otherwise, all data and figures were analyzed and plotted in Igor.

Funding

National Institutes of Health, Award: T32 GM065103:CU Molecular Biophysics Graduate Traineeship

National Science Foundation, Award: MCB-1716033

National Institute of Standards and Technology