Multiple driving forces required for efficient secretion of autotransporter virulence proteins

Abstract

Autotransporter (AT) proteins are a broad class of virulence proteins from Gram-negative bacterial pathogens that require their own C-terminal transmembrane domain to translocate their N-terminal passenger across the bacterial outer membrane (OM). But given the unavailability of ATP or a proton gradient across the OM, it is unknown what energy source(s) drives this process. Here we used a combination of computational and experimental approaches to quantitatively compare proposed AT OM translocation mechanisms. We show directly for the first time that when translocation was blocked an AT passenger remained unfolded in the periplasm. We demonstrate that AT secretion is a kinetically controlled, non-equilibrium process coupled to folding of the passenger and propose a model connecting passenger conformation to secretion kinetics. These results reconcile seemingly contradictory reports regarding the importance of passenger folding as a driving force for OM translocation but also reveal that another energy source is required to initiate translocation.

Keywords: Autotransporter; Bioenergetics; Kinetics; Outer Membrane; Periplasm; Protein Folding; Protein Secretion; Thermodynamics; Translocation; Virulence Factor.

FIGURE 1.

Overview of AT protein structure and secretion mechanism. a, common AT structural features illustrated for pertactin from B. pertussis. The signal sequence (red) is followed by the extracellularly secreted passenger (green). The β-helical fold observed for pertactin (Protein Data Bank code 1DAB (29)) is a characteristic of most AT passengers (8). The C-terminal β-barrel domain (blue; Protein Data Bank code 3QQ2 (53)) forms a pore in the OM. The structure shown is that of BrkA, the nearest pertactin homolog for which a β-barrel structure is available. Not resolved in the crystal structures is a linker that passes through the lumen of the pore to connect the extracellular passenger with the periplasmic face of the β-barrel. b, proposed mechanism of AT secretion. The AT protein is transported across the inner membrane via the Sec translocon guided by the signal sequence. The β-barrel domain is then inserted into the OM in a process assisted by BamA and other periplasmic chaperones. This is followed by translocation of the passenger across the OM, which may also be assisted by chaperones. Once secretion is complete, the passenger can be autocatalytically cleaved. In the case of pertactin, the cleaved passenger remains non-covalently attached to the OM.

FIGURE 2.

Kinetic simulations of AT secretion. a, model used in simulations. The five states correspond to folded (F) or unfolded (U) passenger located in the periplasm (subscript in) or the extracellular space (subscript out). M is the final, proteolytically cleaved form. b, dependence of simulated secretion efficiency (after 60 min) on different rates in the kinetic model (double arrow, fast; double line, slow). Labels on bars correspond to simulation traces shown in c–f for various secretion scenarios. Traces in c–f use the same color-coding scheme as the labels in a. Red boxes highlight key differences in the rate constants used for alternative scenarios. c, passenger cleavage drives secretion. As long as the passenger is able to reach the cell surface, it is irreversibly cleaved, making secretion a unidirectional process. d, passenger folding can drive secretion even in the absence of cleavage. Slow periplasmic folding gives the unfolded passenger time to translocate before folding on the cell surface. If the folded passenger is unable to cross the OM, then it will stay surface-exposed. e, a combination of fast periplasmic folding and slow translocation of a folded passenger results in inefficient secretion. Most of the passenger folds while still in the periplasm and can neither cross the OM nor unfold. f, in the absence of cleavage and major kinetic barriers, the system will establish an equilibrium with only half of the passenger on the cell surface unless the stability of the secreted and periplasmic folded states differs.

FIGURE 3.

In vivo pertactin secretion and passenger conformation. Passenger folding occurs concomitantly with OM translocation. a, pertactin OM translocation kinetics after stopping induction and degrading the mature secreted passenger so that only the periplasmic precursor remains at t = 0. The band labeled “Truncated” is a degradation product (see d) not included in the quantification; one smudged band (*) was also omitted from quantification. ′, minutes. b, quantification of Western blot bands plotted versus time elapsed after ProK treatment. Both folding-driven secretion (solid lines) and cleavage-driven secretion (dashed lines) are compatible with experimental results as long as reactions for the degradation of mature passenger and unfolded precursor are added to the basic model from Fig. 2. AU, arbitrary units. c, effect of medium on pertactin OM translocation kinetics. Resuspending cells into LB after ProK shaving results in efficient secretion, whereas using PBS or PBS supplemented with 4 g liter⁻¹ glucose (Glc) stalls secretion. Stalling is maintained for more than 2 h, but the amount of stalled precursor decreases due to degradation. d, kinetics of pertactin OM translocation in PBS and LB monitored as in a and b. OM translocation stalls in PBS buffer but resumes once cells are resuspended in LB. Precursor levels decrease throughout the experiment as it is either translocated or degraded. Shaded areas represent time spent spinning and resuspending cells in fresh medium. The dotted line indicates the boundary between two separate Western blots. e, ProK susceptibility of cell lysates taken at different times during the process shown in d. ProK-resistant pertactin fragments appear only when the passenger is folded (8). Only LB samples containing the mature secreted passenger show any significant amount of folded, ProK-resistant passenger fragments (arrows); no passenger folding is detected for the pertactin precursor even after >90 min of incubation in PBS.

FIGURE 4.

Comparison between in vitro folding properties of purified wild type (8, 30) and 4K pertactin passengers. a, the pertactin passenger native structure (Protein Data Bank code 1DAB (29)) showing the locations of four residues (Leu¹⁵⁷, Leu²²⁸, Ile⁴³⁵, and Leu⁴⁹⁸; red) replaced with Lys in the unstable, non-native 4K construct. Also shown are loop residues (265–273; blue) replaced with an HA epitope tag. The figure was drawn using UCSF Chimera (54). b, difference in ProK susceptibility between wild type and 4K purified pertactin passengers. Folded wild type passenger produces characteristic ProK-resistant fragments (8), whereas 4K is rapidly degraded. c, far-UV circular dichroism (CD) spectra of wild type and 4K passengers. The wild type passenger displays a characteristic β-sheet signal (a minimum at 218 nm), whereas the 4K mutant does not. deg, degrees. d, thermal denaturation monitored by the change in the CD signal at 218 nm. Two cooperative transitions above 60 °C are characteristic of the wild type passenger (8), whereas 4K displays only a very weak, broad transition at much lower temperatures. e, similar behavior is observed upon chemical denaturation using guanidinium hydrochloride (GdnHCl) monitored by the ratio of fluorescence intensities at 335 and 350 nm.

FIGURE 5.

Folding complementation assay used as a measure of pertactin folding (based on Ref. 34). a, more robust folding of the protein of interest (POI; in our case the pertactin passenger) translates into better folding of the attached Bla domain (left), which results in higher resistance to Amp (34). b, ampicillin resistance measured as the lowest Amp concentration at which less than 1 in 200 cells is able to form colonies. The equal values for cells expressing WT (P.69) and 4K (P.69–4K) pertactin-Bla chimeras suggest that the two passengers adopt a similar conformational state in the periplasm. Error bars correspond to S.D. from at least three biological replicates. c, ProK resistance of pertactin-Bla chimeras. The white arrowhead indicates the full-length pertactin-Bla chimera, and the gray arrowhead indicates ProK-resistant fragments characteristic of a folded pertactin passenger. No such fragments are detected after limited ProK digestion of the two periplasmically localized pertactin-Bla chimeras, confirming that the pertactin passenger is not natively folded. d, chimera expression control. Anti-pertactin passenger and anti-Bla Western blots show uniform expression of both WT and 4K pertactin-Bla chimeras (white arrowhead). The distributions between soluble (S) and pelletable (P) fractions of the whole cell lysate (WCL) are likewise very similar for WT and 4K. Only a negligible amount of functional (soluble) Bla is cleaved from either chimera, so artifacts due to free Bla are not likely to affect the folding complementation assay. The MalE-Bla chimera described previously (34) is loaded for comparison. The gray lines in c and d indicate omission of unrelated lanes from the western blot.

FIGURE 6.

Introduced mutations do not affect pertactin expression or its stability on the cell surface. a, expression test of WT and 4K pertactin and their HA-tagged variants. All four constructs are expressed at about the same level after incubation with 50 μm IPTG for similar amounts of time. IPTG incubation beyond 50 min results primarily in the buildup of additional precursor and truncated fragments rather than substantially increased levels of mature, secreted passenger. b, stability of WT and 4K pertactin under experimental conditions. After IPTG-induced expression of each pertactin construct, the cell cultures were repeatedly spun down and resuspended in fresh PBS over a period of ∼2 h. Despite a moderate decrease of the precursor levels, both the WT and 4K secreted passengers remain stable and securely attached to the cells through several successive cycles of PBS washing. “log,” samples taken in the logarithmic growth phase just before induction; “IPTG,” samples taken immediately after the induction period and before the first PBS wash. EV, empty vector; ′, minutes.

FIGURE 7.

Effects of folding- and/or cleavage-deficient mutants on secretion. a, quantification of pertactin secretion by competition ELISA (36) using whole cells. Secretion of 4K and 4K-NQ mutants is more than 5-fold lower compared with wild type or NQ pertactin. Raw signal was normalized to 0 for empty vector (EV) and 1 for WT, but note that the y axis scale is not linear as indicated by the standard curve for purified pertactin passenger shown on the right-hand y axis. Error bars are S.D. from four separate measurements of the same biological sample. b, flow cytometry of pertactin-expressing cells stained for surface-exposed pertactin. Cells expressing 4K or 4K-NQ exhibit significantly more fluorescence than empty vector (shown as dashed lines) but still secrete about 6–7-fold less pertactin compared with WT or NQ. The labels correspond to the mean fluorescence intensity of the indicated sample. c, summary of the results. Secretion depends on the ability of the passenger to fold regardless of cleavage. AU, arbitrary units.

FIGURE 8.

Secretion of the uncleavable full-length pertactin (P.93EB-NQ) monitored by anti-pertactin immunofluorescence microscopy. Extracellular crowding does not reduce secretion efficiency.

FIGURE 9.

Free energy diagram for pertactin OM translocation. Passage through the narrow translocation channel imposes a conformational constraint on the unfolded passenger, introducing a large free energy barrier early in the secretion process. This barrier may be overcome by coupling to the energetically favorable folding and OM insertion of the β-barrel (gray shaded areas), a process thought to be catalyzed by BamA or TamA. Once the initial hairpin is formed, secretion of additional residues should not result in major energetic penalties, effectively leading to random Brownian diffusion of the passenger in both directions. a, if the passenger C terminus is able to form an early, semistable folding intermediate, this will decrease the free energy and make it unlikely that the passenger will re-enter the periplasm. Under this model, the passenger can escape the conformational restriction of the pore only by going forward, completing secretion. b, in the absence of folding, the passenger is in a high energy state throughout secretion, and only a small portion of the molecules (15%; see Fig. 7) will find their way to the extracellular space through a random walk.