Substrate-activated conformational switch on chaperones encodes a targeting signal in type III secretion

Abstract

The targeting of type III secretion (TTS) proteins at the injectisome is an important process in bacterial virulence. Nevertheless, how the injectisome specifically recognizes TTS substrates among all bacterial proteins is unknown. A TTS peripheral membrane ATPase protein located at the base of the injectisome has been implicated in the targeting process. We have investigated the targeting of the EspA filament protein and its cognate chaperone, CesAB, to the EscN ATPase of the enteropathogenic E. coli (EPEC). We show that EscN selectively engages the EspA-loaded CesAB but not the unliganded CesAB. Structure analysis revealed that the targeting signal is encoded in a disorder-order structural transition in CesAB that is elicited only upon the binding of its physiological substrate, EspA. Abrogation of the interaction between the CesAB-EspA complex and EscN resulted in severe secretion and infection defects. Additionally, we show that the targeting and secretion signals are distinct and that the two processes are likely regulated by different mechanisms.

Figure 1. Structures of CesAB, CesAB–EspA and CesAB^s

(A) Solution structure of the homodimeric CesAB, which adopts a molten-globule-like structure in solution (Chen et al., 2011). (B) ¹H-¹⁵N HSQC spectrum of CesAB. (C) Crystal structure of the heterodimeric CesAB–EspA (Yip et al., 2005). Regions of the proteins that were not crystallographically resolved are represented as dotted lines. (D) ¹H-¹⁵N HSQC spectrum of CesAB–EspA. The CesAB subunit is ¹⁵N labeled whereas the EspA subunit is unlabeled. (E) Solution structure of the CesAB^s variant (D14L/R18D/E20L), determined in this work. (F) ¹H-¹⁵N HSQC spectrum of CesAB^s (G) Superposition of the CesAB subunit of CesAB, CesAB–EspA and CesAB^s.

Figure 2. Interaction of CesAB–EspA with EscN

(A) Overlaid ¹H-¹⁵N HSQC spectra of the titration of U-²H-¹⁵N labeled CesAB–EspA with unlabeled hexameric EscN. Stepwise addition of EscN results in gradual resonance broadening of the interacting residues in CesAB–EspA. Spectra recorded at 10 different titration points are overlaid. The CesAB residues most affected by EscN binding are shown. The resonances not affected by EscN binding even at saturating concentrations of EscN are located in flexible regions of EspA that were crystallographically unresolved. (B) CesAB–EspA residues (shown in red sticks) identified by NMR to be most affected upon binding to EscN. All these residues are located in helices α2 and α3 in CesAB. (C) Superposition of the CesAB subunit of the homodimeric CesAB (blue) and the heterodimeric CesAB–EspA complex (green). The residues identified to mediate the binding to CesAB–EspA to EscN are shown.

Figure 3. Disruption of CesAB–EspA Binding to EscN Gives Rise to Secretion and Functional Defects

(A) Effect of the Y64A/R68A substitution on the interaction of CesAB–EspA with EscN. Overlaid ¹H-¹⁵N HSQC spectra of CesAB^Y64A/R68A–EspA in the absence (blue) and presence (magenta) of EscN. Compared to wild-type CesAB–EspA binding to EscN, the NMR data indicate a significant decrease in the affinity of the ternary complex (Kd is larger than 80 μM). The boxed areas show the corresponding regions of the spectra of CesAB–EspA (green) superimposed on the spectra of its complex with EscN (red). Whereas the majority of the peaks are broadened beyond detection in the CesAB–EspA–EscN complex, they are still present at substantial intensity in the CesAB^Y64A/R68A–EspA–EscN complex. (B) In vivo secretion of EspA from EPECΔcesAB strains complemented with pASK-IBA7 plasmids expressing wild-type or mutated CesAB. The graph reports the total amount of EspA secreted in 90 min following CesAB expression. (C) In vivo infection of HeLa cells from EPECΔcesAB or EPECΔescN strains complemented with pASK-IBA7 plasmids expressing wild-type or mutated CesAB. The graph reports the percentage of HeLa cells infected after 90-min inoculation with the bacteria. (D) Infection of HeLa cells by bacterial EPECΔcesAB strains complemented with plasmids expressing wild-type or mutant CesAB. The results show very little actin pedestal formation, indicating uninfected HeLa cells, after 90-min inoculation with bacteria.

Figure 4. Targeting of CesAB–EspA to the ATPase

Although CesAB carries the targeting signal (colored in red), this is presented to EscN only when EspA is bound to CesAB by means of an induced conformational switch on CesAB. As a result, EscN recognizes the re-structured CesAB region and engages the CesAB–EspA complex.