Structural Features Reminiscent of ATP-Driven Protein Translocases Are Essential for the Function of a Type III Secretion-Associated ATPase

Abstract

Many bacterial pathogens and symbionts utilize type III secretion systems to interact with their hosts. These machines have evolved to deliver bacterial effector proteins into eukaryotic target cells to modulate a variety of cellular functions. One of the most conserved components of these systems is an ATPase, which plays an essential role in the recognition and unfolding of proteins destined for secretion by the type III pathway. Here we show that structural features reminiscent of other ATP-driven protein translocases are essential for the function of InvC, the ATPase associated with a Salmonella enterica serovar Typhimurium type III secretion system. Mutational and functional analyses showed that a two-helix-finger motif and a conserved loop located at the entrance of and within the predicted pore formed by the hexameric ATPase are essential for InvC function. These findings provide mechanistic insight into the function of this highly conserved component of type III secretion machines.

Importance: Type III secretion machines are essential for the virulence or symbiotic relationships of many bacteria. These machines have evolved to deliver bacterial effector proteins into host cells to modulate cellular functions, thus facilitating bacterial colonization and replication. An essential component of these machines is a highly conserved ATPase, which is necessary for the recognition and secretion of proteins destined to be delivered by the type III secretion pathway. Using modeling and structure and function analyses, we have identified structural features of one of these ATPases from Salmonella enterica serovar Typhimurium that help to explain important aspects of its function.

FIG 1

Modeling of the InvC hexamer reveals structural features reminiscent of ATP-driven protein translocases. (A) Schematic representation of InvC. Sequence alignments show the two-helix-finger motifs and the luminal loop domains of type III secretion- and flagellar export-associated ATPases. The sequences used in the alignment are as follows: InvC, SsaN, and FliI from Salmonella enterica serovar Typhimurium, Spa47 from Shigella flexneri, YscN from Yersinia enterocolitica, HrcN from Pseudomonas syringae, EscN from Escherichia coli, and CdsN from Chlamydophila pneumoniae. Conserved residues in the two-helix-finger motif and acidic residues within the luminal loop are shown in red and blue, respectively. (B) Hexamer model of InvC viewed from its C-terminal end. The Tyr385 side chain is highlighted in red. (C) Side view of the InvC hexamer model. (D) The two-helix-finger motif and the luminal loop domain are shown on an InvC monomer. (E) An apo structure of InvC (shown in green) is overlaid on the ATP-bound InvC shown in panel D. A change in angle around the axis is seen at the side chain of Tyr385.

FIG 2

A two-helix-finger motif in InvC is essential for type III secretion. (A) Secretion profile of the Y385A mutant for the indicated T3SS substrates. InvC expression levels in the whole-cell lysates were visualized by Western immunoblotting. Levels of the T3SS substrates in the culture supernatants were also analyzed by Western immunoblotting and quantified with a LICOR-Odyssey system. Values in the lower panel are normalized to the wild type (wt) and represent the mean ± standard deviation from three independent observations. InvJ, early substrate; SipB, SipC, and SipD, middle substrates (translocators); SptP, SopB, SipA, and SopE2, late substrates (effectors). (B) Effect of replacing tyrosine 385 of InvC with the indicated amino acids on type III secretion. Analyses of the levels of InvC in whole-cell lysates and the indicated type III secretion substrates in culture supernatants were carried out as indicated for panel A. (C) Effect of replacing conserved amino acids within the loop of the two-helix-finger motif, individually or in combination, with alanine. Analyses of the levels of InvC in whole-cell lysates and the indicated type III secretion substrates in culture supernatants were carried out as indicated for panel A. (D) Interaction of the indicated InvC mutants with OrgB. His-tagged InvC, coexpressed with OrgB in E. coli, was isolated by Ni affinity chromatography followed by size exclusion chromatography. The SDS-PAGE gel was stained with Coomassie brilliant blue. (E) ATPase activity of InvC wild-type and mutant proteins. ATPase activity was measured by a malachite green assay. Note that the Y385W mutation did not affect secretion, indicating that the level of ATPase activity of the Y385W mutant is sufficient for InvC function.

FIG 3

Effect of mutations on the two-helix-finger motif of InvC on needle complex assembly. (A) Electron micrographs of needle complexes prepared from S. Typhimurium strains expressing the indicated InvC mutants. Apparently normal needle complex structures were seen in strains expressing the InvC loop mutants, whereas only base structures were observed in strains expressing a catalytic mutant (the K165E mutant). (B) Efficiency of the inner rod protein PrgJ incorporation into needle complexes, a measure of the completion of their assembly, obtained from strains expressing the indicated InvC mutants. Fractions containing the needle complexes used for EM analyses were subjected to SDS-PAGE followed by Western immunoblotting. Sample loading was standardized by the amount of the base components (InvG, PrgH, and PrgK).

FIG 4

The luminal loop of the InvC hexameric pore is required for type III secretion. (A) Acidic residues of the luminal loop of InvC were individually replaced with alanine, and the impacts on type III secretion and InvC stability were analyzed as indicated in the Fig. 2 legend. (B and C) Effects of the indicated InvC mutations on its ability to bind OrgB (B) or its catalytic activity (C). These experiments were carried out as indicated in the Fig. 2 legend.

FIG 5

Model of the functional role of the InvC loops. The two-helix finger and the luminal loop (red) may help in engaging the T3SS chaperone/substrate complexes, possibly by interacting with the amino-terminal secretion signal (chaperone, blue; substrate, green). The side chain of Tyr385 is depicted in orange. The movement of the Tyr385 side chain by cycles of ATP binding, hydrolysis, and ADP release may provide mechanical force to release the chaperone from its substrate.