Structural basis of chaperone recognition of type III secretion system minor translocator proteins

Abstract

The type III secretion system (T3SS) is a complex nanomachine employed by many Gram-negative pathogens, including the nosocomial agent Pseudomonas aeruginosa, to inject toxins directly into the cytoplasm of eukaryotic cells. A key component of all T3SS is the translocon, a proteinaceous channel that is inserted into the target membrane, which allows passage of toxins into target cells. In most bacterial species, two distinct membrane proteins (the "translocators") are involved in translocon formation, whereas in the bacterial cytoplasm, however, they remain associated to a common chaperone. To date, the strategy employed by a single chaperone to recognize two distinct translocators is unknown. Here, we report the crystal structure of a complex between the Pseudomonas translocator chaperone PcrH and a short region from the minor translocator PopD. PcrH displays a 7-helical tetratricopeptide repeat fold that harbors the PopD peptide within its concave region, originally believed to be involved in recognition of the major translocator, PopB. Point mutations introduced into the PcrH-interacting region of PopD impede translocator-chaperone recognition in vitro and lead to impairment of bacterial cytotoxicity toward macrophages in vivo. These results indicate that T3SS translocator chaperones form binary complexes with their partner molecules, and the stability of their interaction regions must be strictly maintained to guarantee bacterial infectivity. The PcrH-PopD complex displays homologs among a number of pathogenic strains and could represent a novel, potential target for antibiotic development.

FIGURE 1.

PcrH displays a monomer-dimer equilibrium, except when associated to PopD. A, gel filtration profiles of PcrH at 8.3 mg/ml (solid line), 0.8 mg/ml (dashed line), and 0.08 mg/ml (dotted line). B, gel filtration profiles of PopD-PcrH at 3.3 mg/ml (solid line), 0.33 mg/ml (dashed line), and 0.033 mg/ml (dotted line). Molecular mass standards employed were as follows: 1, blue dextran; 2, albumin (67 kDa); 3, ovalbumin (43 kDa); 4, chymotrypsinogen A (25 kDa); and 5, ribonuclease A (13.7 kDa).

FIGURE 2.

A, PcrH(21–160) displays a 7-helical TPR-like fold. Each one of the TPR domains is shown in a different color, with helix 7 in violet. B, superposition of IpgC (orange), SycD (pink), and PcrH (cyan) reveals very similar overall folds, with differences mostly at the N termini. C, PcrH displays a highly charged surface. Indicated residues point directly into the concave cleft and interact with the partner molecule. D, dimer interactions are made through the convex side of the TPR fold of PcrH.

FIGURE 3.

A, sequence alignment of minor translocon molecules of bacteria harboring T3SS in the Ysc/Psc family. Sequences include PopD (P. aeruginosa), LopD (Photorhabdus luminescens), AopD (Aeromonas hydrophila), YopD (Y. pestis), and VopD (Vibrio harveyi). Green and blue residues display strong/weak similarities, respectively, as determined by ClustalW. Identical residues are indicated in red. The sequence that corresponds to the region selected for crystallization studies is highlighted in yellow, and those of the mutants studied in this work are shown above the alignment. B, PopD(47–56) is located snugly within the charged PcrH concave cleft. Two hydrophobic residues, Val-49 and Leu-51, anchor PopD deeply into the binding site. C, details of the PcrH-PopD interactions. A number of residues from H1, H3, and H5 (in green) interact directly with the peptide, which is shown in yellow. The view has been slightly rotated with respect to B to facilitate analysis.

FIGURE 4.

A, Pseudomonas ΔpopBD cultures or complemented by pIapG-pcrHpopBD carrying either popDwt, popD-4D, or popD-RAE were grown in T3SS-induced conditions (see “Experimental Procedures”). The protein content of both pellet (expression) and supernatant (secretion) were analyzed by Western blotting. B, capacity of the PopD mutants to lyse macrophages was measured by the quantification of lactate dehydrogenase release. Noninfected cells (n.i) were used as negative controls. All values are the average of four measurements. C, stability of PopD mutants was tested with the same culture conditions as for A. At A_{600 nm} = 1 A.U., protein synthesis was interrupted by the addition of chloramphenicol, and protein stability was checked at 1–3 h and analyzed by Western blotting. Expression of PcrV was used as a positive control. D, interaction between PcrH and PopD was analyzed by co-purification on a HisTrap column. E. coli cultures expressing both His₆-PcrH/PopDwt or His₆-PcrH/PopD-4D (Tot) were lysed, and the soluble fractions (Sol) were applied onto a HisTrap column. Proteins bound to the column were eluted with a linear gradient of imidazole (Elution). Only PopDwt co-eluted with His₆-PcrH. The different purification steps were analyzed by SDS-PAGE 15% and colored by Coomassie Blue stain. Lane M, molecular weight marker.

FIGURE 5.

PcrH mutant analysis and surface conservation. A, mapping of the residues mutated by Bröms et al. (14) on the surface of PcrH reveals that mutations that affected both PopB and PopD binding (yellow) are mostly located on the N-terminal region of the chaperone, whereas mutations that affect only PopB recognition (magenta) are mostly located on H5. PcrH from P. aeruginosa strain CHA contains one less amino acid on its N terminus than that from strain PAO1 or Yersinia SycD; thus, by comparison with these molecules, the numbering differs by 1. B, surface mapping of conserved (orange) and identical (red) amino acids in type II chaperone sequences of T3SS in P. aeruginosa, E. coli, Salmonella typhimurium, S. flexneri, and Y. pestis. C, same as B, but from a 180° perspective. The concave region of the TPR “hand” is highly conserved, but the top part of the convex region also displays considerable conservation.