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 PcrHPopD complex displays homologs among a number of pathogenic strains and could represent a novel, potential target for antibiotic development.

Membrane and Chaperone Recognition by the Major Translocator Protein PopB of the Type III Secretion System of Pseudomonas aeruginosa

“…Subsequently, pET15b-PcrH was digested with XbaI-BamHI to maintain the ribosomal binding site and cloned blunt-ended in the BamHI blunt-ended pET15b-PopB. The pETDuet-PopD/ PcrH vector was obtained by cloning popD in the NdeI-XhoI sites present in the second multiple cloning site of pETDuet1-PcrH(1-160) vector, described previously (8).…”

“…Crystallization, Data Collection, and Structure SolutionPcrH (21-160) was expressed and purified as previously described (8). Purified PcrH was mixed with a synthetic peptide corresponding to residues 51-59 of PopB (TGVALTPPS) in molar ratios 1:1, 1:2, and 1:4 and submitted to high throughput crystallization (HTXLab; Partnership for Structural Biology, Grenoble, France) at 4°C.…”

“…Because the internal diameter of the needle is on the order of 25 Å, it is believed that effectors must travel in a semi-unfolded state (2, 3) due to energy provided by an ATPase located at the base of the system (4 -6). Introduction of point mutations into key proteins of the T3SS have shown to be highly deleterious for the cytotoxicity potential of certain pathogens (7)(8)(9)(10)(11), and T3SS-deficient strains display attenuated infectivity in animal models (12)(13)(14), indicating that an understanding of T3SS formation and regulation mechanisms could lead to the development of strategies for infection control.…”

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“…It is highly soluble and very stable and possesses rigid tertiary structure. Till now, only the structures of small peptides or fragments of translocator proteins are available [18,26,27,28]. Therefore, it would Interestingly, these helices do not occur in a sequential manner.…”

“…These pockets are formed by the residues mainly located in the first two TPR regions. Also PcrH (another class II chaperone) interacts with minor translocator PopD using the residues present within its concave cleft [26]. …”