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Structure of the Mycobacterium tuberculosis type VII secretion system chaperone EspG5 in complex with PE25-PPE41 dimer.

ABSTRACT: The growth or virulence of Mycobacterium tuberculosis bacilli depends on homologous type VII secretion systems, ESX-1, ESX-3 and ESX-5, which export a number of protein effectors across membranes to the bacterial surface and environment. PE and PPE proteins represent two large families of highly polymorphic proteins that are secreted by these ESX systems. Recently, it was shown that these proteins require system-specific cytoplasmic chaperones for secretion. Here, we report the crystal structure of M. tuberculosis?ESX-5-secreted PE25-PPE41 heterodimer in complex with the cytoplasmic chaperone EspG(5). EspG(5) represents a novel fold that is unrelated to previously characterized secretion chaperones. Functional analysis of the EspG(5) -binding region uncovered a hydrophobic patch on PPE41 that promotes dimer aggregation, and the chaperone effectively abolishes this process. We show that PPE41 contains a characteristic chaperone-binding sequence, the hh motif, which is highly conserved among ESX-1-, ESX-3- and ESX-5-specific PPE proteins. Disrupting the interaction between EspG(5) and three different PPE target proteins by introducing different point mutations generally affected protein secretion. We further demonstrate that the EspG(5) chaperone plays an important role in the ESX secretion mechanism by keeping aggregation-prone PE-PPE proteins in their soluble state.

SUBMITTER: Korotkova N

PROVIDER: S-EPMC4192059 | biostudies-literature | 2014 Oct

REPOSITORIES: biostudies-literature

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Structure of the Mycobacterium tuberculosis type VII secretion system chaperone EspG5 in complex with PE25-PPE41 dimer.

Korotkova Natalia N Freire Diana D Phan Trang H TH Ummels Roy R Creekmore Christopher C CC Evans Timothy J TJ Wilmanns Matthias M Bitter Wilbert W Parret Annabel H A AH Houben Edith N G EN Korotkov Konstantin V KV

Molecular microbiology 20140915 2

The growth or virulence of Mycobacterium tuberculosis bacilli depends on homologous type VII secretion systems, ESX-1, ESX-3 and ESX-5, which export a number of protein effectors across membranes to the bacterial surface and environment. PE and PPE proteins represent two large families of highly polymorphic proteins that are secreted by these ESX systems. Recently, it was shown that these proteins require system-specific cytoplasmic chaperones for secretion. Here, we report the crystal structure ...[more]

PMID: 25155747

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Project description:CpnT, a NAD+ glycohydrolase, is the only known toxin that is secreted by Mycobacterium tuberculosis CpnT is composed of two domains; the C-terminal domain is the toxin, whereas the N-terminal domain is required for secretion. CpnT shows characteristics of type VII secretion (T7S) substrates, including a predicted helix-turn-helix domain followed by a secretion motif (YxxxE). Disruption of this motif indeed abolished CpnT secretion. By analyzing different mutants, we established that CpnT is specifically secreted by the ESX-5 system in Mycobacterium marinum under axenic conditions and during macrophage infection. Surprisingly, intracellular secretion of CpnT was also dependent on both ESX-1 and ESX-4. These secretion defects could be partially rescued by coinfection with wild-type bacteria, indicating that secreted effectors are involved in this process. In summary, our data reveal that three different type VII secretion systems have to be functional in order to observe intracellular secretion of the toxin CpnT.IMPORTANCE For decades, it was believed that the intracellular pathogen M. tuberculosis does not possess toxins. Only fairly recently it was discovered that CpnT is a potent secreted toxin of M. tuberculosis, causing necrotic cell death in host cells. However, until now the secretion pathway remained unknown. In our study, we were able to identify CpnT as a substrate of the mycobacterial type VII secretion system. Pathogenic mycobacteria have up to five different type VII secretion systems, called ESX-1 to ESX-5, which play distinct roles for the pathogen during growth or infection. We were able to elucidate that CpnT is exclusively secreted by the ESX-5 system in bacterial culture. However, to our surprise we discovered that, during infection studies, CpnT secretion relies on intact ESX-1, ESX-4, and ESX-5 systems. We elucidate for the first time the intertwined interplay of three different and independent secretion systems to secrete one substrate during infection.

Project description:BackgroundType III secretion systems are a common virulence mechanism in many Gram-negative bacterial pathogens. These systems use a nanomachine resembling a molecular needle and syringe to provide an energized conduit for the translocation of effector proteins from the bacterial cytoplasm to the host cell cytoplasm for the benefit of the pathogen. Prior to translocation specialized chaperones maintain proper effector protein conformation. The class II chaperone, Invasion plasmid gene (Ipg) C, stabilizes two pore forming translocator proteins. IpgC exists as a functional dimer to facilitate the mutually exclusive binding of both translocators.ResultsIn this study, we present the 3.3 A crystal structure of an amino-terminally truncated form (residues 10-155, denoted IpgC10-155) of the class II chaperone IpgC from Shigella flexneri. Our structure demonstrates an alternative quaternary arrangement to that previously described for a carboxy-terminally truncated variant of IpgC (IpgC1-151). Specifically, we observe a rotationally-symmetric "head-to- head" dimerization interface that is far more similar to that previously described for SycD from Yersinia enterocolitica than to IpgC1-151. The IpgC structure presented here displays major differences in the amino terminal region, where extended coil-like structures are seen, as opposed to the short, ordered alpha helices and asymmetric dimerization interface seen within IpgC1-151. Despite these differences, however, both modes of dimerization support chaperone activity, as judged by a copurification assay with a recombinant form of the translocator protein, IpaB.ConclusionsFrom primary to quaternary structure, these results presented here suggest that a symmetric dimerization interface is conserved across bacterial class II chaperones. In light of previous data which have described the structure and function of asymmetric dimerization, our results raise the possibility that class II chaperones may transition between asymmetric and symmetric dimers in response to changes in either biochemical modifications (e.g. proteolytic cleavage) or other biological cues. Such transitions may contribute to the broad range of protein-protein interactions and functions attributed to class II chaperones.

Dataset Information

Structure of the Mycobacterium tuberculosis type VII secretion system chaperone EspG5 in complex with PE25-PPE41 dimer.

Publications

Structure of the Mycobacterium tuberculosis type VII secretion system chaperone EspG5 in complex with PE25-PPE41 dimer.

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