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Secretion signal recognition by YscN, the Yersinia type III secretion ATPase.

ABSTRACT: Yersinia type III machines secrete protein substrates across the bacterial envelope. Secretion signals of some substrates have been identified; however, the mechanisms whereby these signals interact with type III machines are not known. Here we show that fusion of YopR, an early secretion substrate, to the N terminus of dihydrofolate reductase (DHFR) or other tightly folded proteins generates impassable hybrids that cannot travel the type III pathway. YopR hybrids capture YscN, the ATPase that provides energy for type III transport reactions, in the bacterial cytoplasm. Eleven N-terminal residues function as the YopR secretion signal, which is required for both binding to YscN and blocking the type III pathway. When expressed during type III machine assembly, YopR-DHFR blocks all secretion. Delayed expression of YopR-DHFR, when yersiniae have already engaged the type III pathway, blocks secretion of early (YscP) but not of late (effector Yops) substrates. These observations support a model whereby type III machines are programmed to secrete a sequence of proteins that can be disrupted when an impassable early substrate interacts with the YscN ATPase and blocks the transport of late substrates.

SUBMITTER: Sorg JA

PROVIDER: S-EPMC1637609 | biostudies-literature | 2006 Oct

REPOSITORIES: biostudies-literature

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Secretion signal recognition by YscN, the Yersinia type III secretion ATPase.

Sorg Joseph A JA Blaylock Bill B Schneewind Olaf O

Proceedings of the National Academy of Sciences of the United States of America 20061018 44

Yersinia type III machines secrete protein substrates across the bacterial envelope. Secretion signals of some substrates have been identified; however, the mechanisms whereby these signals interact with type III machines are not known. Here we show that fusion of YopR, an early secretion substrate, to the N terminus of dihydrofolate reductase (DHFR) or other tightly folded proteins generates impassable hybrids that cannot travel the type III pathway. YopR hybrids capture YscN, the ATPase that p ...[more]

PMID: 17050689

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Project description:Specialized protein translocation systems are used by many bacterial pathogens to deliver effector proteins into host cells that interfere with normal cellular functions. How the host immune system recognizes and responds to this intrusive event is not understood. To address these questions, we determined the mammalian cellular response to the virulence-associated type III secretion system (T3SS) of the human pathogen Yersinia pseudotuberculosis. We found that macrophages devoid of Toll-like receptor (TLR) signaling regulate expression of 266 genes following recognition of the Y. pseudotuberculosis T3SS. This analysis revealed two temporally distinct responses that could be separated into activation of NFkappaB- and type I IFN-regulated genes. Extracellular bacteria were capable of triggering these signaling events, as inhibition of bacterial uptake had no effect on the ensuing innate immune response. The cytosolic peptidoglycan sensors Nod1 and Nod2 and the inflammasome component caspase-1 were not involved in NFkappaB activation following recognition of the Y. pseudotuberculosis T3SS. However, caspase-1 was required for secretion of the inflammatory cytokine IL-1beta in response to T3SS-positive Y. pseudotuberculosis. In order to characterize the bacterial requirements for induction of this novel TLR-, Nod1/2-, and caspase-1-independent response, we used Y. pseudotuberculosis strains lacking specific components of the T3SS. Formation of a functional T3SS pore was required, as bacteria expressing a secretion needle, but lacking the pore-forming proteins YopB or YopD, did not trigger these signaling events. However, nonspecific membrane disruption could not recapitulate the NFkappaB signaling triggered by Y. pseudotuberculosis expressing a functional T3SS pore. Although host cell recognition of the T3SS did not require known translocated substrates, the ensuing response could be modulated by effectors such as YopJ and YopT, as YopT amplified the response, while YopJ dampened it. Collectively, these data suggest that combined recognition of the T3SS pore and YopBD-mediated delivery of immune activating ligands into the host cytosol informs the host cell of pathogenic challenge. This leads to a unique, multifactorial response distinct from the canonical immune response to a bacterium lacking a T3SS.

Project description:Pathogenic Yersinia spp. secrete Yop proteins via the type III pathway. yopQ codons 1 to 15 were identified as a signal necessary and sufficient for the secretion of a fused reporter protein. Frameshift mutations that alter codons 2 to 15 with little alteration of yopQ mRNA sequence do not abolish type III transport, suggesting a model in which yopQ mRNA may provide a signal for secretion (D. M. Anderson and O. Schneewind, Mol. Microbiol. 31:1139-1148, 2001). In a recent study, the yopE signal was truncated to codons 1 to 12. All frameshift mutations introduced within the first 12 codons of yopE abolished secretion. Also, multiple synonymous mutations that changed the mRNA sequence of yopE codons 1 to 12 without altering the amino acid sequence did not affect secretion. These results favor a model whereby an N-terminal signal peptide initiates YopE into the type III pathway (S. A. Lloyd et al., Mol. Microbiol. 39:520-531, 2001). It is reported here that codons 1 to 10 of yopQ act as a minimal secretion signal. Further truncation of yopQ, either at codon 10 or at codon 2, abolished secretion. Replacement of yopQ AUG with either of two other start codons, UUG or GUG, did not affect secretion. However, replacement of AUG with CUG or AAA and initiating translation at the fusion site with npt did not permit Npt secretion, suggesting that the translation of yopQ codons 1 to 15 is a prerequisite for secretion. Frameshift mutations of yopQ codons 1 to 10, 1 to 11, and 1 to 12 abolished secretion signaling, whereas frameshift mutations of yopQ codons 1 to 13, 1 to 14, and 1 to 15 did not. Codon changes at yopQ positions 2 and 10 affected secretion signaling when placed within the first 10 codons but had no effect when positioned in the larger fusion of yopQ codons 1 to 15. An mRNA mutant of yopQ codons 1 to 10, generated by a combination of nine synonymous mutations, was defective in secretion signaling, suggesting that the YopQ secretion signal is not proteinaceous. A model is discussed whereby the initiation of YopQ polypeptide into the type III pathway is controlled by properties of yopQ mRNA.

Dataset Information

Secretion signal recognition by YscN, the Yersinia type III secretion ATPase.

Publications

Secretion signal recognition by YscN, the Yersinia type III secretion ATPase.

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