scholarly journals Yersinia enterocolitica Type III Secretion: Mutational Analysis of the yopQ Secretion Signal

2002 ◽  
Vol 184 (12) ◽  
pp. 3321-3328 ◽  
Author(s):  
Kumaran S. Ramamurthi ◽  
Olaf Schneewind
Keyword(s):  
Type Iii Secretion ◽  
Mutational Analysis ◽  
Mrna Sequence ◽  
Reporter Protein ◽  
Frameshift Mutations ◽  
Type Iii ◽  
Synonymous Mutations ◽  
Secretion Signal ◽  
Fusion Site ◽  
Necessary And Sufficient

ABSTRACT 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.

scholarly journals Impassable YscP Substrates and Their Impact on the Yersinia enterocolitica Type III Secretion Pathway

2008 ◽  
Vol 190 (18) ◽  
pp. 6204-6216 ◽  
Author(s):  
Kelly E. Riordan ◽  
Joseph A. Sorg ◽  
Bryan J. Berube ◽  
Olaf Schneewind
Keyword(s):  
Type Iii Secretion ◽  
Host Cells ◽  
Block Type ◽  
Reporter Protein ◽  
Secrete Protein ◽  
Protein Substrates ◽  
Type Iii ◽  
Secretion Signal ◽  
Secretion Pathway ◽  
Partial Blockade

ABSTRACT Yersinia type III machines secrete protein substrates across the bacterial envelope and, following assembly of their secretion needles, transport effector Yops into host cells. According to their destination during type III secretion, early, middle, and late secretion substrates can be distinguished; however, the signals and mechanisms whereby these proteins are recognized and transported by the secretion machine are not understood. Here, we examine several hybrids between secretion substrates and the impassable reporter protein glutathione S-transferase (GST). YscP-GST and YopR-GST blocked type III secretion; however, YscF-, YopD-, YopN-, and LcrV-GST did not. Unlike YopR-GST, which can block type III machines only during their assembly, expression of YscP-GST led to an immediate and complete block of all secretion. The secretion signal of YscP was mapped to its first 10 codons or amino acids; however, YscPΔ2-15-GST, lacking this secretion signal, imposed a partial blockade. YscP-GST copurified with the type III ATPase complex (YscN, YscL, and YscQ) and with YscO, suggesting that the association of specific machine components with the impassable substrate may cause the block in type III secretion.

scholarly journals A Synonymous Mutation in Yersinia enterocolitica yopE Affects the Function of the YopE Type III Secretion Signal

2005 ◽  
Vol 187 (2) ◽  
pp. 707-715 ◽  
Author(s):  
Kumaran S. Ramamurthi ◽  
Olaf Schneewind
Keyword(s):  
Amino Acid ◽  
Amino Acid Sequence ◽  
Type Iii Secretion ◽  
Predicted Amino Acid Sequence ◽  
Nucleotide Position ◽  
Reporter Protein ◽  
Single Nucleotide ◽  
Type Iii ◽  
Secretion Signal ◽  
Nucleotide Mutation

ABSTRACT Yersinia spp. inject virulence proteins called Yops into the cytosol of target eukaryotic cells in an effort to evade phagocytic killing via a dedicated protein-sorting pathway termed type III secretion. Previous studies have proposed that, unlike other protein translocation mechanisms, Yops are not recognized as substrates for secretion via a solely proteinaceous signal. Rather, at least some of this information may be encoded within yop mRNA. Herein, we report that the first seven codons of yopE, when fused to the reporter protein neomycin phosphotransferase (Npt), are sufficient for the secretion of YopE1-7-Npt when type III secretion is induced in vitro. Systematic mutagenesis of yopE codons 1 to 7 reveals that, like yopQ, codons 2, 3, 5, and 7 are sensitive to mutagenesis, thereby defining the first empirical similarity between the secretion signals of two type III secreted substrates. Like that of yopQ, the secretion signal of yopE exhibits a bipartite nature. This is manifested by the ability of codons 8 to 15 to suppress point mutations in the minimal secretion signal that change the amino acid specificities of particular codons or that induce alterations in the reading frame. Further, we have identified a single nucleotide position in codon 3 that, when mutated, conserves the predicted amino acid sequence of the YopE1-7-Npt but abrogates secretion of the reporter protein. When introduced into the context of the full-length yopE gene, the single-nucleotide mutation reduces the type III injection of YopE into HeLa cells, even though the predicted amino acid sequence remains the same. Thus, yopE mRNA appears to encode a property that mediates the type III injection of YopE.

scholarly journals The Secretion Signal of YopN, a Regulatory Protein of the Yersinia enterocolitica Type III Secretion Pathway

2004 ◽  
Vol 186 (18) ◽  
pp. 6320-6324 ◽  
Author(s):  
John W. Goss ◽  
Joseph A. Sorg ◽  
Kumaran S. Ramamurthi ◽  
Hung Ton-That ◽  
Olaf Schneewind
Keyword(s):  
Yersinia Enterocolitica ◽  
Type Iii Secretion ◽  
Regulatory Protein ◽  
Signal Function ◽  
Type Iii ◽  
Synonymous Mutations ◽  
Secretion Signal ◽  
Secretion Pathway ◽  
Hybrid Proteins ◽  
Fusion Approach

ABSTRACT The type III secretion signal of Yersinia enterocolitica YopN was mapped using a gene fusion approach. yopN codons 1 to 12 were identified as critical for signal function. Several synonymous mutations that abolish secretion of hybrid proteins without altering the codon specificity of yopN mRNA were identified.

scholarly journals Molecular and functional analysis of the type III secretion signal of the Salmonella enterica InvJ protein

2002 ◽  
Vol 46 (3) ◽  
pp. 769-779 ◽  
Author(s):  
Holger Rüssmann ◽  
Tomoko Kubori ◽  
Jeannette Sauer ◽  
Jorge E. Galán
Keyword(s):  
Functional Analysis ◽  
Type Iii Secretion ◽  
Salmonella Enterica ◽  
Type Iii ◽  
Secretion Signal

scholarly journals An Amino-Terminal Secretion Signal Is Required for YplA Export by the Ysa, Ysc, and Flagellar Type III Secretion Systems of Yersinia enterocolitica Biovar 1B

2005 ◽  
Vol 187 (17) ◽  
pp. 6075-6083 ◽  
Author(s):  
Sasha M. Warren ◽  
Glenn M. Young
Keyword(s):  
Yersinia Enterocolitica ◽  
Type Iii Secretion ◽  
Distinct Type ◽  
Host Cells ◽  
Amino Acid Residues ◽  
Secretion Systems ◽  
Type Iii ◽  
Secretion Signal ◽  
Amino Terminal ◽  
Type Iii Secretion Systems

ABSTRACT Yersinia enterocolitica biovar 1B maintains three distinct type III secretion (TTS) systems, which independently operate to target proteins to extracellular sites. The Ysa and Ysc systems are prototypical contact-dependent TTS systems that translocate toxic effectors to the cytosols of targeted eukaryotic host cells during infection. The flagellar TTS system is utilized during the assembly of the flagellum and is required for secretion of the virulence-associated phospholipase YplA to the bacterial milieu. When ectopically produced, YplA is also a secretion substrate for the Ysa and Ysc TTS systems. In this study, we define elements that allow YplA recognition and export by the Ysa, Ysc, and flagellar TTS systems. Fusion of various amino-terminal regions of YplA to Escherichia coli alkaline phosphatase (PhoA) lacking its native secretion signal demonstrated that the first 20 amino acids or corresponding mRNA codons of YplA were sufficient for export of YplA-PhoA chimeras by each TTS system. Export of native YplA by each of the three TTS systems was also found to depend on the integrity of its amino terminus. Introduction of a frameshift mutation or deletion of yplA sequences encoding the amino-terminal 20 residues negatively impacted YplA secretion. Deletion of other yplA regions was tolerated, including that resulting in the removal of amino acid residues 30 through 40 of the polypeptide and removal of the 5′ untranslated region of the mRNA. This work supports a model in which independent and distantly related TTS systems of Y. enterocolitica recognize protein substrates by a similar mechanism.

scholarly journals Regulated Secretion of YopN by the Type III Machinery of Yersinia enterocolitica

2001 ◽  
Vol 183 (18) ◽  
pp. 5293-5301 ◽  
Author(s):  
Luisa W. Cheng ◽  
Olga Kay ◽  
Olaf Schneewind
Keyword(s):  
Yersinia Enterocolitica ◽  
Type Iii Secretion ◽  
Calcium Concentration ◽  
Environmental Changes ◽  
Environmental Concentration ◽  
Type Iii ◽  
Hybrid Proteins ◽  
Low Calcium ◽  
Membrane Envelope ◽  
Necessary And Sufficient

ABSTRACT During infection, Yersinia enterocoliticaexports Yop proteins via a type III secretion pathway. Secretion is activated when the environmental concentration of calcium ions is below 100 μM (low-calcium response). Yersiniae lacking yopN (lcrE), yscB, sycN, or tyeA do not inactivate the type III pathway even when the concentration of calcium is above 100 μM (calcium-blind phenotype). Purified YscB and SycN proteins form cytoplasmic complexes that bind a region including amino acids 16 to 100 of YopN, whereas TyeA binds YopN residues 101 to 294. Translational fusion of yopN gene sequences to the 5′ end of thenpt reporter generates hybrid proteins that are transported by the type III pathway. The signal necessary and sufficient for the type III secretion of hybrid proteins is located within the first 15 codons of yopN. Expression of plasmid-borneyopN, but not ofyopN 1–294-npt, complements the calcium-blind phenotype of yopN mutants. Surprisingly,yopN mutants respond to environmental changes in calcium concentration and secrete YopN1–294-Npt in the absence but not in the presence of calcium. tyeA is required for the low-calcium regulation of YopN1–294-Npt secretion, whereassycN and yscB mutants fail to secrete YopN1–294-Npt in the presence of calcium. Experiments withyopN-npt fusions identified two other signals that regulate the secretion of YopN. yopN codons 16 to 100 prevent the entry of YopN into the type III pathway, a negative regulatory effect that is overcome by expression of yscB andsycN. The portion of YopN encoded by codons 101 to 294 prevents transport of the polypeptide across the bacterial double membrane envelope in the presence of functional tyeA. These data support a model whereby YopN transport may serve as a regulatory mechanism for the activity of the type III pathway. YscB/SycN binding facilitates the initiation of YopN into the type III pathway, whereas TyeA binding prevents transport of the polypeptide across the bacterial envelope. Changes in the environmental calcium concentration relieve the TyeA-mediated regulation, triggering YopN transport and activating the type III pathway.

scholarly journals A Bacterial Type III Secretion Assay for Delivery of Fungal Effector Proteins into Wheat

2014 ◽  
Vol 27 (3) ◽  
pp. 255-264 ◽  
Author(s):  
Narayana M. Upadhyaya ◽  
Rohit Mago ◽  
Brian J. Staskawicz ◽  
Michael A. Ayliffe ◽  
Jeffrey G. Ellis ◽  
...  
Keyword(s):  
Pseudomonas Fluorescens ◽  
Delivery System ◽  
Type Iii Secretion ◽  
Large Scale ◽  
Rust Fungus ◽  
Effector Proteins ◽  
In Planta ◽  
Host Genotype ◽  
Reporter Protein ◽  
Type Iii

Large numbers of candidate effectors from fungal pathogens are being identified through whole-genome sequencing and in planta expression studies. Although Agrobacterium-mediated transient expression has enabled high-throughput functional analysis of effectors in dicot plants, this assay is not effective in cereal leaves. Here, we show that a nonpathogenic Pseudomonas fluorescens engineered to express the type III secretion system (T3SS) of P. syringae and the wheat pathogen Xanthomonas translucens can deliver fusion proteins containing T3SS signals from P. syringae (AvrRpm1) and X. campestris (AvrBs2) avirulence (Avr) proteins, respectively, into wheat leaf cells. A calmodulin-dependent adenylate cyclase reporter protein was delivered effectively into wheat and barley by both bacteria. Absence of any disease symptoms with P. fluorescens makes it more suitable than X. translucens for detecting a hypersensitive response (HR) induced by an effector protein with avirulence activity. We further modified the delivery system by removal of the myristoylation site from the AvrRpm1 fusion to prevent its localization to the plasma membrane which could inhibit recognition of an Avr protein. Delivery of the flax rust AvrM protein by the modified delivery system into transgenic tobacco leaves expressing the corresponding M resistance protein induced a strong HR, indicating that the system is capable of delivering a functional rust Avr protein. In a preliminary screen of effectors from the stem rust fungus Puccinia graminis f. sp. tritici, we identified one effector that induced a host genotype-specific HR in wheat. Thus, the modified AvrRpm1:effector–Pseudomonas fluorescens system is an effective tool for large-scale screening of pathogen effectors for recognition in wheat.

scholarly journals Salmonella pathogenicity island 1 (SPI-1) type III secretion of SopD involves N- and C-terminal signals and direct binding to the InvC ATPase

Microbiology ◽  
2010 ◽  
Vol 156 (6) ◽  
pp. 1805-1814 ◽  
Author(s):  
R. Boonyom ◽  
M. H. Karavolos ◽  
D. M. Bulmer ◽  
C. M. A. Khan
Keyword(s):  
Protein Interactions ◽  
Type Iii Secretion ◽  
Pathogenicity Island ◽  
Effector Proteins ◽  
Amino Acid Residues ◽  
Type Iii ◽  
Protein Protein Interaction ◽  
Secretion Signal ◽  
Serovar Typhimurium ◽  
Bacterial Cytoplasm

Salmonella enterica serovar Typhimurium (S. Typhimurium) is an important pathogen and a causative agent of gastroenteritis. During infection, S. Typhimurium assembles molecular-needle complexes termed type III secretion (T3S) systems to translocate effector proteins from the bacterial cytoplasm directly into the host cell. The T3S signals that direct the secretion of effectors still remain enigmatic. SopD is a key T3S effector contributing to the systemic virulence of S. Typhimurium and the development of gastroenteritis. We have scrutinized the distribution of the SopD T3S signals using in silico analysis and a targeted deletion approach. We show that amino acid residues 6–10 act as the N-terminal secretion signal for Salmonella pathogenicity island 1 (SPI-1) T3S. Furthermore, we show that two putative C-terminal helical regions of SopD are essential for its secretion and also help prevent erroneous secretion through the flagellar T3S machinery. In addition, using protein–protein interaction assays, we have identified an association between SopD and the SPI-1 T3S system ATPase, InvC. These findings demonstrate that T3S of SopD involves multiple signals and protein interactions, providing important mechanistic insights into effector protein secretion.

scholarly journals SepD/SepL-Dependent Secretion Signals of the Type III Secretion System Translocator Proteins in Enteropathogenic Escherichia coli

2015 ◽  
Vol 197 (7) ◽  
pp. 1263-1275 ◽  
Author(s):  
Wanyin Deng ◽  
Hong B. Yu ◽  
Yuling Li ◽  
B. Brett Finlay
Keyword(s):  
Escherichia Coli ◽  
Protein Secretion ◽  
Type Iii Secretion ◽  
Bacterial Pathogens ◽  
Secretion System ◽  
Content Type ◽  
Type Iii ◽  
Secretion Signal ◽  
Protein Secretion System ◽  
Type Iii Protein Secretion

ABSTRACTThe type III protein secretion system (T3SS) encoded by the locus of enterocyte effacement (LEE) is essential for the pathogenesis of attaching/effacing bacterial pathogens, including enteropathogenicEscherichia coli(EPEC), enterohemorrhagicE. coli(EHEC), andCitrobacter rodentium. These pathogens use the T3SS to sequentially secrete three categories of proteins: the T3SS needle and inner rod protein components; the EspA, EspB, and EspD translocators; and many LEE- and non-LEE-encoded effectors. SepD and SepL are essential for translocator secretion, and mutations in either lead to hypersecretion of effectors. However, how SepD and SepL control translocator secretion and secretion hierarchy between translocators and effectors is poorly understood. In this report, we show that the secreted T3SS components, the translocators, and both LEE- and non-LEE-encoded effectors all carry N-terminal type III secretion and translocation signals. These signals all behave like those of the effectors and are sufficient for mediating type III secretion and translocation by wild-type EPEC and hypersecretion by thesepDandsepLmutants. Our results extended previous observations and suggest that the secretion hierarchy of the different substrates is determined by a signal other than the N-terminal secretion signal. We identified a domain located immediately downstream of the N-terminal secretion signal in the translocator EspB that is required for SepD/SepL-dependent secretion. We further demonstrated that this EspB domain confers SepD/SepL- and CesAB-dependent secretion on the secretion signal of effector EspZ. Our results thus suggest that SepD and SepL control and regulate secretion hierarchy between translocators and effectors by recognizing translocator-specific export signals.IMPORTANCEMany bacterial pathogens use a syringe-like protein secretion apparatus, termed the type III protein secretion system (T3SS), to secrete and inject numerous proteins directly into the host cells to cause disease. The secreted proteins perform different functions at various stages during infection and are classified into three substrate categories (T3SS components, translocators, and effectors). They all contain secretion signals at their N termini, but how their secretion hierarchy is determined is poorly understood. Here, we show that the N-terminal secretion signals from different substrate categories all behave the same and do not confer substrate specificity. We further characterize the secretion signals of the translocators and identify a translocator-specific signal, demonstrating that substrate-specific secretion signals are required in regulating T3SS substrate hierarchy.

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