scholarly journals Gezielte Injektion von Effektoren durch Kontrolle der Proteindynamik

BIOspektrum ◽  
2021 ◽  
Vol 27 (7) ◽  
pp. 697-700
Author(s):  
Stephan Wimmi ◽  
Florian Lindner ◽  
Andreas Diepold
Keyword(s):  
Yersinia Enterocolitica ◽  
Type Iii Secretion ◽  
Direct Injection ◽  
Low Ph ◽  
Secretion System ◽  
Effector Proteins ◽  
Eukaryotic Cells ◽  
Site Of Action ◽  
Rapid Activation ◽  
Spatiotemporal Control

AbstractThe type III secretion system (T3SS) enables direct injection of bacterial effector proteins into eukaryotic cells. We found that the dynamic cytosolic interface of the system allows Yersinia enterocolitica to suppress premature secretion at low pH, ensuring rapid activation at the site of action. Exploiting this principle, we developed a light-controlled T3SS based on optogenetic interaction switches, which provides unprecedented spatiotemporal control of protein secretion and translocation.

scholarly journals SpiC Is Required for Translocation of Salmonella Pathogenicity Island 2 Effectors and Secretion of Translocon Proteins SseB and SseC

2002 ◽  
Vol 184 (18) ◽  
pp. 4971-4980 ◽  
Author(s):  
Jeremy A. Freeman ◽  
Catherine Rappl ◽  
Volker Kuhle ◽  
Michael Hensel ◽  
Samuel I. Miller
Keyword(s):  
Type Iii Secretion ◽  
Pathogenicity Island ◽  
Secretion System ◽  
Effector Proteins ◽  
Mutant Phenotype ◽  
Eukaryotic Cells ◽  
Large Contribution ◽  
Salmonella Pathogenicity Island ◽  
Virulence Phenotype ◽  
Serovar Typhimurium

ABSTRACT The Salmonella pathogenicity island 2 (SPI2) type III secretion system (TTSS) promotes Salmonella enterica serovar Typhimurium virulence for mice and increased survival and replication within eukaryotic cells. After phagocytosis, Salmonella serovar Typhimurium assembles the SPI2 TTSS to translocate over a dozen effector proteins across the phagosome membrane. SpiC has been previously shown to be a translocated effector with a large contribution to virulence (K. Uchiya, M. A. Barbieri, K. Funato, A. H. Shah, P. D. Stahl, and E. A. Groisman, EMBO J. 18:3924-3933, 1999). This report demonstrates by competitive index that the virulence phenotype of a spiC mutant is equivalent to that of a secretion component mutant. In addition, translocation of SPI2 effector proteins was shown to require SpiC. Thus, the severe virulence phenotype resulting from deletion of spiC is likely due to the inability to translocate all SPI2 effectors. SpiC was also required to secrete translocon proteins SseB and SseC but not translocated effector SseJ, indicating that lack of assembly of the translocon explains the spiC mutant phenotype.

scholarly journals Cryoelectron-microscopy structure of the enteropathogenic Escherichia coli type III secretion system EspA filament

2021 ◽  
Vol 118 (2) ◽  
pp. e2022826118
Author(s):  
Weili Zheng ◽  
Alejandro Peña ◽  
Aravindan Ilangovan ◽  
Jasmine Naemi-Baghshomali Clark ◽  
Gad Frankel ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Type Iii Secretion ◽  
Rational Design ◽  
Type Iii Secretion System ◽  
Secretion System ◽  
Effector Proteins ◽  
Eukaryotic Cells ◽  
Bacterial Membranes ◽  
Type Iii ◽  
Functional Conservation

Enteropathogenic Escherichia coli (EPEC) and enterohemorrhagic Escherichia coli (EHEC) utilize a macromolecular type III secretion system (T3SS) to inject effector proteins into eukaryotic cells. This apparatus spans the inner and outer bacterial membranes and includes a helical needle protruding into the extracellular space. Thus far observed only in EPEC and EHEC and not found in other pathogenic Gram-negative bacteria that have a T3SS is an additional helical filament made by the EspA protein that forms a long extension to the needle, mediating both attachment to eukaryotic cells and transport of effector proteins through the intestinal mucus layer. Here, we present the structure of the EspA filament from EPEC at 3.4 Å resolution. The structure reveals that the EspA filament is a right-handed 1-start helical assembly with a conserved lumen architecture with respect to the needle to ensure the seamless transport of unfolded cargos en route to the target cell. This functional conservation is despite the fact that there is little apparent overall conservation at the level of sequence or structure with the needle. We also unveil the molecular details of the immunodominant EspA epitope that can now be exploited for the rational design of epitope display systems.

scholarly journals Comparative Analysis of Locus of Enterocyte Effacement Pathogenicity Islands of Atypical Enteropathogenic Escherichia coli

2004 ◽  
Vol 72 (11) ◽  
pp. 6722-6728 ◽  
Author(s):  
Julia F. Gärtner ◽  
M. Alexander Schmidt
Keyword(s):  
Escherichia Coli ◽  
Type Iii Secretion ◽  
Functional Expression ◽  
Secretion System ◽  
Effector Proteins ◽  
Eukaryotic Cells ◽  
Pathogenicity Islands ◽  
E Coli ◽  
Enterocyte Effacement ◽  
Pedestal Formation

ABSTRACT The pathogenicity of enteropathogenic Escherichia coli (EPEC) is linked to the locus of enterocyte effacement, or LEE, encoding a type III secretion system (T3SS) that directly transfers bacterial effector proteins into eukaryotic cells. Atypical diffusely adhering EPEC (DA-EPEC) strains that harbor homologues of the LEE but lack the EPEC adherence factor plasmid have been increasingly associated with outbreaks of diarrhea. In this study, we have completely sequenced and functionally characterized LEE pathogenicity islands derived from the clinical DA-EPEC isolates 3431 (O8:H−) and 0181 (O119:H9:K61). LEE3431 and LEE0181 exhibit genetic organization analogous to that of the prototype LEEE2348/69. Genes constituting the T3SS apparatus are highly conserved. However, LEE-encoded effector proteins exhibit major differences. Transfer and functional expression of LEE0181 in an E. coli XL1 blue MR background demonstrated that LEE0181 contains all the information for signal transduction and pedestal formation.

scholarly journals Pseudomonas syringae Strains Naturally Lacking the Classical P. syringae hrp/hrc Locus Are Common Leaf Colonizers Equipped with an Atypical Type III Secretion System

2010 ◽  
Vol 23 (2) ◽  
pp. 198-210 ◽  
Author(s):  
Christopher R. Clarke ◽  
Rongman Cai ◽  
David J. Studholme ◽  
David S. Guttman ◽  
Boris A. Vinatzer
Keyword(s):  
Pseudomonas Syringae ◽  
Type Iii Secretion ◽  
Plant Cells ◽  
Type Iii Secretion System ◽  
Ice Nucleation ◽  
Secretion System ◽  
Effector Proteins ◽  
Population Densities ◽  
Type Iii ◽  
Effector Genes

Pseudomonas syringae is best known as a plant pathogen that causes disease by translocating immune-suppressing effector proteins into plant cells through a type III secretion system (T3SS). However, P. syringae strains belonging to a newly described phylogenetic subgroup (group 2c) are missing the canonical P. syringae hrp/hrc cluster coding for a T3SS, flanking effector loci, and any close orthologue of known P. syringae effectors. Nonetheless, P. syringae group 2c strains are common leaf colonizers and grow on some tested plant species to population densities higher than those obtained by other P. syringae strains on nonhost species. Moreover, group 2c strains have genes necessary for the production of phytotoxins, have an ice nucleation gene, and, most interestingly, contain a novel hrp/hrc cluster, which is only distantly related to the canonical P. syringae hrp/hrc cluster. This hrp/hrc cluster appears to encode a functional T3SS although the genes hrpK and hrpS, present in the classical P. syringae hrp/hrc cluster, are missing. The genome sequence of a representative group 2c strain also revealed distant orthologues of the P. syringae effector genes avrE1 and hopM1 and the P. aeruginosa effector genes exoU and exoY. A putative life cycle for group 2c P. syringae is discussed.

scholarly journals Use of the Galleria mellonella Caterpillar as a Model Host To Study the Role of the Type III Secretion System in Pseudomonas aeruginosa Pathogenesis

2003 ◽  
Vol 71 (5) ◽  
pp. 2404-2413 ◽  
Author(s):  
Sachiko Miyata ◽  
Monika Casey ◽  
Dara W. Frank ◽  
Frederick M. Ausubel ◽  
Eliana Drenkard
Keyword(s):  
Pseudomonas Aeruginosa ◽  
Type Iii Secretion ◽  
Galleria Mellonella ◽  
Type Iii Secretion System ◽  
Secretion System ◽  
Effector Proteins ◽  
Triple Mutant ◽  
Type Iii ◽  
Wax Moth

ABSTRACT Nonvertebrate model hosts represent valuable tools for the study of host-pathogen interactions because they facilitate the identification of bacterial virulence factors and allow the discovery of novel components involved in host innate immune responses. In this report, we determined that the greater wax moth caterpillar Galleria mellonella is a convenient nonmammalian model host for study of the role of the type III secretion system (TTSS) in Pseudomonas aeruginosa pathogenesis. Based on the observation that a mutation in the TTSS pscD gene of P. aeruginosa strain PA14 resulted in a highly attenuated virulence phenotype in G. mellonella, we examined the roles of the four known effector proteins of P. aeruginosa (ExoS, ExoT, ExoU, and ExoY) in wax moth killing. We determined that in P. aeruginosa strain PA14, only ExoT and ExoU play a significant role in G. mellonella killing. Strain PA14 lacks the coding sequence for the ExoS effector protein and does not seem to express ExoY. Moreover, using ΔexoU ΔexoY, ΔexoT ΔexoY, and ΔexoT ΔexoU double mutants, we determined that individual translocation of either ExoT or ExoU is sufficient to obtain nearly wild-type levels of G. mellonella killing. On the other hand, data obtained with a ΔexoT ΔexoU ΔexoY triple mutant and a ΔpscD mutant suggested that additional, as-yet-unidentified P. aeruginosa components of type III secretion are involved in virulence in G. mellonella. A high level of correlation between the results obtained in the G. mellonella model and the results of cytopathology assays performed with a mammalian tissue culture system validated the use of G. mellonella for the study of the P. aeruginosa TTSS.

scholarly journals Pseudomonas aeruginosa injects NDK into host cells through a type III secretion system

Microbiology ◽  
2014 ◽  
Vol 160 (7) ◽  
pp. 1417-1426 ◽  
Author(s):  
Dennis Neeld ◽  
Yongxin Jin ◽  
Candace Bichsel ◽  
Jinghua Jia ◽  
Jianhui Guo ◽  
...  
Keyword(s):  
Pseudomonas Aeruginosa ◽  
Type Iii Secretion ◽  
Type Iii Secretion System ◽  
Secretion System ◽  
Host Cells ◽  
Eukaryotic Cells ◽  
Mammalian Host ◽  
Type I ◽  
Dependent Manner ◽  
Type Iii

Pseudomonas aeruginosa is a Gram-negative opportunistic human pathogen possessing a type III secretion system (T3SS) which injects toxic effector proteins into mammalian host cells. In previous studies, P. aeruginosa strains lacking all of the known type III effectors were shown to cause cytotoxicity upon prolonged infection time. In this study, we report the identification of a new cytotoxin, nucleoside diphosphate kinase (NDK), which is injected into eukaryotic cells in a T3SS-dependent manner. Injection of NDK is inhibited by the presence of previously known effectors of the T3SS, with an effectorless strain injecting the highest amount, suggesting active competition with the known T3SS effectors. NDK is shown to cause a cytotoxic response when expressed in eukaryotic cells, and P. aeruginosa strains harbouring NDK also show a greater toxicity than strains lacking it. Interestingly, the cytotoxic effect of intracellular NDK is independent of its kinase activity. In previous studies, NDK was shown to be secreted into culture supernatants via a type I secretion system and cause cytotoxicity in a kinase-dependent manner. Therefore, the current study highlights an alternative route of NDK secretion as well as two different cytotoxic mechanisms of NDK, depending on the extra- or intra-cellular location of the protein.

scholarly journals Evidence for Targeting of Yop Effectors by the Chromosomally Encoded Ysa Type III Secretion System of Yersinia enterocolitica

2002 ◽  
Vol 184 (20) ◽  
pp. 5563-5571 ◽  
Author(s):  
Briana M. Young ◽  
Glenn M. Young
Keyword(s):  
Yersinia Enterocolitica ◽  
Type Iii Secretion ◽  
Immunoblot Analysis ◽  
Effector Proteins ◽  
Secretion Systems ◽  
Necrosis Factor Alpha ◽  
Type Iii ◽  
Type Iii Secretion Systems ◽  
Molecular Masses ◽  
Factor Alpha

ABSTRACT Yersinia enterocolitica O:8 has two contact-dependent type III secretion systems (TTSSs). The Ysa TTSS is encoded by a set of genes located on the chromosome and exports Ysp proteins. The Ysc TTSS and the Yop effector proteins it exports are encoded by genes located on plasmid pYVe8081. In this study, secretion of YspG, YspH, and YspJ by the Ysa TTSS was shown to require pYVe8081. Furthermore, mutations that blocked the function of the Ysc TTSS did not affect YspG, YspH, and YspJ production. This indicated that YspG, YspH, and YspJ are encoded by genes located on pYVe8081 and that they may correspond to Yops. A comparison of Ysps with Yop effectors secreted by Y. enterocolitica indicated that YspG, YspH, and YspJ have apparent molecular masses similar to those of YopN, YopP, and YopE, respectively. Immunoblot analysis demonstrated that antibodies directed against YopN, YopP, and YopE recognized YspG, YspH, and YspJ. Furthermore, mutations in yopN, yopP, and yopE specifically blocked YopN, YopP, and YopE secretion by the Ysc TTSS and YspG, YspH, and YspJ secretion by the Ysa TTSS. These results indicate YspG, YspH, and YspJ are actually YopN, YopP, and YopE. Additional analysis demonstrated that YopP and YspH secretion was restored to yopP mutants by complementation in trans with a wild-type copy of the yopP gene. Examination of Y. enterocolitica-infected J774A.1 macrophages revealed that both the Ysc and Ysa TTSSs contribute to YopP-dependent suppression of tumor necrosis factor alpha production. This indicates that both the Ysa and Ysc TTSSs are capable of targeting YopP and that they influence Y. enterocolitica interactions with macrophages. Taken together, these results suggest that the Ysa and Ysc TTSSs contribute to Y. enterocolitica virulence by exporting both unique and common subsets of effectors.

scholarly journals Salmonella-based platform for efficient delivery of functional binding proteins to the cytosol

2020 ◽  
Vol 3 (1) ◽  
Author(s):  
Antoine Chabloz ◽  
Jonas V. Schaefer ◽  
Ivona Kozieradzki ◽  
Shane J. F. Cronin ◽  
Daniel Strebinger ◽  
...  
Keyword(s):  
Pathogenic Bacteria ◽  
Basic Research ◽  
Secretion System ◽  
Effector Proteins ◽  
Eukaryotic Cells ◽  
Ras Signaling ◽  
Avirulent Strain ◽  
Affinity Reagents ◽  
Efficient Delivery ◽  
Intracellular Targets

AbstractProtein-based affinity reagents (like antibodies or alternative binding scaffolds) offer wide-ranging applications for basic research and therapeutic approaches. However, whereas small chemical molecules efficiently reach intracellular targets, the delivery of macromolecules into the cytosol of cells remains a major challenge; thus cytosolic applications of protein-based reagents are rather limited. Some pathogenic bacteria have evolved a conserved type III secretion system (T3SS) which allows the delivery of effector proteins into eukaryotic cells. Here, we enhance the T3SS of an avirulent strain of Salmonella typhimurium to reproducibly deliver multiple classes of recombinant proteins into eukaryotic cells. The efficacy of the system is probed with both DARPins and monobodies to functionally inhibit the paradigmatic and largely undruggable RAS signaling pathway. Thus, we develop a bacterial secretion system for potent cytosolic delivery of therapeutic macromolecules.

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