scholarly journals LITESEC-T3SS - Light-controlled protein delivery into eukaryotic cells with high spatial and temporal resolution

2019 ◽  
Author(s):  
Florian Lindner ◽  
Bailey Milne-Davies ◽  
Katja Langenfeld ◽  
Andreas Diepold
Keyword(s):  
Temporal Resolution ◽  
Type Iii Secretion ◽  
Protein Delivery ◽  
Protein Translocation ◽  
Eukaryotic Cell ◽  
Host Cells ◽  
Eukaryotic Cells ◽  
Reporter Protein ◽  
Effector Secretion ◽  
Control Protein

AbstractMany bacteria employ a type III secretion system (T3SS), also called injectisome, to translocate proteins into eukaryotic host cells through a hollow extracellular needle. The system can efficiently transport heterologous cargo, which makes it a uniquely suited tool for the translocation of proteins into eukaryotic cells. However, the injectisome indiscriminately injects proteins into any adjoining eukaryotic cell, and this lack of target specificity currently limits its application in biotechnology and healthcare. In this study, we exploit the dynamic nature of the T3SS to control protein secretion and translocation into eukaryotic cells by light. By combining optogenetic interaction switches with the dynamic cytosolic T3SS component SctQ, the cytosolic availability of SctQ and in consequence T3SS-dependent effector secretion can be regulated by external light. The resulting system, which we call LITESEC-T3SS (Light-induced translocation of effectors through sequestration of endogenous components of the T3SS), allows rapid, specific, and reversible activation or deactivation of the T3SS upon illumination. We demonstrate the application of the system for light-regulated translocation of a heterologous reporter protein into cultured eukaryotic cells. LITESEC-T3SS represents a new method to achieve unparalleled spatial and temporal resolution for the controlled protein translocation into eukaryotic host cells.

scholarly journals Bacterial tail anchors can target to the mitochondrial outer membrane

2017 ◽  
Author(s):  
Güleycan Lutfullahoğlu Bal ◽  
Abdurrahman Keskin ◽  
Ayşe Bengisu Seferoğlu ◽  
Cory D. Dunn
Keyword(s):  
Outer Membrane ◽  
Protein Translocation ◽  
Mitochondrial Protein ◽  
Eukaryotic Cell ◽  
Eukaryotic Cells ◽  
Mitochondrial Outer Membrane ◽  
Bacterial Proteins ◽  
Rate Limiting Step ◽  
Protein Entry ◽  
Rate Limiting

ABSTRACTDuring the generation and evolution of the eukaryotic cell, a proteobacterial endosymbiont was refashioned into the mitochondrion, an organelle that appears to have been present in the ancestor of all present-day eukaryotes. Mitochondria harbor proteomes derived from coding information located both inside and outside the organelle, and the rate-limiting step toward the formation of eukaryotic cells may have been development of an import apparatus allowing protein entry to mitochondria. Currently, a widely conserved translocon allows proteins to pass from the cytosol into mitochondria, but how proteins encoded outside of mitochondria were first directed to these organelles at the dawn of eukaryogenesis is not clear. Because several proteins targeted by a carboxyl-terminal tail anchor (TA) appear to have the ability to insert spontaneously into the mitochondrial outer membrane (OM), it is possible that self-inserting, tail-anchored polypeptides obtained from bacteria might have formed the first gate allowing proteins to access mitochondria from the cytosol. Here, we tested whether bacterial TAs are capable of targeting to mitochondria. In a survey of proteins encoded by the proteobacterium Escherichia coli, predicted TA sequences were directed to specific subcellular locations within the yeast Saccharomyces cerevisiae. Importantly, TAs obtained from DUF883 family members ElaB and YqjD were abundantly localized to and inserted at the mitochondrial OM. Our results support the notion that eukaryotic cells are able to utilize membrane-targeting signals present in bacterial proteins obtained by lateral gene transfer, and our findings make plausible a model in which mitochondrial protein translocation was first driven by tail-anchored proteins.

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 EspZ of Enteropathogenic and Enterohemorrhagic Escherichia coli Regulates Type III Secretion System Protein Translocation

mBio ◽  
2012 ◽  
Vol 3 (5) ◽  
Author(s):  
Cedric N. Berger ◽  
Valerie F. Crepin ◽  
Kobi Baruch ◽  
Aurelie Mousnier ◽  
Ilan Rosenshine ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Type Iii Secretion ◽  
Protein Translocation ◽  
Type Iii Secretion System ◽  
Secretion System ◽  
Enterohemorrhagic Escherichia Coli ◽  
Host Cells ◽  
Dependent Manner ◽  
Content Type ◽  
Type Iii

ABSTRACTTranslocation of effector proteins via a type III secretion system (T3SS) is a widespread infection strategy among Gram-negative bacterial pathogens. Each pathogen translocates a particular set of effectors that subvert cell signaling in a way that suits its particular infection cycle. However, as effector unbalance might lead to cytotoxicity, the pathogens must employ mechanisms that regulate the intracellular effector concentration. We present evidence that the effector EspZ controls T3SS effector translocation from enteropathogenic (EPEC) and enterohemorrhagic (EHEC)Escherichia coli. Consistently, an EPECespZmutant is highly cytotoxic. Following ectopic expression, we found that EspZ inhibited the formation of actin pedestals as it blocked the translocation of Tir, as well as other effectors, including Map and EspF. Moreover, during infection EspZ inhibited effector translocation following superinfection. Importantly, while EspZ of EHEC O157:H7 had a universal “translocation stop” activity, EspZ of EPEC inhibited effector translocation from typical EPEC strains but not from EHEC O157:H7 or its progenitor, atypical EPEC O55:H7. We found that the N and C termini of EspZ, which contains two transmembrane domains, face the cytosolic leaflet of the plasma membrane at the site of bacterial attachment, while the extracellular loop of EspZ is responsible for its strain-specific activity. These results show that EPEC and EHEC acquired a sophisticated mechanism to regulate the effector translocation.IMPORTANCEEnteropathogenicEscherichia coli(EPEC) and enterohemorrhagicE. coli(EHEC) are important diarrheal pathogens responsible for significant morbidity and mortality in developing countries and the developed world, respectively. The virulence strategy of EPEC and EHEC revolves around a conserved type III secretion system (T3SS), which translocates bacterial proteins known as effectors directly into host cells. Previous studies have shown that when cells are infected in two waves with EPEC, the first wave inhibits effector translocation by the second wave in a T3SS-dependent manner, although the factor involved was not known. Importantly, we identified EspZ as the effector responsible for blocking protein translocation following a secondary EPEC infection. Interestingly, we found that while EspZ of EHEC can block protein translocation from both EPEC and EHEC strains, EPEC EspZ cannot block translocation from EHEC. These studies show that EPEC and EHEC employ a novel infection strategy to regulate T3SS translocation.

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 Salmonella Type III Secretion-Associated Protein InvE Controls Translocation of Effector Proteins into Host Cells

2002 ◽  
Vol 184 (17) ◽  
pp. 4699-4708 ◽  
Author(s):  
Tomoko Kubori ◽  
Jorge E. Galán
Keyword(s):  
Type Iii Secretion ◽  
Protein Translocation ◽  
Secretion System ◽  
Effector Proteins ◽  
Host Cells ◽  
Loss Of Function ◽  
Cellular Functions ◽  
Type Iii ◽  
Bacterial Proteins ◽  
Serovar Typhimurium

ABSTRACT Salmonella enterica encodes a type III secretion system (TTSS) within a pathogenicity island located at centisome 63 (SPI-1), which is essential for its pathogenicity. This system mediates the transfer of a battery of bacterial proteins into the host cell with the capacity to modulate cellular functions. The transfer process is dependent on the function of protein translocases SipB, SipC, and SipD. We report here that Salmonella protein InvE, which is also encoded within SPI-1, is essential for the translocation of bacterial proteins into host cells. An S. enterica serovar Typhimurium mutant carrying a loss-of-function mutation in invE shows reduced secretion of SipB, SipC, and SipD while exhibiting increased secretion of other TTSS effector proteins. We also demonstrate that InvE interacts with a protein complex formed by SipB, SipC, and their cognate chaperone, SicA. We propose that InvE controls protein translocation by regulating the function of the Sip protein translocases.

scholarly journals Delivery of Heterologous Proteins, Enzymes, and Antigens via the Bacterial Type III Secretion System

2020 ◽  
Vol 8 (5) ◽  
pp. 777 ◽  
Author(s):  
Heather A. Pendergrass ◽  
Aaron E. May
Keyword(s):  
Type Iii Secretion ◽  
Protein Delivery ◽  
Type Iii Secretion System ◽  
Secretion System ◽  
Host Cells ◽  
Heterologous Proteins ◽  
Communication Tool ◽  
Type Iii ◽  
Heterologous Protein Secretion ◽  
Multimeric Protein

The Type III Secretion System (T3SS) is a multimeric protein complex composed of over 20 different proteins, utilized by Gram-negative bacteria to infect eukaryotic host cells. The T3SS has been implicated as a virulence factor by which pathogens cause infection and has recently been characterized as a communication tool between bacteria and plant cells in the rhizosphere. The T3SS has been repurposed to be used as a tool for the delivery of non-native or heterologous proteins to eukaryotic cells or the extracellular space for a variety of purposes, including drug discovery and drug delivery. This review covers the methodology of heterologous protein secretion as well as multiple cases of utilizing the T3SS to deliver heterologous proteins or artificial materials. The research covered in this review will serve to outline the scope and limitations of utilizing the T3SS as a tool for protein delivery.

scholarly journals In Vitro Translocation Experiments with RxLR-Reporter Fusion Proteins of Avr1b from Phytophthora sojae and AVR3a from Phytophthora infestans Fail to Demonstrate Specific Autonomous Uptake in Plant and Animal Cells

2013 ◽  
Vol 26 (5) ◽  
pp. 528-536 ◽  
Author(s):  
Stephan Wawra ◽  
Armin Djamei ◽  
Isabell Albert ◽  
Thorsten Nürnberger ◽  
Regine Kahmann ◽  
...  
Keyword(s):  
Fusion Proteins ◽  
Fluorescent Protein ◽  
Phytophthora Sojae ◽  
Host Cells ◽  
Eukaryotic Cells ◽  
Large Set ◽  
Animal Cells ◽  
Reporter Protein ◽  
Rxlr Effectors ◽  
Translocation Experiments

Plant-pathogenic oomycetes have a large set of secreted effectors that can be translocated into their host cells during infection. One group of these effectors are the RxLR effectors for which it has been shown, in a few cases, that the RxLR motif is important for their translocation. It has been suggested that the RxLR-leader sequences alone are enough to translocate the respective effectors into eukaryotic cells through binding to surface-exposed phosphoinositol-3-phosphate. These conclusions were primary based on translocation experiments conducted with recombinant fusion proteins whereby the RxLR leader of RxLR effectors (i.e., Avr1b from Phytophthora sojae) were fused to the green fluorescent protein reporter-protein. However, we failed to observe specific cellular uptake for a comparable fusion protein where the RxLR leader of the P. infestans AVR3a was fused to monomeric red fluorescent protein. Therefore, we reexamined the ability of the reported P. sojae AVR1b RxLR leader to enter eukaryotic cells. Different relevant experiments were performed in three independent laboratories, using fluorescent reporter fusion constructs of AVR3a and Avr1b proteins in a side-by-side comparative study on plant tissue and human and animal cells. We report that we were unable to obtain conclusive evidence for specific RxLR-mediated translocation.

scholarly journals CesL Regulates Type III Secretion Substrate Specificity of the Enteropathogenic E. coli Injectisome

2021 ◽  
Vol 9 (5) ◽  
pp. 1047
Author(s):  
Miguel Díaz-Guerrero ◽  
Meztlli O. Gaytán ◽  
Eduardo Soto ◽  
Norma Espinosa ◽  
Elizabeth García-Gómez ◽  
...  
Keyword(s):  
Substrate Specificity ◽  
Type Iii Secretion ◽  
Pathogenic Bacteria ◽  
Effector Proteins ◽  
Host Cells ◽  
Target Cells ◽  
Remarkable Feature ◽  
Type Iii ◽  
Pairwise Interactions ◽  
Effector Secretion

The type III secretion system (T3SS) is a complex molecular device used by several pathogenic bacteria to translocate effector proteins directly into eukaryotic host cells. One remarkable feature of the T3SS is its ability to secrete different categories of proteins in a hierarchical manner, to ensure proper assembly and timely delivery of effectors into target cells. In enteropathogenic Escherichia coli, the substrate specificity switch from translocator to effector secretion is regulated by a gatekeeper complex composed of SepL, SepD, and CesL proteins. Here, we report a characterization of the CesL protein using biochemical and genetic approaches. We investigated discrepancies in the phenotype among different cesL deletion mutants and showed that CesL is indeed essential for translocator secretion and to prevent premature effector secretion. We also demonstrated that CesL engages in pairwise interactions with both SepL and SepD. Furthermore, while association of SepL to the membrane does not depended on CesL, the absence of any of the proteins forming the heterotrimeric complex compromised the intracellular stability of each component. In addition, we found that CesL interacts with the cytoplasmic domains of the export gate components EscU and EscV. We propose a mechanism for substrate secretion regulation governed by the SepL/SepD/CesL complex.

scholarly journals ExoS Controls the Cell Contact-Mediated Switch to Effector Secretion in Pseudomonas aeruginosa

2007 ◽  
Vol 190 (8) ◽  
pp. 2726-2738 ◽  
Author(s):  
Michelle Cisz ◽  
Pei-Chung Lee ◽  
Arne Rietsch
Keyword(s):  
Pseudomonas Aeruginosa ◽  
Plasma Membrane ◽  
Host Cell ◽  
Type Iii Secretion ◽  
Host Cells ◽  
Cell Contact ◽  
Cell Plasma Membrane ◽  
Type Iii ◽  
Cell Plasma ◽  
Effector Secretion

ABSTRACT Type III secretion is used by many gram-negative bacterial pathogens to directly deliver protein toxins (effectors) into targeted host cells. In all cases, secretion of effectors is triggered by host cell contact, although the mechanism is unclear. In Pseudomonas aeruginosa, expression of all type III secretion-related genes is up-regulated when secretion is triggered. We were able to visualize this process using a green fluorescent protein reporter system and to use it to monitor the ability of bacteria to trigger effector secretion on cell contact. Surprisingly, the action of one of the major type III secreted effectors, ExoS, prevented triggering of type III secretion by bacteria that subsequently attached to cells, suggesting that triggering of secretion is feedback regulated. Evidence is presented that translocation (secretion of effectors across the host cell plasma membrane) of ExoS is indeed self-regulated and that this inhibition of translocation can be achieved by either of its two enzymatic activities. The translocator proteins PopB, PopD, and PcrV are secreted via the type III secretion system and are required for pore formation and translocation of effectors across the host cell plasma membrane. Here we present data that secretion of translocators is in fact not controlled by calcium, implying that triggering of effector secretion on cell contact represents a switch in secretion specificity, rather than a triggering of secretion per se. The requirement for a host cell cofactor to control effector secretion may help explain the recently observed phenomenon of target cell specificity in both the Yersinia and P. aeruginosa type III secretion systems.

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