scholarly journals Molecular analysis of protein secretion through type III secretion system from enteropathogenic E. coli

2015 ◽  
Author(s):  
Αθηνά Πορτάλιου
Keyword(s):  
Molecular Analysis ◽  
Protein Secretion ◽  
Type Iii Secretion ◽  
Type Iii Secretion System ◽  
Secretion System ◽  
E Coli ◽  
Type Iii

Το σύστημα έκκρισης τύπου ΙΙΙ (Τ3SS) είναι ένα ευρεία διαδεδομένο σύστημα που χρησιμοποιείται από πολλά παθογόνα, κατά Gram αρνητικά βακτήρια. Το σύστημα τύπου ΙΙΙ, είναι μια εξειδικευμένη μικρο-μηχανή που χρησιμοποιείται για την μεταφορά των μολυσματικών παραγόντων του βακτηρίου από το κυτταρόπλασμα του κατευθείαν μέσα στο κύταρο ξεωιστή, διαπερνώντας τρεις μεμβρανικές δομές, δυο βακτηριακές και μία του ευκαρυώτη. Το σύστημα έκκρισης τύπου ΙΙΙ σχηματίζει μια δομή στο χώρο που μπορεί να παρομοιαστεί με βελόνα (injectisome). Για την δημιουργία και ενεργοποίηση του συστήματος περίπου 50 πρωτεΐνες πρέπει να συντονιστούν ώστε το σύστημα να απόκτηση τη σωστή διαμόρφωση στο χώρο και οι πρωτεΐνες που πρόκειται να εκκριθούν από αυτό να μεταφερθούν εκεί, η όλοι διαδικασία υπόκειται πολύπλοκο και σύνθετη έλεγχο από διάφορους παράγοντες σε διάφορα επίπεδα κατά το μονοπάτι εξόδου των πρωτεϊνών από το κύτταρο. Παρόλο που πάρα πολλές δομικές και βιοχημικές μελέτες έχουν συμβάλει στην κατανόηση και δομική ανάλυση του συστήματος, ελάχιστες πληροφορίες σχετικά με το μονοπάτι που ακολουθούν οι πρωτεΐνες με στόχο την έξοδό τους από το κύτταρο και την ρύθμιση αυτού είναι γνωστές. Βασικός στόχος της παρούσας διδακτορικής διατριβής είναι η κατανόηση και αποσαφήνιση του μονοπατιού που ακολουθούν οι πρωτεΐνες οι οποίες πρόκειται να εκκριθούν, κατά την μετατόπιση αυτών από το βακτηριακό κυτταρόπλασμα μέχρι την μεμβράνη, στον πόρο εξόδου του συστήματος έκκρισης τύπου ΙΙΙ. Μέλημά μας είναι ο εντοπισμός και χαρακτηρισμός των αλληλεπιδράσεων που συμβαίνουν ανάμεσα στις πρωτεΐνες του συστήματος και η χαρτογράφηση αυτών με στόχο την διασαφήνιση του μηχανισμού που ακολουθείται κατά την στόχευση των πρωτεΐνων στην μεμβράνη.

scholarly journals An optimized growth medium for increased recombinant protein secretion titer via the type III secretion system

2021 ◽  
Vol 20 (1) ◽  
Author(s):  
Lisa Ann Burdette ◽  
Han Teng Wong ◽  
Danielle Tullman-Ercek
Keyword(s):  
Protein Secretion ◽  
Salt Concentration ◽  
Type Iii Secretion ◽  
Heterologous Protein ◽  
Carbon Sources ◽  
Type Iii Secretion System ◽  
Secretion System ◽  
Type Iii ◽  
Heterologous Protein Secretion ◽  
Extracellular Environment

Abstract Background Protein secretion in bacteria is an attractive strategy for heterologous protein production because it retains the high titers and tractability of bacterial hosts while simplifying downstream processing. Traditional intracellular production strategies require cell lysis and separation of the protein product from the chemically similar cellular contents, often a multi-step process that can include an expensive refolding step. The type III secretion system of Salmonella enterica Typhimurium transports proteins from the cytoplasm to the extracellular environment in a single step and is thus a promising solution for protein secretion in bacteria. Product titer is sensitive to extracellular environmental conditions, however, and T3SS regulation is integrated with essential cellular functions. Instead of attempting to untangle a complex web of regulatory input, we took an “outside-in” approach to elucidate the effect of growth medium components on secretion titer. Results We dissected the individual and combined effects of carbon sources, buffers, and salts in a rich nutrient base on secretion titer. Carbon sources alone decreased secretion titer, secretion titer increased with salt concentration, and the combination of a carbon source, buffer, and high salt concentration had a synergistic effect on secretion titer. Transcriptional activity measured by flow cytometry showed that medium composition affected secretion system activity, and prolonged secretion system activation correlated strongly with increased secretion titer. We found that an optimal combination of glycerol, phosphate, and sodium chloride provided at least a fourfold increase in secretion titer for a variety of proteins. Further, the increase in secretion titer provided by the optimized medium was additive with strain enhancements. Conclusions We leveraged the sensitivity of the type III secretion system to the extracellular environment to increase heterologous protein secretion titer. Our results suggest that maximizing secretion titer via the type III secretion system is not as simple as maximizing secreted protein expression—one must also optimize secretion system activity. This work advances the type III secretion system as a platform for heterologous protein secretion in bacteria and will form a basis for future engineering efforts.

scholarly journals Escherichia coli type III secretion system 2 (ETT2) is widely distributed in avian pathogenic Escherichia coli isolates from Eastern China

2016 ◽  
Vol 144 (13) ◽  
pp. 2824-2830 ◽  
Author(s):  
S. WANG ◽  
X. LIU ◽  
X. XU ◽  
Y. ZHAO ◽  
D. YANG ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Type Iii Secretion ◽  
Bacterial Infections ◽  
Eastern China ◽  
Type Iii Secretion System ◽  
Secretion System ◽  
Secretion Systems ◽  
E Coli ◽  
Type Iii ◽  
System 2

SUMMARYPathogens utilize type III secretion systems to deliver effector proteins, which facilitate bacterial infections. The Escherichia coli type III secretion system 2 (ETT2) which plays a crucial role in bacterial virulence, is present in the majority of E. coli strains, although ETT2 has undergone widespread mutational attrition. We investigated the distribution and characteristics of ETT2 in avian pathogenic E. coli (APEC) isolates and identified five different ETT2 isoforms, including intact ETT2, in 57·6% (141/245) of the isolates. The ETT2 locus was present in the predominant APEC serotypes O78, O2 and O1. All of the ETT2 loci in the serotype O78 isolates were degenerate, whereas an intact ETT2 locus was mostly present in O1 and O2 serotype strains, which belong to phylogenetic groups B2 and D, respectively. Interestingly, a putative second type III secretion-associated locus (eip locus) was present only in the isolates with an intact ETT2. Moreover, ETT2 was more widely distributed in APEC isolates and exhibited more isoforms compared to ETT2 in human extraintestinal pathogenic E. coli, suggesting that APEC might be a potential risk to human health. However, there was no distinct correlation between ETT2 and other virulence factors in APEC.

scholarly journals A Degenerate Type III Secretion System from Septicemic Escherichia coli Contributes to Pathogenesis

2005 ◽  
Vol 187 (23) ◽  
pp. 8164-8171 ◽  
Author(s):  
Diana Ideses ◽  
Uri Gophna ◽  
Yossi Paitan ◽  
Roy R. Chaudhuri ◽  
Mark J. Pallen ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Gene Cluster ◽  
Type Iii Secretion ◽  
Type Iii Secretion System ◽  
Gene Clusters ◽  
Secretion System ◽  
Effector Proteins ◽  
E Coli ◽  
Type Iii

ABSTRACT The type III secretion system (T3SS) is an important virulence factor used by several gram-negative bacteria to deliver effector proteins which subvert host cellular processes. Enterohemorrhagic Escherichia coli O157 has a well-defined T3SS involved in attachment and effacement (ETT1) and critical for virulence. A gene cluster potentially encoding an additional T3SS (ETT2), which resembles the SPI-1 system in Salmonella enterica, was found in its genome sequence. The ETT2 gene cluster has since been found in many E. coli strains, but its in vivo role is not known. Many of the ETT2 gene clusters carry mutations and deletions, raising the possibility that they are not functional. Here we show the existence in septicemic E. coli strains of an ETT2 gene cluster, ETT2sepsis, which, although degenerate, contributes to pathogenesis. ETT2sepsis has several premature stop codons and a large (5 kb) deletion, which is conserved in 11 E. coli strains from cases of septicemia and newborn meningitis. A null mutant constructed to remove genes coding for the putative inner membrane ring of the secretion complex exhibited significantly reduced virulence. These results are the first demonstration of the importance of ETT2 for pathogenesis.

scholarly journals Dynamic relocalization of the cytosolic type III secretion system components prevents premature protein secretion at low external pH

2019 ◽  
Author(s):  
Stephan Wimmi ◽  
Alexander Balinovic ◽  
Hannah Jeckel ◽  
Lisa Selinger ◽  
Dimitrios Lampaki ◽  
...  
Keyword(s):  
Protein Secretion ◽  
Type Iii Secretion ◽  
Type Iii Secretion System ◽  
Secretion System ◽  
Host Cells ◽  
Type Iii ◽  
Acidic Environments ◽  
Fast Activation ◽  
Partial Dissociation ◽  
External Ph

AbstractMany bacterial pathogens use a type III secretion system (T3SS) to manipulate host cells. Protein secretion by the T3SS injectisome is activated upon contact to any host cell, and it has been unclear how premature secretion is prevented during infection. We found that in gastrointestinal pathogens, cytosolic injectisome components are temporarily released from the proximal interface of the injectisome at low external pH, preventing protein secretion in acidic environments, such as the stomach. In Yersinia enterocolitica, low external pH is detected in the periplasm and leads to a partial dissociation of the inner membrane injectisome component SctD, which in turn causes the dissociation of the cytosolic T3SS components. This effect is reversed upon restoration of neutral pH, allowing a fast activation of the T3SS at the native target regions within the host. These findings indicate that the cytosolic components form an adaptive regulatory interface, which regulates T3SS activity in response to environmental conditions.

scholarly journals Enterococcus faecalis Enhances Expression and Activity of the Enterohemorrhagic Escherichia coli Type III Secretion System

mBio ◽  
2019 ◽  
Vol 10 (6) ◽  
Author(s):  
Elizabeth A. Cameron ◽  
Vanessa Sperandio ◽  
Gary M. Dunny
Keyword(s):  
Enterococcus Faecalis ◽  
Type Iii Secretion ◽  
Type Iii Secretion System ◽  
Secretion System ◽  
Host Cells ◽  
Cell Contact ◽  
Content Type ◽  
E Coli ◽  
Type Iii ◽  
Expression And Activity

ABSTRACT The gut microbiota can significantly impact invading pathogens and the disease they cause; however, many of the mechanisms that dictate commensal-pathogen interactions remain unclear. Enterohemorrhagic Escherichia coli (EHEC) is a potentially lethal human intestinal pathogen that uses microbiota-derived molecules as cues to efficiently regulate virulence factor expression. Here, we investigate the interaction between EHEC and Enterococcus faecalis, a common human gut commensal, and show that E. faecalis affects both expression and activity of the EHEC type III secretion system (T3SS) via two distinct mechanisms. First, in the presence of E. faecalis there is increased transcription of genes encoding the EHEC T3SS. This leads to increased effector translocation and ultimately greater numbers of pedestals formed on host cells. The same effect was observed with several strains of enterococci, suggesting that it is a general characteristic of this group. In a mechanism separate from E. faecalis-induced transcription of the T3SS, we report that an E. faecalis-secreted protease, GelE, cleaves a critical structural component of the EHEC T3SS, EspB. Our data suggest that this cleavage actually increases effector translocation by the T3SS, supporting a model where EspB proteolysis promotes maximum T3SS activity. Finally, we report that treatment of EHEC with E. faecalis-conditioned cell-free medium is insufficient to induce increased T3SS expression, suggesting that this effect relies on cell contact between E. faecalis and EHEC. This work demonstrates a complex interaction between a human commensal and pathogen that impacts both expression and function of a critical virulence factor. IMPORTANCE This work reveals a complex and multifaceted interaction between a human gut commensal, Enterococcus faecalis, and a pathogen, enterohemorrhagic E. coli. We demonstrate that E. faecalis enhances expression of the enterohemorrhagic E. coli type III secretion system and that this effect likely depends on cell contact between the commensal and the pathogen. Additionally, the GelE protease secreted by E. faecalis cleaves a critical structural component of the EHEC type III secretion system. In agreement with previous studies, we find that this cleavage actually increases effector protein delivery into host cells by the secretion system. This work demonstrates that commensal bacteria can significantly shape expression and activity of pathogen virulence factors, which may ultimately shape the progression of disease.

scholarly journals The ETT2 Gene Cluster, Encoding a Second Type III Secretion System from Escherichia coli, Is Present in the Majority of Strains but Has Undergone Widespread Mutational Attrition

2004 ◽  
Vol 186 (11) ◽  
pp. 3547-3560 ◽  
Author(s):  
Chuan-Peng Ren ◽  
Roy R. Chaudhuri ◽  
Amanda Fivian ◽  
Christopher M. Bailey ◽  
Martin Antonio ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Gene Cluster ◽  
Type Iii Secretion ◽  
Type Iii Secretion System ◽  
Secretion System ◽  
Genome Sequences ◽  
Phylogenetic Structure ◽  
E Coli ◽  
Type Iii ◽  
K 12

ABSTRACT ETT2 is a second cryptic type III secretion system in Escherichia coli which was first discovered through the analysis of genome sequences of enterohemorrhagic E. coli O157:H7. Comparative analyses of Escherichia and Shigella genome sequences revealed that the ETT2 gene cluster is larger than was previously thought, encompassing homologues of genes from the Spi-1, Spi-2, and Spi-3 Salmonella pathogenicity islands. ETT2-associated genes, including regulators and chaperones, were found at the same chromosomal location in the majority of genome-sequenced strains, including the laboratory strain K-12. Using a PCR-based approach, we constructed a complete tiling path through the ETT2 gene cluster for 79 strains, including the well-characterized E. coli reference collection supplemented with additional pathotypes. The ETT2 gene cluster was found to be present in whole or in part in the majority of E. coli strains, whether pathogenic or commensal, with patterns of distribution and deletion mirroring the known phylogenetic structure of the species. In almost all strains, including enterohemorrhagic E. coli O157:H7, ETT2 has been subjected to varying degrees of mutational attrition that render it unable to encode a functioning secretion system. A second type III secretion system-associated locus that likely encodes the ETT2 translocation apparatus was found in some E. coli strains. Intact versions of both ETT2-related clusters are apparently present in enteroaggregative E. coli strain O42.

scholarly journals Identification of translocation inhibitors targeting the type III secretion system of enteropathogenic E. coli

2021 ◽  
Author(s):  
Sabrina Mühlen ◽  
Viktor Zapolskii ◽  
Ursula Bilitewski ◽  
Petra Dersch
Keyword(s):  
Type Iii Secretion ◽  
Type Iii Secretion System ◽  
Secretion System ◽  
Effector Proteins ◽  
Host Cells ◽  
Immune Recognition ◽  
E Coli ◽  
Type Iii ◽  
Compound Libraries ◽  
High Throughput Screening Assay

Infections with enteropathogenic E. coli (EPEC) cause severe diarrhea in children. The non-invasive bacteria adhere to enterocytes of the small intestine and use a type III secretion system (T3SS) to inject effector proteins into host cells to modify and exploit cellular processes in favor of bacterial survival and replication. Several studies have shown that the T3SSs of bacterial pathogens are essential for virulence. Furthermore, the loss of T3SS-mediated effector translocation results in increased immune recognition and clearance of the bacteria. The T3SS is, therefore, considered a promising target for antivirulence strategies and novel therapeutics development. Here, we report the results of a high-throughput screening assay based on the translocation of the EPEC effector protein Tir. Using this assay, we screened more than 13,000 small molecular compounds of six different compound libraries and identified three substances which showed a significant dose-dependent effect on translocation without adverse effects on bacterial or eukaryotic cell viability. Additionally, these substances reduced bacterial binding to host cells, effector-dependent cell detachment and abolished A/E lesion formation without affecting the expression of components of the T3SS or associated effector proteins. Moreover, no effects of the inhibitors on bacterial motility or Shiga-toxin expression were observed. In summary, we have identified three new compounds that strongly inhibit T3SS-mediated translocation of effectors into mammalian cells, which could be valuable as lead substances for treating EPEC and EHEC infections.

scholarly journals Characterization of Nops, Nodulation Outer Proteins, Secreted Via the Type III Secretion System of NGR234

2003 ◽  
Vol 16 (9) ◽  
pp. 743-751 ◽  
Author(s):  
Corinne Marie ◽  
William J. Deakin ◽  
Virginie Viprey ◽  
Joanna Kopciñska ◽  
Wladyslaw Golinowski ◽  
...  
Keyword(s):  
Protein Secretion ◽  
Type Iii Secretion ◽  
Type Iii Secretion System ◽  
Symbiotic Bacterium ◽  
Secretion System ◽  
Nodule Development ◽  
Nodule Formation ◽  
Dependent Manner ◽  
Double Mutation ◽  
Type Iii

The nitrogen-fixing symbiotic bacterium Rhizobium species NGR234 secretes, via a type III secretion system (TTSS), proteins called Nops (nodulation outer proteins). Abolition of TTSS-dependent protein secretion has either no effect or leads to a change in the number of nodules on selected plants. More dramatically, Nops impair nodule development on Crotalaria juncea roots, resulting in the formation of nonfixing pseudonodules. A double mutation of nopX and nopL, which code for two previously identified secreted proteins, leads to a phenotype on Pachyrhizus tuberosus differing from that of a mutant in which the TTSS is not functional. Use of antibodies and a modification of the purification protocol revealed that NGR234 secretes additional proteins in a TTSS-dependent manner. One of them was identified as NopA, a small 7-kDa protein. Single mutations in nopX and nopL were also generated to assess the involvement of each Nop in protein secretion and nodule formation. Mutation of nopX had little effect on NopL and NopA secretion but greatly affected the interaction of NGR234 with many plant hosts tested. NopL was not necessary for the secretion of any Nops but was required for efficient nodulation of some plant species. NopL may thus act as an effector protein whose recognition is dependent upon the hosts' genetic background.

scholarly journals An Optimized Growth Medium for Increased Recombinant Protein Secretion Titer via the Type III Secretion System

2020 ◽  
Author(s):  
Lisa Burdette ◽  
Han Teng Wong ◽  
Danielle Tullman-Ercek
Keyword(s):  
Protein Secretion ◽  
Type Iii Secretion ◽  
Heterologous Protein ◽  
Carbon Sources ◽  
Type Iii Secretion System ◽  
Secretion System ◽  
Medium Composition ◽  
Type Iii ◽  
Heterologous Protein Secretion ◽  
Extracellular Environment

Abstract BackgroundProtein secretion in bacteria is an attractive strategy for heterologous protein production because it retains the high titers and tractability of bacterial hosts while simplifying downstream processing. Traditional intracellular production strategies require cell lysis and separation of the protein product from the chemically similar cellular contents, often a multi-step process that can include an expensive refolding step. The type III secretion system of Salmonella enterica transports proteins from the cytoplasm to the extracellular environment in a single step and is thus a promising solution for protein secretion in bacteria. Product titer is sensitive to extracellular environmental conditions, however, and is therefore not robust. We investigated growth medium composition to provide a favorable environment for secretion that produces consistently high secretion titers, advancing the type III secretion system as a heterologous protein production platform.ResultsWe investigated the effect of carbon sources, buffers, and salts in a rich nutrient base on secretion titer. Carbon sources alone decreased secretion titer, secretion titer increased with salt concentration, and the combination of a carbon source, buffer, and high salt concentration had a synergistic effect on secretion titer. Transcriptional activity measured by flow cytometry showed that medium composition affected secretion system activity, and prolonged secretion system activation correlated strongly with increased secretion titer. We found that an optimal combination of glycerol, phosphate, and sodium chloride provided at least a fourfold increase in secretion titer for a variety of proteins. Further, the increase in secretion titer provided by the optimized medium was additive with strain enhancements.ConclusionsWe leveraged the sensitivity of the type III secretion system to the extracellular environment to increase heterologous protein secretion titer. Our results suggest that maximizing secretion titer via the type III secretion system is not as simple as maximizing secreted protein expression—one must also optimize secretion system activity. This work advances the type III secretion system as a platform for heterologous protein secretion in bacteria and will form a basis for future engineering efforts.

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