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.

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 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 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 Analysis of Erwinia chrysanthemi EC16 pelE∷uidA, pelL∷uidA, and hrpN∷uidA Mutants Reveals Strain-Specific Atypical Regulation of the Hrp Type III Secretion System

2004 ◽  
Vol 17 (2) ◽  
pp. 184-194 ◽  
Author(s):  
Jong Hyun Ham ◽  
Yaya Cui ◽  
James R. Alfano ◽  
Pablo Rodríguez-Palenzuela ◽  
Clemencia M. Rojas ◽  
...  
Keyword(s):  
Growth Phase ◽  
Type Iii Secretion ◽  
Type Iii Secretion System ◽  
Secretion System ◽  
Medium Composition ◽  
Erwinia Chrysanthemi ◽  
Type Ii ◽  
Logarithmic Growth Phase ◽  
Direct Production ◽  
Type Iii

The plant pathogen Erwinia chrysanthemi produces a variety of factors that have been implicated in its ability to cause soft-rot diseases in various hosts. These include HrpN, a harpin secreted by the Hrp type III secretion system; PelE, one of several major pectate lyase isozymes secreted by the type II system; and PelL, one of several secondary Pels secreted by the type II system. We investigated these factors in E. chrysanthemi EC16 with respect to the effects of medium composition and growth phase on gene expression (as determined with uidA fusions and Northern analyses) and effects on virulence. pelE was induced by polygalacturonic acid, but pelL was not, and hrpN was expressed unexpectedly in nutrient-rich King's medium B and in minimal salts medium at neutral pH. In contrast, the effect of medium composition on hrp expression in E. chrysanthemi CUCPB1237 and 3937 was like that of many other phytopathogenic bacteria in being repressed in complex media and induced in acidic pH minimal medium. Northern blot analysis of hrpN and hrpL expression by the wild-type and hrpL∷ΩCmr and hrpS∷ΩCmr mutants revealed that hrpN expression was dependent on the HrpL alternative sigma factor, whose expression, in turn, was dependent on the HrpS putative σ54 enhancer binding protein. The expression of pelE and hrpN increased strongly in late logarithmic growth phase. To test the possible role of quorum sensing in this expression pattern, the expI/expR locus was cloned in Escherichia coli on the basis of its ability to direct production of acyl-homoserine lactone and then used to construct expI mutations in pelE∷uidA, pelL∷uidA, and hrpN∷uidA Erwinia chrysanthemi strains. Mutation of expI had no apparent effect on the growth-phase-dependent expression of hrpN and pelE, or on the virulence of E. chrysanthemi in witloof chicory leaves. Overexpression of hrpN in E. chrysanthemi resulted in approximately 50% reduction of lesion size on chicory leaves without an effect on infection initiation.

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 A Novel Periplasmic Protein, VrpA, Contributes to Efficient Protein Secretion by the Type III Secretion System in Xanthomonas spp.

2015 ◽  
Vol 28 (2) ◽  
pp. 143-153 ◽  
Author(s):  
Xiaofeng Zhou ◽  
Xiufang Hu ◽  
Jinyun Li ◽  
Nian Wang
Keyword(s):  
Protein Secretion ◽  
Type Iii Secretion ◽  
Type Iii Secretion System ◽  
Secretion System ◽  
Effector Proteins ◽  
Periplasmic Protein ◽  
Transcription Activator ◽  
Type Iii Effector ◽  
Type Iii

Efficient secretion of type III effector proteins from the bacterial cytoplasm to host cell cytosol via a type III secretion system (T3SS) is crucial for virulence of plant-pathogenic bacterium. Our previous study revealed a conserved hypothetical protein, virulence-related periplasm protein A (VrpA), which was identified as a critical virulence factor for Xanthomonas citri subsp. citri. In this study, we demonstrate that mutation of vrpA compromises X. citri subsp. citri virulence and hypersensitive response induction. This deficiency is also observed in the X. campestris pv. campestris strain, suggesting a functional conservation of VrpA in Xanthomonas spp. Our study indicates that VrpA is required for efficient protein secretion via T3SS, which is supported by multiple lines of evidence. A CyaA reporter assay shows that VrpA is involved in type III effector secretion; quantitative reverse-transcription polymerase chain reaction analysis suggests that the vrpA mutant fails to activate citrus-canker-susceptible gene CsLOB1, which is transcriptionally activated by transcription activator-like effector PthA4; in vitro secretion study reveals that VrpA plays an important role in secretion of T3SS pilus, translocon, and effector proteins. Our data also indicate that VrpA in X. citri subsp. citri localizes to bacterial periplasmic space and the periplasmic localization is required for full function of VrpA and X. citri subsp. citri virulence. Protein–protein interaction studies show that VrpA physically interacts with periplasmic T3SS components HrcJ and HrcC. However, the mutation of VrpA does not affect T3SS gene expression. Additionally, VrpA is involved in X. citri subsp. citri tolerance of oxidative stress. Our data contribute to the mechanical understanding of an important periplasmic protein VrpA in Xanthomonas spp.

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.

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