scholarly journals Genetic code expansion enables visualization of Salmonella type three secretion system components and secreted effectors

eLife ◽  
2021 ◽  
Vol 10 ◽  
Author(s):  
Moirangthem Kiran Singh ◽  
Parisa Zangoui ◽  
Yuki Yamanaka ◽  
Linda J Kenney
Keyword(s):  
Genetic Code ◽  
Unnatural Amino Acids ◽  
Click Reaction ◽  
Host Cells ◽  
Secretion Systems ◽  
Cellular Functions ◽  
Small Proteins ◽  
Type Three Secretion System ◽  
Genetic Code Expansion ◽  
Type Three Secretion

Type three secretion systems enable bacterial pathogens to inject effectors into the cytosol of eukaryotic hosts to reprogram cellular functions. It is technically challenging to label effectors and the secretion machinery without disrupting their structure/function. Herein, we present a new approach for labeling and visualization of previously intractable targets. Using genetic code expansion, we site-specifically labeled SsaP, the substrate specificity switch, and SifA, a here-to-fore unlabeled secreted effector. SsaP was secreted at later infection times; SsaP labeling demonstrated the stochasticity of injectisome and effector expression. SifA was labeled after secretion into host cells via fluorescent unnatural amino acids or non-fluorescent labels and a subsequent click reaction. We demonstrate the superiority of imaging after genetic code expansion compared to small molecule tags. It provides an alternative for labeling proteins that do not tolerate N- or C-terminal tags or fluorophores and thus is widely applicable to other secreted effectors and small proteins.

scholarly journals Transferability of N-terminal mutations of pyrrolysyl-tRNA synthetase in one species to that in another species on unnatural amino acid incorporation efficiency

Amino Acids ◽  
2020 ◽  
Author(s):  
Thomas L. Williams ◽  
Debra J. Iskandar ◽  
Alexander R. Nödling ◽  
Yurong Tan ◽  
Louis Y. P. Luk ◽  
...  
Keyword(s):  
Amino Acids ◽  
Amino Acid ◽  
Genetic Code ◽  
Unnatural Amino Acids ◽  
Trna Synthetase ◽  
Unnatural Amino Acid ◽  
Amino Acid Incorporation ◽  
Acid Incorporation ◽  
Genetic Code Expansion ◽  
Incorporation Efficiency

AbstractGenetic code expansion is a powerful technique for site-specific incorporation of an unnatural amino acid into a protein of interest. This technique relies on an orthogonal aminoacyl-tRNA synthetase/tRNA pair and has enabled incorporation of over 100 different unnatural amino acids into ribosomally synthesized proteins in cells. Pyrrolysyl-tRNA synthetase (PylRS) and its cognate tRNA from Methanosarcina species are arguably the most widely used orthogonal pair. Here, we investigated whether beneficial effect in unnatural amino acid incorporation caused by N-terminal mutations in PylRS of one species is transferable to PylRS of another species. It was shown that conserved mutations on the N-terminal domain of MmPylRS improved the unnatural amino acid incorporation efficiency up to five folds. As MbPylRS shares high sequence identity to MmPylRS, and the two homologs are often used interchangeably, we examined incorporation of five unnatural amino acids by four MbPylRS variants at two temperatures. Our results indicate that the beneficial N-terminal mutations in MmPylRS did not improve unnatural amino acid incorporation efficiency by MbPylRS. Knowledge from this work contributes to our understanding of PylRS homologs which are needed to improve the technique of genetic code expansion in the future.

scholarly journals Chemical Inhibitors of the Type Three Secretion System: Disarming Bacterial Pathogens

2012 ◽  
Vol 56 (11) ◽  
pp. 5433-5441 ◽  
Author(s):  
Miles C. Duncan ◽  
Roger G. Linington ◽  
Victoria Auerbuch
Keyword(s):  
Bacterial Infections ◽  
Pathogenic Bacteria ◽  
Bacterial Pathogens ◽  
Secretion System ◽  
Effector Proteins ◽  
Host Cells ◽  
Magic Bullet ◽  
Antimicrobial Drugs ◽  
Type Three Secretion System ◽  
Type Three Secretion

ABSTRACTThe recent and dramatic rise of antibiotic resistance among bacterial pathogens underlies the fear that standard treatments for infectious disease will soon be largely ineffective. Resistance has evolved against nearly every clinically used antibiotic, and in the near future, we may be hard-pressed to treat bacterial infections previously conquered by “magic bullet” drugs. While traditional antibiotics kill or slow bacterial growth, an important emerging strategy to combat pathogens seeks to block the ability of bacteria to harm the host by inhibiting bacterial virulence factors. One such virulence factor, the type three secretion system (T3SS), is found in over two dozen Gram-negative pathogens and functions by injecting effector proteins directly into the cytosol of host cells. Without T3SSs, many pathogenic bacteria are unable to cause disease, making the T3SS an attractive target for novel antimicrobial drugs. Interdisciplinary efforts between chemists and microbiologists have yielded several T3SS inhibitors, including the relatively well-studied salicylidene acylhydrazides. This review highlights the discovery and characterization of T3SS inhibitors in the primary literature over the past 10 years and discusses the future of these drugs as both research tools and a new class of therapeutic agents.

scholarly journals Genetic Code Expansion of Vibrio natriegens

2021 ◽  
Vol 9 ◽  
Author(s):  
Eden Ozer ◽  
Lital Alfonta
Keyword(s):  
Genetic Code ◽  
Unnatural Amino Acids ◽  
Unnatural Amino Acid ◽  
Negative Bacterium ◽  
Amino Acid Incorporation ◽  
Gram Negative ◽  
E Coli ◽  
Acid Incorporation ◽  
Genetic Code Expansion ◽  
First Time

Escherichia coli has been considered as the most used model bacteria in the majority of studies for several decades. However, a new, faster chassis for synthetic biology is emerging in the form of the fast-growing gram-negative bacterium Vibrio natriegens. Different methodologies, well established in E. coli, are currently being adapted for V. natriegens in the hope to enable a much faster platform for general molecular biology studies. Amongst the vast technologies available for E. coli, genetic code expansion, the incorporation of unnatural amino acids into proteins, serves as a robust tool for protein engineering and biorthogonal modifications. Here we designed and adapted the genetic code expansion methodology for V. natriegens and demonstrate an unnatural amino acid incorporation into a protein for the first time in this organism.

scholarly journals Simplified Methodology for a Modular and Genetically Expanded Protein Synthesis in Cell-Free Systems

2019 ◽  
Author(s):  
Yonatan Chemla ◽  
Eden Ozer ◽  
Michael Shaferman ◽  
Ben Zaad ◽  
Rambabu Dandela ◽  
...  
Keyword(s):  
Amino Acids ◽  
Genetic Code ◽  
Unnatural Amino Acids ◽  
High Efficiency ◽  
Living Cells ◽  
Trna Synthetase ◽  
Complex Nature ◽  
Reporter Protein ◽  
A Cell ◽  
Genetic Code Expansion

ABSTRACTGenetic code expansion, which enables the site-specific incorporation of unnatural amino acids into proteins, has emerged as a new and powerful tool for protein engineering. Currently, it is mainly utilized inside living cells for a myriad of applications. However, utilization of this technology in a cell-free, reconstituted platform has several advantages over living systems. The common limitations to the employment of these systems are the laborious and complex nature of its preparation and utilization. Herein, we describe a simplified method for the preparation of this system from Escherichia coli cells, which is specifically adapted for the expression of the components needed for cell-free genetic code expansion. In addition, we propose and demonstrate a modular approach to its utilization. By this approach, it is possible to prepare and store different extracts, harboring various translational components, and mix and match them as needed for more than four years retaining its high efficiency. We demonstrate this with the simultaneous incorporation of two different unnatural amino acids into a reporter protein. Finally, we demonstrate the advantage of cell-free systems over living cells for the incorporation of δ-thio-boc-lysine into ubiquitin by using the methanosarcina mazei wild-type pyrrolysyl tRNACUA and tRNA-synthetase pair, which can not be achieved in a living cell.

scholarly journals Sterilization of drug-resistant influenza virus through genetic interference: use of unnatural amino acid-engineered virions

2021 ◽  
Author(s):  
Xuesheng Wu ◽  
Zhetao Zheng ◽  
Hongmin Chen ◽  
Haishuang Lin ◽  
Yuelin Yang ◽  
...  
Keyword(s):  
Influenza Virus ◽  
Amino Acid ◽  
Genetic Code ◽  
Unnatural Amino Acids ◽  
Influenza A ◽  
Antiviral Agent ◽  
Unnatural Amino Acid ◽  
Drug Resistant ◽  
Genetic Code Expansion

AbstractThe frequent emergence of drug resistance during the treatment of influenza A virus (IAV) infections highlights a need for effective antiviral countermeasures. Here, we present an antiviral method that utilizes unnatural amino acid-engineered drug-resistant (UAA-DR) virus. The engineered virus is generated through genetic code expansion to combat emerging drug-resistant viruses. The UAA-DR virus has unnatural amino acids incorporated into its drug-resistant protein and its polymerase complex for replication control. The engineered virus can undergo genomic segment reassortment with normal virus and produce sterilized progenies due to artificial amber codons in the viral genome. We validate in vitro that UAA-DR can provide a broad-spectrum antiviral strategy for several H1N1 strains, different DR-IAV strains, multidrug-resistant (MDR) strains, and even antigenically distant influenza strains (e.g., H3N2). Moreover, a minimum dose of neuraminidase (NA) inhibitors for influenza virus can further enhance the sterilizing effect when combating inhibitor-resistant strains, partly due to the promoted superinfection of unnatural amino acid-modified virus in cellular and animal models. We also exploited the engineered virus to achieve adjustable efficacy after external UAA administration, for mitigating DR virus infection on transgenic mice harboring the pair, and to have substantial elements of the genetic code expansion technology, which further demonstrated the safety and feasibility of the strategy. We anticipate that the use of the UAA-engineered DR virion, which is a novel antiviral agent, could be extended to combat emerging drug-resistant influenza virus and other segmented RNA viruses.

scholarly journals Role for the Burkholderia pseudomallei Type Three Secretion System Cluster 1bpscNGene in Virulence

2011 ◽  
Vol 79 (9) ◽  
pp. 3659-3664 ◽  
Author(s):  
Tanya D'Cruze ◽  
Lan Gong ◽  
Puthayalai Treerat ◽  
Georg Ramm ◽  
John D. Boyce ◽  
...  
Keyword(s):  
Mammalian Cells ◽  
Burkholderia Pseudomallei ◽  
Gene Clusters ◽  
Secretion System ◽  
Host Cells ◽  
Infection Model ◽  
Content Type ◽  
Type Three Secretion System ◽  
Type Three Secretion

ABSTRACTBurkholderia pseudomallei, the causal agent of melioidosis, employs a number of virulence factors during its infection of mammalian cells. One such factor is the type three secretion system (TTSS), which is proposed to mediate the transport and secretion of bacterial effector molecules directly into host cells. TheB. pseudomalleigenome contains three TTSS gene clusters (designated TTSS1, TTSS2, and TTSS3). Previous research has indicated that neither TTSS1 nor TTSS2 is involved inB. pseudomalleivirulence in a hamster infection model. We have characterized aB. pseudomalleimutant lacking expression of the predicted TTSS1 ATPase encoded bybpscN. This mutant was significantly attenuated for virulence in a respiratory melioidosis mouse model of infection. In addition, analysesin vitroshowed diminished survival and replication in RAW264.7 cells and an increased level of colocalization with the autophagy marker protein LC3 but an unhindered ability to escape from phagosomes. Taken together, these data provide evidence that the TTSS1bpscNgene product plays an important role in the intracellular survival ofB. pseudomalleiand the pathogenesis of murine infection.

scholarly journals Single-molecule FRET and conformational analysis of beta-arrestin-1 through genetic code expansion and Se-Click reaction

2021 ◽  
Author(s):  
Ming-Jie Han ◽  
Qing-Tao He ◽  
Mengyi Yang ◽  
Chao Chen ◽  
Yirong Yao ◽  
...  
Keyword(s):  
Energy Transfer ◽  
Conformational Analysis ◽  
Genetic Code ◽  
Single Molecule ◽  
Click Reaction ◽  
Dynamic Properties ◽  
Resonance Energy Transfer ◽  
Resonance Energy ◽  
Single Molecule Fret ◽  
Genetic Code Expansion

Single-molecule Förster resonance energy transfer (smFRET) is a powerful tool for investigating the dynamic properties of biomacromolecules. However, the success of protein smFRET relies on the precise and efficient labeling...

scholarly journals Using genetically incorporated unnatural amino acids to control protein functions in mammalian cells

2019 ◽  
Vol 63 (2) ◽  
pp. 237-266 ◽  
Author(s):  
Alexander R. Nödling ◽  
Luke A. Spear ◽  
Thomas L. Williams ◽  
Louis Y.P. Luk ◽  
Yu-Hsuan Tsai
Keyword(s):  
Amino Acids ◽  
Genetic Code ◽  
Protein Function ◽  
Unnatural Amino Acids ◽  
Mammalian Cells ◽  
Structural Investigations ◽  
Cellular Translation ◽  
Protein Functions ◽  
Canonical Amino Acid ◽  
Genetic Code Expansion

Abstract Genetic code expansion allows unnatural (non-canonical) amino acid incorporation into proteins of interest by repurposing the cellular translation machinery. The development of this technique has enabled site-specific incorporation of many structurally and chemically diverse amino acids, facilitating a plethora of applications, including protein imaging, engineering, mechanistic and structural investigations, and functional regulation. Particularly, genetic code expansion provides great tools to study mammalian proteins, of which dysregulations often have important implications in health. In recent years, a series of methods has been developed to modulate protein function through genetically incorporated unnatural amino acids. In this review, we will first discuss the basic concept of genetic code expansion and give an up-to-date list of amino acids that can be incorporated into proteins in mammalian cells. We then focus on the use of unnatural amino acids to activate, inhibit, or reversibly modulate protein function by translational, optical or chemical control. The features of each approach will also be highlighted.

scholarly journals Antimicrobial Peptides and Proteins: From Nature’s Reservoir to the Laboratory and Beyond

2021 ◽  
Vol 9 ◽  
Author(s):  
Tanumoy Sarkar ◽  
Monikha Chetia ◽  
Sunanda Chatterjee
Keyword(s):  
Antimicrobial Peptides ◽  
Targeted Delivery ◽  
Unnatural Amino Acids ◽  
De Novo ◽  
Multidrug Resistant ◽  
Host Cells ◽  
Natural Sources ◽  
Small Proteins ◽  
The Future ◽  
Synthetic Designs

Rapid rise of antimicrobial resistance against conventional antimicrobials, resurgence of multidrug resistant microbes and the slowdown in the development of new classes of antimicrobials, necessitates the urgent development of alternate classes of therapeutic molecules. Antimicrobial peptides (AMPs) are small proteins present in different lifeforms in nature that provide defense against microbial infections. They have been effective components of the host defense system for a very long time. The fact that the development of resistance by the microbes against the AMPs is relatively slower or delayed compared to that against the conventional antibiotics, makes them prospective alternative therapeutics of the future. Several thousands of AMPs have been isolated from various natural sources like microorganisms, plants, insects, crustaceans, animals, humans, etc. to date. However, only a few of them have been translated commercially to the market so far. This is because of some inherent drawbacks of the naturally obtained AMPs like 1) short half-life owing to the susceptibility to protease degradation, 2) inactivity at physiological salt concentrations, 3) cytotoxicity to host cells, 4) lack of appropriate strategies for sustained and targeted delivery of the AMPs. This has led to a surge of interest in the development of synthetic AMPs which would retain or improve the antimicrobial potency along with circumventing the disadvantages of the natural analogs. The development of synthetic AMPs is inspired by natural designs and sequences and strengthened by the fusion with various synthetic elements. Generation of the synthetic designs are based on various strategies like sequence truncation, mutation, cyclization and introduction of unnatural amino acids and synthons. In this review, we have described some of the AMPs isolated from the vast repertoire of natural sources, and subsequently described the various synthetic designs that have been developed based on the templates of natural AMPs or from de novo design to make commercially viable therapeutics of the future. This review entails the journey of the AMPs from their natural sources to the laboratory.

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