Name: Super-Resolution Imaging of Bacterial Secreted Proteins Using Genetic Code Expansion (Video) | JoVE Journal
Uploaded: 2023-02-10T13:23:00.000+00:00
Duration: 13 min 11 s
Description: Learn genetic code expansion for site-specific labeling of Salmonella secreted effectors using dSTORM super-resolution microscopy in HeLa cells. This method enables precise visualization of bacterial protein localization during host-pathogen interactions.

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This work was supported by start-up funds from the University of Texas Medical branch, Galveston, TX, and a Texas STAR award to L.J.K. We thank Prof. Edward Lemke (European Molecular Biology Laboratory, Heidelberg, Germany) for plasmid pEVOL-PylRS-AF. Images in Figure 1 were created using BioRender.

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</ol>

The authors declare that they have no competing financial interests.

The approach described herein was used to track the precise location of effector proteins injected into the host cell by the bacterial T3SS after infection. T3SSs are employed by intracellular pathogens such as Salmonella, Shigella, and Yersinia to transport virulence components into the host. The development of super-resolution imaging technologies has made it possible to visualize virulence factors at a previously unimaginable resolution12,...

Bacterial infections have long been regarded as a serious hazard to human health. Pathogens use highly evolved, extremely powerful, and intricate defense systems, as well as a variety of bacterial virulence factors (referred to as effector proteins) to evade host immune responses and establish infections1,2. However, the molecular mechanisms underlying these systems and the role of individual effector proteins are still largely unknown due to the dearth of suitable approaches for directly following the crucial protein components and effectors in host cells during pathogenesis.
One typical example is Salmonella enterica serovar Typhimurium, which causes acute gastroenteritis. Salmonella Typhimurium uses type three secretion systems (T3SS) to inject a variety of effector proteins directly into host cells. As soon as Salmonella enters the host cell, it resides in an acidic membrane-bound compartment, termed the Salmonella-containing vacuole (SCV)3,4. The acid pH of the SCV activates the Salmonella pathogenicity island 2 (SPI-2)-encoded T3SS and translocates a volley of 20 or more effector proteins across the vacuolar membrane into the host cytosol5,6,7,8. Inside the host, these complex cocktails of effector proteins coordinately manipulate host cell signaling pathways, resulting in the formation of highly dynamic, complex tubular membrane structures extended from the SCV along microtubules, termed Salmonella-induced filaments (SIFs), that enable Salmonella to survive and replicate within the host cells9,10,11.
Methods to visualize, track, and monitor bacterial effector localizations, and examine their trafficking and interactions inside host cells, provides critical insight into the mechanisms underpinning bacterial pathogenesis. Labeling and localization of Salmonella secreted T3SS effector proteins inside host cells has proven to be a technological challenge12,13; Nonetheless, the development of genetically-encoded fluorescent proteins has transformed our ability to study and visualize proteins within living systems. However, the size of fluorescent proteins (~25-30 kDa)15 is often comparable to or even greater than that of the protein of interest (POI; e.g., 13.65 kDa for SsaP, 37.4 kDa for SifA). In fact, fluorescent protein labeling of effectors often blocks the secretion of the labeled effector and jams the T3SS14.
Furthermore, fluorescent proteins are less stable and emit a low number of photons before photobleaching, limiting their use in super-resolution microscopic techniques16,17,18, particularly in photoactivation localization microscopy (PALM), STORM, and stimulated emission depletion (STED) microscopy. While the photophysical properties of organic fluorescent dyes are superior to those of fluorescent proteins, methods/techniques such as CLIP/SNAP19,20, Split-GFP21, ReAsH/FlAsH22,23, and HA-Tags24,25 require an additional protein or peptide appendage that may impair the structure-function of the effector protein of interest by interfering with post-translational modification or trafficking. An alternative method that minimizes necessary protein modification involves the incorporation of ncAAs into a POI during translation through GCE. The ncAAs are either fluorescent or can be made fluorescent via click chemistry12,13,26,27,28.
Using GCE, ncAAs with tiny, functional, bio-orthogonal groups (such as an azide, cyclopropene, or cyclooctyne group) can be introduced at nearly any location in a target protein. In this strategy, a native codon is swapped with a rare codon such as an amber (TAG) stop codon at a specified position in the gene of the POI. The modified protein is subsequently expressed in cells alongside an orthogonal aminoacyl-tRNA synthetase/tRNA pair. The tRNA synthetase active site is designed to receive only one particular ncAA, which is then covalently attached to the 3&#39;-end of the tRNA that recognizes the amber codon. The ncAA is simply introduced into the growth medium, but it must be taken up by the cell and reach the cytosol where the aminoacyl-tRNA synthetase (aaRS) can link it to the orthogonal tRNA; it is then incorporated into the POI at the specified location (see Figure 1)12. Thus, GCE enables site-specific incorporation of a plethora of bio-orthogonal reactive groups such as ketone, azide, alkyne, cyclooctyne, transcyclooctene, tetrazine, norbonene, &#945;, &#946;-unsaturated amide, and bicyclo [6.1.0]-nonyne into a POI, potentially overcoming the limitations of conventional protein labeling methods12,26,27,28.
Recent emerging trends in super-resolution imaging techniques have opened up new avenues to investigate biological structures at the molecular level. In particular, STORM, a single-molecule, localization-based, super-resolution technique, has become an invaluable tool to visualize cellular structures down to ~20-30 nm and is able to investigate biological processes one molecule at a time, thereby discovering the roles of intracellular molecules that are yet unknown in traditional ensemble-averaged studies13. Single-molecule and super-resolution techniques require a small tag with bright, photostable organic fluorophores for the best resolution. We recently demonstrated that GCE can be used for incorporating suitable probes for super-resolution imaging12.
Two of the best choices for protein labeling in cells are bicyclo [6.1.0] nonynelysine (BCN) and trans-cyclooctene-lysine (TCO; shown in Figure 1), which may be genetically encoded using a variant of the tRNA/synthetase pair (here termed tRNAPyl/PylRSAF), where Pyl represents pyrrolysine, and AF represents a rationally designed double mutant (Y306A, Y384F) derived from Methanosarcina mazei that naturally encodes pyrrolysine12,29,30,31. Through the strain-promoted inverse electron-demand Diels-Alder cycloaddition (SPIEDAC) reaction, these amino acids react chemoselectively with tetrazine conjugates (Figure 1)12,30,31. Such cycloaddition reactions are exceptionally fast and compatible with living cells; they may also be fluorogenic, if an appropriate fluorophore is functionalized with the tetrazine moiety12,26,32. This paper presents an optimized protocol for monitoring the dynamics of bacterial effectors delivered into host cells using GCE, followed by subcellular localization of secreted proteins in HeLa cells using dSTORM. The results indicate that incorporation of an ncAA via GCE, followed by a click reaction with fluorogenic tetrazine-bearing dyes, represents a versatile method for selective labeling, visualization of secreted proteins, and subsequent sub-cellular localization in the host. All the components and procedures detailed here, however, can be adjusted or substituted so that the GCE system can be adapted to investigate other biological questions.

<table><tbody><tr><td>10x Tris/Glycine SDS running buffer</td><td>BIO-RAD</td><td>1610732</td><td></td></tr><tr><td>Ammonium chloride</td><td>Fischer Scientific</td><td>A661-500</td><td></td></tr><tr><td>Ammonium sulphate</td><td>Fischer Scientific</td><td>BP212R-1</td><td></td></tr><tr><td>Ampicillin Sodium</td><td>Sigma-Aldrich</td><td>A0166</td><td>5 g</td></tr><tr><td>Antibiotic-Antimycotic (100x)</td><td>ThermiFischer Scientific</td><td>15240096</td><td></td></tr><tr><td>Arabinose</td><td>Sigma-Aldrich</td><td>A3256</td><td>500 g</td></tr><tr><td>Avanti J-26XP (High-Performance Centrifuge)</td><td>Beckman Coulter</td><td></td><td></td></tr><tr><td>Bacto-Agar</td><td>BD Diagnostics, Franklin Lakes, USA</td><td>214010</td><td></td></tr><tr><td>BDP-FL-tetrazine</td><td>Lumiprobe (USA)</td><td>2.14E+02</td><td></td></tr><tr><td>&amp;beta;-mercaptoethanol</td><td>Millipore</td><td>444203</td><td>250 mL</td></tr><tr><td>Bromophenol blue</td><td>Sigma-Aldrich</td><td>B8026</td><td></td></tr><tr><td>BSA</td><td>Sigma-Aldrich</td><td>A4503</td><td>500 g</td></tr><tr><td>Casein</td><td>Sigma-Aldrich</td><td>C8654</td><td></td></tr><tr><td>Catalase</td><td>Sigma-Aldrich</td><td>C9322</td><td></td></tr><tr><td>Chloramphenicol</td><td>Sigma-Aldrich</td><td>C1919</td><td></td></tr><tr><td>Click Amino Acid / trans-Cyclooct-2-en &amp;ndash; L - Lysine (TCO*A)</td><td>SiChem GmbH</td><td>SC-8008</td><td>Size: 500 mg</td></tr><tr><td>DAPI (Hoechst33342)</td><td>Invitrogen</td><td>H3570</td><td></td></tr><tr><td>DeNovix DS-11+ Spectrophotometer</td><td>DeNovix</td><td></td><td></td></tr><tr><td>DMEM*</td><td>Corning</td><td>10-013-CV</td><td>* Used for maintaining HeLa Cell</td></tr><tr><td>DMEM**</td><td>Gibco</td><td>11965-092</td><td>**Used for bacterial infection in presence of ncAA, see section 5.4.</td></tr><tr><td>DMSO</td><td>Sigma-Aldrich</td><td>D8418</td><td>250 g</td></tr><tr><td>Donkey anti-rabbit Alexa fluoro555 secondary antibody</td><td>Invitrogen</td><td>A-31572</td><td></td></tr><tr><td>DPBS, 1x</td><td>Corning</td><td>21-031-CV</td><td></td></tr><tr><td>E. coli strain BL21 (DE3)</td><td>Novagen (Madison, WI)</td><td></td><td></td></tr><tr><td>EMCCD Camera</td><td>Andor</td><td>iXon Ultra 897-BV</td><td></td></tr><tr><td>Eppendorf Safe-Lock Tube 1.5 mL (PCR clean)</td><td>Eppendorf, Hamburg, Germany</td><td>30123.328</td><td></td></tr><tr><td>Fetal Bovine Serum (FBS)</td><td>Fischer</td><td>10082147</td><td></td></tr><tr><td>Fisherbrand Syringe Filters - Sterile (PVDF 0.22 &amp;micro;m)</td><td>Fischer Scientific</td><td>97203</td><td>Pack of 100</td></tr><tr><td>Gene Pulser Xcell Electroporator</td><td>BIO-RAD</td><td>1652660</td><td></td></tr><tr><td>Gentamycin</td><td>Sigma-Aldrich</td><td>G1272</td><td>10 mL</td></tr><tr><td>Gibco L-Glutamine (200 mM), 100x</td><td>Fischer Scientific</td><td>25-030-081</td><td></td></tr><tr><td>Glucose oxidase</td><td>Sigma-Aldrich</td><td>G7141-50KU</td><td></td></tr><tr><td>Glycerol</td><td>Fischer Scientific</td><td>BF229-4</td><td></td></tr><tr><td>HeLa cells</td><td>ATTC</td><td>CCL-2</td><td></td></tr><tr><td>HEPES Buffer</td><td>Corning</td><td>25-060-C1</td><td>100 mL</td></tr><tr><td>Hydrocloric acid</td><td>Fischer Scientific</td><td>A144-212</td><td></td></tr><tr><td>ImageJ</td><td></td><td></td><td>Image processing and analysis:&amp;nbsp; http://rsbweb.nih.gov/ij</td></tr><tr><td>IntantBlue</td><td>Expedeon</td><td>ISB1L</td><td>Coomassie-based stain</td></tr><tr><td>Isopropyl &amp;beta;-D-1-thiogalactopyranoside (IPTG)</td><td>Sigma-Aldrich</td><td>I5502</td><td></td></tr><tr><td>Janelia Fluoro 646-tetrazine</td><td>Tocris Bioscience</td><td>7279</td><td></td></tr><tr><td>Kanamycin</td><td>Sigma-Aldrich</td><td>60615</td><td>5 g</td></tr><tr><td>LB Broth</td><td>BD Difco</td><td>244620</td><td>500 g</td></tr><tr><td>Lysozyme</td><td>Sigma-Aldrich</td><td>L6876</td><td></td></tr><tr><td>&amp;mu;Manager (v. 1.4.2)</td><td></td><td></td><td>https://micro-manager.org/Download_Micro-Manager_Latest_Release</td></tr><tr><td>MES</td><td>Sigma-Aldrich</td><td>M3671</td><td>250 g</td></tr><tr><td>Micro pulser cuvette</td><td>BIO-RAD</td><td>165-2086</td><td>0.2 cm electrode gap, pkg. of 50</td></tr><tr><td>Nikon N-STORM</td><td>Nikon Instruments Inc.</td><td></td><td>https://www.microscope.healthcare.nikon.com/products/super-resolution-microscopes/n-storm-super-resolution</td></tr><tr><td>Nunc EasYFlask Cell Culture Flasks</td><td>ThermiFischer Scientific</td><td>156499</td><td></td></tr><tr><td>Omnipur Casamino Acid</td><td>Calbiochem</td><td>2240</td><td>500 g</td></tr><tr><td>Paraformaldehhyde (PFA)</td><td>Electron Microscopy Sciences&amp;nbsp;</td><td>15710</td><td></td></tr><tr><td>PBS (10x)</td><td>Roche</td><td>11666789001</td><td></td></tr><tr><td>Penicillin-Streptomycin Solution (100x)</td><td>GenDEPOT&amp;nbsp;</td><td>CA005-010</td><td>100 mL</td></tr><tr><td>pEVOL-PylRS-AF</td><td></td><td></td><td>For plasmid construction and map see following references&lt;br/&gt; 1. Angew Chem Int Ed Engl. 2011, 50(17), 3878-81.&lt;br/&gt; 2. Angew Chem Int Ed Engl., 2012, 51, 4166-70.</td></tr><tr><td>Plasmid Mini-prep Kit</td><td>Qiagen</td><td>27106</td><td></td></tr><tr><td>plasmid pWSK29-sseJ10TAG-HA</td><td></td><td></td><td>Ref.: elife. 2021, 10, e67789.</td></tr><tr><td>plasmid pWSK29-sseJ-HA</td><td></td><td></td><td>Ref.: elife. 2021, 10, e67789.&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp; Vector map of PWSK29: https://www.addgene.org/172972/</td></tr><tr><td>Pluronic F-127</td><td>Millipore</td><td>540025</td><td>Protein grade, 10% Solution</td></tr><tr><td>Potassium phosphate monobasic</td><td>Fischer Scientific</td><td>P285-500</td><td></td></tr><tr><td>Potassium sulphate</td><td>Acros Organic</td><td>424220250</td><td></td></tr><tr><td>Protease Inhibitor Cocktail Set I - Calbiochem</td><td>Sigma-Aldrich</td><td>539131</td><td>100x Solution</td></tr><tr><td>Rabbit anti-HA primary antibody</td><td>Sigma-Aldrich</td><td>H6908</td><td></td></tr><tr><td>S. enterica. serovar Typhimurium 14028s</td><td></td><td></td><td>Ref.: PLoS Biol. 2015, 13, e1002116.</td></tr><tr><td>Saponin</td><td>Sigma-Aldrich</td><td>47036-50G-F</td><td></td></tr><tr><td>Sodium dodecyl sulfate (SDS)</td><td>Sigma-Aldrich</td><td>L3771</td><td></td></tr><tr><td>Sodium hydroxide</td><td>Fischer Scientific</td><td>SS255-1</td><td></td></tr><tr><td>Sodium Pyruvate (100 mM), 100x</td><td>Corning</td><td>25-060-C1</td><td>100 mL</td></tr><tr><td>Sonic Dismembrator Model 100</td><td>Fischer Scientific</td><td>24932</td><td></td></tr><tr><td>STED microsocpe (Leica TCS SP8 STED 3X system)</td><td>Leica Microsystems, Wetzlar, Germany</td><td></td><td>https://www.leica-microsystems.com/products/confocal-microscopes/p/leica-tcs-sp8-sted-one/</td></tr><tr><td>ThunderSTORM</td><td></td><td></td><td>https://zitmen.github.io/thunderstorm/</td></tr><tr><td>Trizma Base</td><td>Sigma-Aldrich</td><td>T1503</td><td></td></tr><tr><td>Trypsin-EDTA (1x), 0.25%&amp;nbsp;&amp;nbsp;</td><td>GenDEPOT&amp;nbsp;</td><td>CA014-010</td><td></td></tr><tr><td>Tween-20</td><td>Sigma-Aldrich</td><td>P9416</td><td>100 mL</td></tr><tr><td>X-well tissue culture chamber slides</td><td>SARSTEDT</td><td>94.6190.802</td><td></td></tr></tbody></table>

super-resolution imaging of bacterial secreted proteins using genetic code expansion

Super-Resolution Imaging of Bacterial Secreted Proteins Using Genetic Code Expansion

Type three secretion systems (T3SSs) enable gram-negative bacteria to inject a battery of effector proteins directly into the cytosol of eukaryotic host cells. Upon entry, the injected effector proteins cooperatively modulate eukaryotic signaling pathways and reprogram cellular functions, enabling bacterial entry and survival. Monitoring and localizing these secreted effector proteins in the context of infections provides a footprint for defining the dynamic interface of host-pathogen interactions. However, labeling and imaging bacterial proteins in host cells without disrupting their structure/function is technically challenging. 
Constructing fluorescent fusion proteins does not resolve this problem, because the fusion proteins jam the secretory apparatus and thus are not secreted. To overcome these obstacles, we recently employed a method for site-specific fluorescent labeling of bacterial secreted effectors, as well as other difficult-to-label proteins, using genetic code expansion (GCE). This paper provides a complete step-by-step protocol to label Salmonella secreted effectors using GCE site-specifically, followed by directions for imaging the subcellular localization of secreted proteins in HeLa cells using direct stochastic optical reconstruction microscopy (dSTORM) 
Recent findings suggest that the incorporation of non-canonical amino acids (ncAAs) via GCE, followed by bio-orthogonal labeling with tetrazine-containing dyes, is a viable technique for selective labeling and visualization of bacterial secreted proteins and subsequent image analysis in the host. The goal of this article is to provide a straightforward and clear protocol that can be employed by investigators interested in conducting super-resolution imaging using GCE to study various biological processes in bacteria and viruses, as well as host-pathogen interactions.

This protocol paper describes a GCE-based method for site-specific fluorescent labeling and visualization of Salmonella secreted effectors, as depicted in Figure 1. Chemical structure of the ncAA bearing trans-cyclooctene bioorthogonal group (TCO) and the fluorescent dye are shown in Figure 1A. SseJ labeling was achieved by genetic incorporation of bioorthogonal ncAAs at an amber stop codon (see Figu...

This article provides a straightforward and clear protocol to label Salmonella secreted effectors using genetic code expansion (GCE) site-specifically and image the subcellular localization of secreted proteins in HeLa cells using direct stochastic optical reconstruction microscopy (dSTORM)

Type three secretion systems (T3SSs) enable gram-negative bacteria to inject a battery of effector proteins directly into the cytosol of eukaryotic host ...

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Research

JoVE Journal

Biology

Affinity purification of tagged proteins in combination with mass spectrometry (APMS) is a powerful method for the systematic mapping of protein interaction networks and for investigating the mechanistic basis of biological processes. Here, we describe an optimized sequential peptide affinity (SPA) APMS procedure developed for the bacterium Escherichia coli that can be used to isolate and characterize stable multi-protein complexes to near homogeneity even starting from low copy numbers per...

Identification of Protein Complexes in Escherichia coli using Sequential Peptide Affinity Purification in Combination with Tandem Mass Spectrometry

The design of a synthetic operon encoding both the secretory apparatus and the structural monomers of curli fibers is described. Overproduction of these amyloids and adherent polymers allows a measurable gain of adherence of the E. coli chassis1. Easy ways to visualize and quantify adherence are explained.

Engineering Adherent Bacteria by Creating a Single Synthetic Curli Operon

We describe a fluorescence microscopy method, Co-Translational Activation by Cleavage (CoTrAC), to image the production of protein molecules in live cells with single-molecule precision without perturbing the protein's functionality. This method has been used to follow the stochastic expression dynamics of a transcription factor, the λ repressor CI 1.

Single-molecule Imaging of Gene Regulation In vivo Using Cotranslational Activation by Cleavage (CoTrAC)

We describe a super-resolution imaging method to probe the structural organization of the bacterial FtsZ-ring, an essential apparatus for cell division.  This method is based on quantitative analyses of photoactivated localization microscopy (PALM) images and can be applied to other bacterial cytoskeletal proteins.

Super-resolution Imaging of the Bacterial Division Machinery

Spatiotemporal information about dynamic proteins inside live cells is crucial for understanding biology. A type of super-resolution microscopy called fast 3D-structured illumination microscopy (f3D-SIM) reveals unique information about the cytokinetic Z ring in bacteria: both its bead-like appearance and the rapid dynamics of FtsZ within the ring.

Super-resolution Imaging of the Cytokinetic Z Ring in Live Bacteria Using Fast 3D-Structured Illumination Microscopy (f3D-SIM)

Synthetic biology enables the engineering of proteins with unprecedented properties using the co-translational insertion of non-canonical amino acids. Here, we presented how a spectrally red-shifted variant of a GFP-type fluorophore with novel fluorescence spectroscopic properties, termed "gold" fluorescent protein (GdFP), is produced in E. coli via selective pressure incorporation (SPI).

Engineering 'Golden' Fluorescence by Selective Pressure Incorporation of Non-canonical Amino Acids and Protein Analysis by Mass Spectrometry and Fluorescence

Here we present a protocol to produce gram-negative Escherichia coli (E. coli) spheroplasts and gram-positive Bacillus megaterium (B. megaterium) protoplasts to clearly visualize and rapidly characterize peptide-bacteria interactions. This provides a systematic method to define membrane localizing and translocating peptides.

Production and Visualization of Bacterial Spheroplasts and Protoplasts to Characterize Antimicrobial Peptide Localization

This protocol explains how to prepare and mount bacterial samples for live three-dimensional imaging and how to reconstruct the three-dimensional shape of E. coli from those images.

Three-dimensional Imaging of Bacterial Cells for Accurate Cellular Representations and Precise Protein Localization

Imaging of bacterial cells is an emerging systems biology approach focused on defining static and dynamic processes that dictate the function of large macromolecular machines. Here, integration of quantitative live cell imaging and cryo-electron tomography is used to study Legionella pneumophila type IV secretion system architecture and functions.

Applying Live Cell Imaging and Cryo-Electron Tomography to Resolve Spatiotemporal Features of the Legionella pneumophila Dot/Icm Secretion System

The presented method enables visualization of fluorescently labeled cellular proteins with expansion microscopy leading to a resolution of 70 nm on a conventional microscope.

Imaging of Podocytic Proteins Nephrin, Actin, and Podocin with Expansion Microscopy

This article presents a comprehensive protocol for PmbExM, an Expansion Microscopy technique designed specifically for Proteus mirabilis biofilms. PmbExM utilizes a stepwise enzymatic treatment of biofilm samples to achieve an isotropic, 4.3-fold expansion, enabling super-resolution analysis of the spatial organization of cellular and subcellular structures within these sessile microbial communities.

Super-resolution Imaging of Proteus mirabilis Biofilm by Expansion Microscopy

Learn genetic code expansion for site-specific labeling of Salmonella secreted effectors using dSTORM super-resolution microscopy in HeLa cells. This method enables precise visualization of bacterial protein localization during host-pathogen interactions.

Super-Resolution Imaging of Bacterial Secreted Proteins Using Genetic Code Expansion

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