Name: split green fluorescent protein system to visualize effectors delivered from bacteria during infection
Uploaded: 2018-05-24T14:30:00.000+00:00
Duration: 7 min 25 s
Description: Watch this Scientific Journal Video about Split Green Fluorescent Protein System to Visualize Effectors Delivered from Bacteria During Infection at JoVE.com

This research was supported by Basic Science Research Program through the National Research Foundation of Korea (NRF) funded by the Ministry of Science, ICT and future Planning (NRF-2018R1A2A1A05019892) to DC and by a grant from Plant Molecular Breeding Center (PMBC) of the Next Generation Biogreen 21 program of the Rural Development Administration (PJ013201) to EP.&#160;We thank the imaging center of the National Instrumentation Center for Environmental Management to provide a confocal microscope for filming.

Note: All steps are performed at room temperature, unless stated otherwise.
1. Preparation of Plant Materials (4 Weeks)
<ol>
	<li>Preparation for the N. benthamiana plants
	<ol>
		<li>Sow 2 seeds of N. benthamiana on the soil surface of each pot, cover the tray with a plastic dome, and allow seeds to germinate in a 25 &#176;C, 60% humidity growth chamber with a 16/8-h light/dark photoperiod cycle.</li>
		<li>After two weeks, pick out and discard the smallest seedling in each pot and continue to grow plants under the same growth conditions as applied for the germination in step 1.1.1. Add 1 L of water per tray every two days. 
		Note: The growth conditions for plants may vary across labs. Therefore, follow the regular watering protocol to grow the plant in a healthy condition.</li>
		<li>In a week, transfer the plants to a new tray and arrange them with adequate space for further growth. Keep growing the plants under the conditions described in step 1.1.1 until they are ready to be infiltrated at 4 weeks of age. 
		Note: Plant growth may differ depending on the growth condition across labs. Usually, we find that the 4-week-old N. benthamiana plants bear about six leaves.</li>
	</ol>
	</li>
	<li>Preparation for A. thaliana transgenic plants
	<ol>
		<li>Refer to the Table of Materials and order the transgenic Arabidopsis seeds.</li>
		<li>Soak ~50 -&#160;100 transgenic Arabidopsis seeds in 1 mL of distilled water and store them at 4 &#176;C for 3 days in the dark to synchronize the onset of germination.</li>
		<li>Sow ~2 - 3 seeds on the soil surface of a plug plant tray and cover the tray with a plastic dome. Allow seeds to germinate at 23&#160;&#176;C, 60% humidity with a 10/14-h light/dark photoperiod cycle. 
		Note: The seeds should be homozygotes. However, we recommend reconfirming the presence of the transgene in the batch. In this case, sterilize the seeds by washing with 70% ethanol for 2 min, 50% bleach (about 2% hypochlorite) containing 0.05% triton X-100 for 5 min. Follow by washing 5 - 6 times with sterile double distilled water (ddH2O). After sterilization, stratify at 4 &#176;C for 3 days and plate them on plant germination media containing 25 &#181;g/L of hygromycin B to select the transgenic plants.</li>
		<li>After a week, leave only one plant per plug and continue growing plants under the same growth conditions used for step 1.2.3. 
		Note: Four-week-old plants were used for the P. syringae infection. Water plants every other day to keep plants healthy.</li>
	</ol>
	</li>
</ol>
2. Preparation of Pseudomonas Culture (~1 Week)
<ol>
	<li>Construction of the plasmid for Pseudomonas transformation
	<ol>
		<li>Refer to the Table of Materials and order the desired vector(s) of the T3SS-based effector delivery system vector25.</li>
		<li>Insert the effector gene of interest into the effector delivery vector using site-specific recombination cloning25. 
		Note: When monitoring subcellular localization of full-length effector protein, put the full-length gene into pBK-GW-1-2 or pBG-GW-1-2. It is also possible to choose pBK-GW-1-4 or pBG-GW-1-4 containing 2x sfGFP11 for increased fluorescence signal. In the case of a partial effector lacking signal peptide, use pBK-GW-2-2 or pBK-GW-2-4. Refer to Park et al. for detailed information about effector delivery vectors25.</li>
	</ol>
	</li>
	<li>Transform the plasmid carrying an effector fused to the sfGFP11 tag to P. syringae pv. Tomato (Pst) CUCPB5500 using standard electroporation29. 
	Note: Other Pseudomonas strains can be used if necessary. The sfGFP11 tag system is constructed for a broad range of vectors30 and the gene expression of the effector is regulated by the AvrRpm1 promoter, which is comparable with, e.g., Pseudomonas fluorescens (EthAn)31.</li>
	<li>Spread the transformed bacterial cells gently over the surface of the King&#8217;s B agar plates containing 100 &#181;g/mL rifampicin and 25 &#181;g/mL kanamycin or 25 &#181;g/mL gentamycin. Incubate at 28 &#176;C for 2 days.</li>
	<li>Inoculate one colony into King&#8217;s B liquid media with antibiotics appropriate for the vector used, and grow the cells overnight at 28 &#176;C with shaking at 200 rpm.</li>
	<li>Make a glycerol stock. Add autoclaved glycerol to a final concentration of 50% and store at -80 &#176;C.</li>
</ol>
3. Transient Expression of Organelle-targeted sfGFP1-10OPT in N. benthamiana (4 Days)
<ol>
	<li>Preparation of Agrobacterium culture
	<ol>
		<li>Order the desired vector(s) of organelle-targeted sfGFP1-10OPT plasmid(s) (refer to the Table of Materials).</li>
		<li>Transform the plasmid(s) into Agrobacterium tumefaciens strain GV3101 cells32. Grow the cells on Luria-Bertani (LB) agar medium supplemented with 50 &#181;g/mL kanamycin and 50 &#181;g/mL rifampicin at 28 &#176;C for 2 days.</li>
		<li>From a single colony on the LB agar medium, inoculate the cells into 5 mL of liquid LB media supplemented with 50 &#181;g/mL kanamycin and 50 &#181;g/mL rifampicin. Grow the cells overnight at 28 &#176;C with shaking at 200 rpm.</li>
		<li>Harvest the cells by centrifugation at 3,000 x g for 10 min. Pour off the supernatant media and resuspend the pellet in 1 mL of freshly made infiltration buffer.</li>
		<li>Measure the quantity of Agrobacterium by obtaining the optical density (OD) value at an absorbance of 600 nm (Abs 600 nm). Adjust the OD600 of the bacteria to 0.5 with infiltration buffer. 
		Note: 1 mL of suspension is enough to infiltrate on two spots.</li>
		<li>Leave the culture at room temperature on a gentle rocker for 1 - 5 h before infiltration.</li>
		<li>Poke a hole into the center of the leaves to be infiltrated with a 10 &#181;L tip. Use a 1-mL needleless syringe to infiltrate the Agrobacterium suspensions. Carefully and slowly inject about 500 &#181;L of the suspensions prepared from step 3.1.5 into the leaf adaxial side via the syringe. Repeat the infiltration on at least three different plants for experimental replicates. 
		Note: For health and safety reasons, eye protection should be worn during infiltration.</li>
		<li>Wipe off the remaining bacterial suspension on the leaves and mark the boundary of the infiltrated region.</li>
		<li>Keep the infiltrated plants under the same growth conditions used for step 1.1.1 for 2 days.</li>
	</ol>
	</li>
</ol>
4. Inoculation of Pseudomonas (4 Days)
<ol>
	<li>Streak the transformed Pseudomonas strain from the glycerol stock in step 2.5 on King&#8217;s B agar media with the appropriate antibiotics at 28 &#176;C for 2 days. 
	Note: The health of Pseudomonas is very critical. If colonies do not form well, streak the cells again or propagate the cells in the King&#8217;s liquid media prior to proceeding.</li>
	<li>Inoculate a loopful of Pseudomonas cells in Mannitol-Glutamate (MG) liquid media at 28 &#176;C with shaking at 200 rpm for overnight.</li>
	<li>Harvest the cells by centrifugation at 3,000 x g for 10 min. Pour off the supernatant media, resuspend the pellet in 10 mM MgCl2, and adjust the OD600 to 0.02 (1 x 107 cfu/mL) for N. benthamiana leaves and to 0.002 (1 x 106 cfu/mL) for Arabidopsis leaves.</li>
	<li>For the N. benthamiana, infiltrate the Pseudomonas suspension into the area of the leaves where the Agrobacterium carrying sfGFP1-10OPT construct was infiltrated 2 days previously (as in step 3.1.7). For the sfGFP1-10OPT transgenic Arabidopsis, infiltrate the Pseudomonas suspension into two 4-week-old short day-grown leaves. 
	Note: At least three plants are needed for experimental replicates.</li>
</ol>
5. Observation of sfGFP Signal via Confocal Microscopy (1 Day)
<ol>
	<li>Cut out the leaf disc from the Pseudomonas-inoculated leaves. At specific time points after infiltration of Pseudomonas, image two 2-cm2 leaf discs from the single plant using a laser scanning confocal system with 40X/1.2 NA C-Apochromat water immersion objective or 63X/0.8 NA C-Apochromat oil immersion objective. To avoid dead cells killed by wounding, observe the cells away from the infiltration hole.</li>
	<li>Start the observation at a low power setting of the 488-nm argon laser. Increase the laser power to detect sfGFP. 
	Note: We usually use 2 - 15% of the laser intensity to detect the fluorescence signal. However, the laser power and detection setting should be adjusted based upon the user&#8217;s microscopy system. Here, the emission filters were set to 520 - 550 nm. The dead cells often emit auto-fluorescence under the 488-nm laser excitation. Therefore, as a negative control, the same effector without the sfGFP11 tag should be infiltrated and observed under the same observation conditions. In addition, high laser excitation can induce chlorophyll autofluorescence. Therefore, adjust the laser intensity using the control plant cells so as&#160;not to induce chlorophyll autofluorescence.
	<ol>
		<li>For a counterstaining of the cell wall, infiltrate 20 mM propidium iodide (PI) into the leaf disc at 5 - 10 min prior to observation.</li>
		<li>For the nucleus staining, submerge the leaf discs into 0.1% paraformaldehyde for 5 min followed by washing with water. Then, infiltrate the 10 mM PI into the leaf disc at 5 - 10 min before microscopic observation. Repeat the experiments at least three times. 
		Note: This step is not critical but helpful to define the effector localization at the plasma membrane or the nucleus. If the effectors of interest showed their localization in a specific organelle, use a marker for the given organelle to confirm the localization of the effector.</li>
	</ol>
	</li>
</ol>

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Q. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Direct+visualization+of+Agrobacterium-delivered+VirE2+in+recipient+cells.">Direct visualization of Agrobacterium-delivered VirE2 in recipient cells.</a> Plant J. 77 (3), 487-495 (2014).</li><li>Tanaka, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Experimental+approaches+to+investigate+effector+translocation+into+host+cells+in+the+Ustilago+maydis/maize+pathosystem.">Experimental approaches to investigate effector translocation into host cells in the Ustilago maydis/maize pathosystem.</a> Eur J Cell Biol. 94 (7-9), 349-358 (2015).</li></ol>

The authors have no conflicts of interest to disclose.

The protocol described here is used for monitoring the accurate localization of the effector proteins injected by the bacterial T3SS into the host plant cell upon infection. Previously, the split GFP system was used as a tool to study the subcellular localization of mammalian proteins23,36, Salmonella T3E localization, and Agrobacterium VirE2 delivery through the T4SS into the plant cells37. To apply this system in the plant cells, previous studies used a transgenic maize plant that constitutively expresses the cytoplasmic GFP1-10 and transgenic fungi, Ustilago maydis, that expresses the GFP11-tagged effector protein. However, the maize-U. maydis system has not been successful because the expression levels of the translocated effector proteins were weak and high level of background autofluorescence impeded the detection of the reconstituted GFP signal38. To remedy this problem, we used a sfGFP1-10 variant, sfGFP1-10OPT, that significantly improves the solubility and fluorescence intensity21,22.
Despite the advantages of the split sfGFP system, there are some limitations to the study of the effector localization and dynamics. First, the observed reconstituted sfGFP fluorescent signal is relatively weak. This could be due to a small amount of effector molecules delivered directly from P. syringae. This weak signal could be improved by using the multimerizing sfGFP11 tag. The vectors carrying 2x sfGFP11 tag are also available from the sources listed in the Table of Materials. Second, the cytosolic sfGFP1-10 OPT failed to be reconstituted with sfGFP11 targeted to Golgi, ER, and plastid25. Co-expression of mitochondria-targeted sfGFP11 and cytosolic sfGFP1-10OPT resulted in mislocalization of the re-constituted sfGFP to the cytosol and nucleus25. Therefore, localization of the effector of interest should be re-validated by applying the appropriate organelle-targeted sfGFP1-10OPT. When any GFP signal is not detected in the infected cells expressing cytosolic sfGFP1-10OPT, we recommend using N. benth expressing Golgi, ER, and plastid targeted sfGFP1-10OPT or using the corresponding organelle targeted sfGFP1-10OPT transgenic Arabidopsis25.
This method demonstrated the use of the split fluorescent protein system as a useful tool to examine the temporal and spatial dynamics of effectors in host plants25. Future research will focus on the advances in single-molecule imaging, which may enable detailed studies of the dynamics of plasma membrane-localized effectors. Furthermore, a secretion assay using a bacterial system may be useful alternative to study localization of fungal effectors; it prevents the aggregation of effectors and high autofluorescence at the fungal penetration site.
It should be noted that there are some key points in this method. Firstly, both plant materials and bacteria should be healthy and fresh. To ensure visualization of the effector signal, both the sfGFP1-10OPT from plants and the sfGFP11 from Pseudomonas should be strongly expressed. Therefore, it is important to grow plants under the optimal growth conditions and protect them from environmental stressors, such as pests. We also recommend that all bacteria used for infiltration be taken from the 2 day-grown cells upon the agar plates and not from glycerol stocks. Secondly, in the case of transient expression in N. benthamiana, the length of time required for the maturation of the organelle-targeted sfGFP1-10OPT&#160;might vary for each organelle marker protein. For example, maturation of PM-sfGFP1-10OPT requires more than 48 h after infiltration. Lastly, the time point and the expression levels of the reconstituted sfGFP signals depend upon the type of effector proteins that are required to optimize some experimental conditions.

Plants are sessile organisms that encounter numerous invading pathogens including bacteria, fungi, viruses, insects, and nematodes throughout their life cycle. Among the phytopathogens, the gram-negative bacterial pathogens such as Pseudomonas spp. and Ralstonia spp., infect their host plants by entering through wounds or natural openings, such as the stomata and hydathode1. To successfully colonize host plants, bacterial pathogens have evolved to develop a variety of virulence factors2. When bacteria invade a host plant, they inject a series of virulence proteins &#8212; known as effectors &#8212; directly into the plant cells to promote their pathogenicity. These effectors suppress or modulate the plant innate immunity, and manipulate the host cellular processes to result in bacterial survival3.
Pathogenic bacteria mainly use a T3SS to deliver effector proteins directly into host cells4. The T3SS resembles a molecular syringe with a needle-like channel connecting from a scaffold protein structure across the inner and the outer bacterial membranes to the injection site of the host cell5. This T3SS-mediated effector (T3E) secretion mechanism is well-conserved in various gram-negative bacterial pathogens of the plant as well as human. One of the representative plant pathogens, the P. syringae pv. Tomato DC3000 hrcC mutant which typically has a defective T3SS, has restricted growth in plants likely due to the inability of this mutant to fully suppress the plant immunity (by injecting effector proteins)6. Upon translocation into the host cells, effectors target various host proteins that are important for the host cell system, including plant defense responses, gene transcription, cell death, proteasome, vesicle trafficking, and hormone pathways7,8,9,10. Therefore, tracking of the cellular localization of the effector proteins in the host cells is an attractive target to understand their functions with respect to modulation of the plant immunity.
Most of the localization studies of the T3Es have employed Agrobacterium-mediated overexpression with a large fluorescence protein in the host plant9. However, the heterologous expression method for genes that are introduced in other species has been shown to be mis-localized or occasionally non-functional11,12,13. In addition, several studies revealed that bacterial effectors undergo modification for proper targeting in the host cells14,15,16,17. Therefore, transiently expressed effectors in the cytosol of the plant cells may not be functionally or quantitatively identical to the effectors which are delivered by the T3SS upon pathogen infection18. Moreover, the fusion of large fluorescent tags to effector proteins may disrupt the proper effector delivery and visualization18,19. Therefore, these approaches to assay the T3E function may not fully reflect the native localization of the T3SS-secreted effectors.
A green fluorescent protein (GFP) is composed of an 11-stranded &#946;-barrel enclosing a central strand that includes a chromophore20. Waldo et al. reported a novel split-GFP system that consists of a small component (GFP &#946; strand 11; GFP11) and a large complementary fragment (GFP &#946; strand 1-10; GFP1-10)21. The fragments do not fluoresce by themselves but fluoresce upon their self-association when both fragments are in close proximity with each other. For the optimization of the protein folding efficiency, robust folding variants of the GFP, i.e., sfGFP and sfGFPOPT, were subsequently developed for the split GFP system20,21,22. Recently, single amino acid mutated variants of sfGFP1-10OPT- sfYFP1-10OPT and sfCFP1-10OPT- that can reconstitute with a sfGFP11 fragment, and show yellow and cyan fluorescence respectively, were generated23. Moreover, sfCherry, a derivative of mCherry, can be split into sfCherry1-10 and sfCherry11 fragments in the same manner as sfGFP23.
This system has been adapted to label and track the T3SS effectors in HeLa cells during infection using the effectors from Salmonella24. However, it was previously not optimized for the host plant-bacterial pathogen system. Recently, we optimized the split GFP system based on the improved sfGFP1-10OPT to monitor the subcellular localization of the T3Es delivered from P. syringae into plant cells25. To facilitate the localization studies of the T3Es to different subcellular compartments in the plant cells, a set of transgenic Arabidopsis thaliana plants were generated to express sfGFP1-10OPT in the various subcellular compartments25. Moreover, the plasmids carrying a variety of organelle-targeted sfGFP1-10OPT for the Agrobacterium-mediated transient overexpression and the sfGFP11-tagged vectors for the T3SS-based effector delivery were also generated. The seeds of various transgenic Arabidopsis lines and the plasmids to express the T3Es of interest can be obtained from sources mentioned in the Table of Materials26,27.
In the following protocol, we describe an optimized system to monitor the dynamics of effectors delivered by bacteria in the host cells using the split sfGFP system. Infection of plants expressing sfGFP1-10OPT with transgenic Pseudomonas carrying recombinant sfGFP11 plasmid results in a delivery of the sfGFP11-tagged effector from Pseudomonas into the host cell. Consequently, these proteins are reconstituted and translocate to the specific effector target compartment(s). The Pseudomonas syringae pv. tomato CUCPB5500 strain in which 18 effectors are deleted, was used because this strain showed low or no cell death in both A. thaliana and N. benthamianas28. However, all of the materials and steps described here can be replaced or modified to adapt the split sfGFP system for investigation of other biological questions or optimization in the given laboratory conditions.

<table><tbody><tr><td>&lt;strong&gt;Arabidopsis transgenic lines&lt;/strong&gt;</td><td></td><td></td><td>Park, E., Lee, H. Y., Woo, J., Choi, D. &amp;amp; Dinesh-Kumar, S. P. Spatiotemporal Monitoring of Pseudomonas syringae Effectors via Type III Secretion Using Split Fluorescent Protein Fragments. Plant Cell. 29 (7), 1571-1584 (2017)</td></tr><tr><td>CYTO-sfGFP1-10</td><td>ABRC</td><td>CS69831</td><td></td></tr><tr><td>NU-sfGFP1-10</td><td>ABRC</td><td>CS69832</td><td></td></tr><tr><td>PT-sfGFP1-10</td><td>ABRC</td><td>CS69833</td><td></td></tr><tr><td>MT-sfGFP1-10</td><td>ABRC</td><td>CS69834</td><td></td></tr><tr><td>PX-sfGFP1-10</td><td>ABRC</td><td>CS69835</td><td></td></tr><tr><td>ER-sfGFP1-10</td><td>ABRC</td><td>CS69836</td><td></td></tr><tr><td>GO-sfGFP1-10</td><td>ABRC</td><td>CS69837</td><td></td></tr><tr><td>PM-sfGFP1-10</td><td>ABRC</td><td>CS69838</td><td></td></tr><tr><td>&lt;strong&gt;Organelle-targeted sfGFP1-10OPT plasmid&lt;/strong&gt;</td><td></td><td></td><td>Park, E., Lee, H. Y., Woo, J., Choi, D. &amp;amp; Dinesh-Kumar, S. P. Spatiotemporal Monitoring of Pseudomonas syringae Effectors via Type III Secretion Using Split Fluorescent Protein Fragments. Plant Cell. 29 (7), 1571-1584 (2017)</td></tr><tr><td>CYTO-sfGFP1-10</td><td>Addgene</td><td>97387</td><td></td></tr><tr><td>NU-sfGFP1-10</td><td>Addgene</td><td>97388</td><td></td></tr><tr><td>PT-sfGFP1-10</td><td>Addgene</td><td>97389</td><td></td></tr><tr><td>MT-sfGFP1-10</td><td>Addgene</td><td>97390</td><td></td></tr><tr><td>PX-sfGFP1-10</td><td>Addgene</td><td>97391</td><td></td></tr><tr><td>ER-sfGFP1-10</td><td>Addgene</td><td>97392</td><td></td></tr><tr><td>GO-sfGFP1-10</td><td>Addgene</td><td>97393</td><td></td></tr><tr><td>PM-sfGFP1-10</td><td>Addgene</td><td>97394</td><td></td></tr><tr><td>ER-sfCherry1-10</td><td>Addgene</td><td>97403</td><td></td></tr><tr><td>ER-sfYFP1-10</td><td>Addgene</td><td>97404</td><td></td></tr><tr><td>CYTO-sfCFP1-10</td><td>Addgene</td><td>97405</td><td></td></tr><tr><td>&lt;strong&gt;sfGFP11-tagged Gateway compatible vector for T3SS-based effector delivery system&lt;/strong&gt;</td><td>Park, E., Lee, H. Y., Woo, J., Choi, D. &amp;amp; Dinesh-Kumar, S. P. Spatiotemporal Monitoring of Pseudomonas syringae Effectors via Type III Secretion Using Split Fluorescent Protein Fragments. Plant Cell. 29 (7), 1571-1584 (2017)</td><td></td><td></td></tr><tr><td>pBK-GW-1-2</td><td>Addgene</td><td>98250</td><td>pAvrRpm1:GW:HA-sfGFP11:AvrRpm1t; Resistant to Kanamycin (25 ug/ml)</td></tr><tr><td>pBK-GW-1-4</td><td>Addgene</td><td>98251</td><td>pAvrRpm1:GW:HA-2xsfGFP11:AvrRpm1t; Resistant to Kanamycin (25 ug/ml)</td></tr><tr><td>pBK-GW-2-2</td><td>Addgene</td><td>98252</td><td>pAvrRpm1:AvrRPM1sp:GW:HA-sfGFP11:AvrRpm1t; Resistant to Kanamycin (25 ug/ml)</td></tr><tr><td>pBK-GW-2-4</td><td>Addgene</td><td>98253</td><td>pAvrRpm1:AvrRPM1sp:GW:HA-2xsfGFP11:AvrRpm1t; Resistant to Kanamycin (25 ug/ml)</td></tr><tr><td>pBG-GW-1-2</td><td>Addgene</td><td>98254</td><td>pAvrRpm1:GW:HA-sfGFP11:AvrRpm1t; Resistant to Gentamycin (25 ug/ml)</td></tr><tr><td>pBG-GW-1-4</td><td>Addgene</td><td>98255</td><td>pAvrRpm1:GW:HA-2xsfGFP11:AvrRpm1t; Resistant to Gentamycin (25 ug/ml)</td></tr><tr><td>pBG-GW-2-2</td><td>Addgene</td><td>98256</td><td>pAvrRpm1:AvrRPM1sp:GW:HA-sfGFP11:AvrRpm1t; Resistant to Gentamycin (25 ug/ml)</td></tr><tr><td>pBG-GW-2-4</td><td>Addgene</td><td>98257</td><td>pAvrRpm1:AvrRPM1sp:GW:HA-2xsfGFP11:AvrRpm1t; Resistant to Gentamycin (25 ug/ml)</td></tr><tr><td>&lt;strong&gt;Bacterial strains&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>&lt;em&gt;Agrobacterium tumefaciens &lt;/em&gt;GV3101</td><td></td><td></td><td>Csaba Koncz and Jeff Schell, The promoter of TL-DNA gene 5 controls the tissue-specific expression of chimaeric genes carried by a novel type of Agrobacterium binary vector. Mol Gen Genet. 204,383-396 (1986); Resistant to gentamycin (50 ug/ml) and rifampicin (50 ug/ml)</td></tr><tr><td>&lt;em&gt;Pseudomonas syringae&lt;/em&gt; pv. &lt;em&gt;Tomato&lt;/em&gt; CUCPB5500</td><td></td><td></td><td>Kvitko, B. H. et al. Deletions in the repertoire of Pseudomonas syringae pv. tomato DC3000 type III secretion effector genes reveal functional overlap among effectors. PLoS Pathog. 5 (4) (2009).; Resistant to rifampicin (100 ug/ml)</td></tr><tr><td>&lt;strong&gt;Media components&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>&lt;strong&gt;Plant germination media&lt;/strong&gt;</td><td></td><td></td><td>Add 2.165g/L Murashige &amp;amp; Skoog powder, 10 g/L sucrose to water. Adjust to pH 5.8 and add 2.2 g/L phytagel. Autocalve.</td></tr><tr><td>Murashige &amp;amp; Skoog medium including vitamins</td><td>Duchefa Biochemie</td><td>M0222</td><td>Store at 4 &amp;deg;C.</td></tr><tr><td>Sucrose</td><td>Duchefa Biochemie</td><td>S0809</td><td></td></tr><tr><td>Phytagel</td><td>Sigma-Aldrich</td><td>P8169</td><td></td></tr><tr><td>&lt;strong&gt;LB media&lt;/strong&gt;</td><td></td><td></td><td>Add 10 g/L tryptone, 5 g/L yeast extract, 10 g/L NaCl to water. For solid media, add 15 g/L micro agar. Autoclave.&amp;nbsp; Allow solution to cool to 55 &amp;deg;C, and add antibiotic if needed.</td></tr><tr><td>Tryptone</td><td>BD Bioscience</td><td>211705</td><td></td></tr><tr><td>Yeast extract</td><td>BD Bioscience</td><td>212750</td><td></td></tr><tr><td>NaCl</td><td>Duchefa Biochemie</td><td>S0520</td><td></td></tr><tr><td>Micro agar</td><td>Duchefa Biochemie</td><td>M1002</td><td></td></tr><tr><td>&lt;strong&gt;King&#039;s B media&lt;/strong&gt;</td><td></td><td></td><td>10 g/L protease peptone #2, 1.5 g/L anhydrous K&lt;sub&gt;2&lt;/sub&gt;HPO&lt;sub&gt;4&lt;/sub&gt;, 15 g/L of agar to water. Autoclave. Cool down to 55 &amp;deg;C and add sterile 15 ml/L glycerol, 5 ml/L MgSO&lt;sub&gt;4&lt;/sub&gt; to the medium. Add antibiotics if needed.</td></tr><tr><td>Proteose peptone</td><td>BD Bioscience</td><td>212120</td><td></td></tr><tr><td>Anhydrous K&lt;sub&gt;2&lt;/sub&gt;HPO&lt;sub&gt;4&lt;/sub&gt;</td><td>Sigma-Aldrich</td><td>1551128 USP</td><td></td></tr><tr><td>Glycerol</td><td>Duchefa Biochemie</td><td>G1345</td><td></td></tr><tr><td>MgSO&lt;sub&gt;4&lt;/sub&gt;</td><td>Sigma-Aldrich</td><td>M7506</td><td></td></tr><tr><td>Bacto Agar</td><td>BD Bioscience</td><td>214010</td><td></td></tr><tr><td>&lt;strong&gt;Mannitol-Glutamate (MG) liquid media &lt;/strong&gt;</td><td></td><td></td><td>Add 10 g/L of mannitol, 2 g/L of L-glutamic acid, 0.5 g/L of KH&lt;sub&gt;2&lt;/sub&gt;PO&lt;sub&gt;4&lt;/sub&gt;, 0.2 g/L of NaCl, and 0.2 g/L of MgSO&lt;sub&gt;4&lt;/sub&gt; to water. Adjust to pH 7</td></tr><tr><td>Mannitol</td><td>Duchefa Biochemie</td><td>M0803</td><td></td></tr><tr><td>L-glutamic acid</td><td>Duchefa Biochemie</td><td>G0707</td><td></td></tr><tr><td>KH&lt;sub&gt;2&lt;/sub&gt;PO&lt;sub&gt;4&lt;/sub&gt;</td><td>Sigma-Aldrich</td><td>NIST200B</td><td></td></tr><tr><td>Infiltration buffer</td><td></td><td></td><td>10 mM MES (2-(N-morpholino)-ethane sulfonic acid), 10 mM MgCl2, 150 &amp;micro;M acetosyringone. pH 5.6; Prepare a fresh buffer before use.</td></tr><tr><td>MES</td><td>Duchefa Biochemie</td><td>M1503</td><td>Prepare 100 mM (pH 5.6) stock in water. Filter sterilize.</td></tr><tr><td>MgCl&lt;sub&gt;2&lt;/sub&gt;</td><td>Sigma-Aldrich</td><td>M8266</td><td>Prepare 100 mM stock in water. Autoclave.</td></tr><tr><td>Acetosyringone</td><td>Sigma-Aldrich</td><td>D134406</td><td>Prepare 150 mM stock in DMSO.</td></tr><tr><td>Confocal microscope equipments/materials</td><td></td><td></td><td></td></tr><tr><td>710 laser scanning confocal system</td><td>Carl Zeiss</td><td></td><td></td></tr><tr><td>Axio observer Z1 inverted microscope</td><td>Carl Zeiss</td><td></td><td></td></tr><tr><td>Propidium iodide</td><td>ThermoFisher</td><td>P1304MP</td><td></td></tr></tbody></table>

split green fluorescent protein system to visualize effectors delivered from bacteria during infection

Bacteria, one of the most important causative agents of various plant diseases, secrete a set of effector proteins into the host plant cell to subvert the plant immune system. During infection cytoplasmic effectors are delivered to the host cytosol via a type III secretion system (T3SS). After delivery into the plant cell, the effector(s) targets the specific compartment(s) to modulate host cell processes for survival and replication of the pathogen. Although there has been some research on the subcellular localization of effector proteins in the host cells to understand their function in pathogenicity by using fluorescent proteins, investigation of the dynamics of effectors directly injected from bacteria has been challenging due to the incompatibility between the T3SS and fluorescent proteins.
Here, we describe our recent method of an optimized split superfolder green fluorescent protein system (sfGFPOPT) to visualize the localization of effectors delivered via the bacterial T3SS in the host cell. The sfGFP11 (11th &#946;-strand of sfGFP)-tagged effector secreted through the T3SS can be assembled with a specific organelle targeted sfGFP1-10OPT (1-10th &#946;-strand of sfGFP) leading to fluorescence emission at the site. This protocol provides a procedure to visualize the reconstituted sfGFP fluorescence signal with an effector protein from Pseudomonas syringae in a particular organelle in the Arabidopsis and Nicotiana benthamiana plants.

The &#946;-barrel structure of GFP is composed of eleven &#946; strands and can be divided into two fragments, the 1 - 10th strand (GFP1-10OPT) and the 11th (GFP11) strand. Although neither of two fragments fluorescent by themselves, self-assembled sfGFP can emit the fluorescence when the two fragments exist in close proximity (Figure 1A). In this system, sfGFP1-10OPT-expressing Arabidopsis or N. benthamiana plants are inoculated with Pseudomonas carrying a sfGFP11 tagged effector. The sfGFP11 tagged effector delivered by Pseudomonas into the cytosol of the host cell is reconstituted to the sfGFP1-10OPT expressed in the cytosol and then together translocate into the target compartment (Figure 1B). Figure 2 represents the overall procedure, from the preparation of the plant materials and Pst to the detection of the fluorescence signal in the plant cell.
As an example of our method, we used the P. syringae effector protein, AvrB, which is delivered into the host plant cells thorough the T3SS during infection. In Arabidopsis, AvrB is recognized by a corresponding resistance protein, RPM1, and triggers immune responses including hypersensitive cell death33. In the previous study, the GFP reporter assay and the biochemical assay revealed that AvrB and RPM1 are localized in the plasma membrane of the plant cell33,34,35. To examine the localization of AvrB using our method, Agrobacterium harboring the CYTO sfGFP1-10OPT gene was infiltrated in N. benthamiana. In two days, AvrB-sfGFP11-transformed Pseudomonas cells were inoculated into the Agrobacterium-infiltrated region. The complemented sfGFP fluorescence signals were observed in the Pst CUCPB5500 containing AvrB-sfGFP11 at the plasma membrane (Figure 3B). In contrast, no signals were found in the infected cell by Pst carrying native AvrB (Figure 3A).
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/57719/57719fig1.jpg" /> 
Figure 1. The optimized split GFP system. (A) The &#946;-barrel structure of GFP is made of eleven &#946;-strands and can be split into the 1-10th &#946;-strand (GFP1-10) and the 11th &#946;-strand (GFP11). (B) In this method, the sfGFP1-10OPT fragments were expressed in plant cells, while the sfGFP11 strand was fused to AvrB and transformed into Pseudomonas. Only plants cell infected by Pseudomonas containing sfGFP11 will show the sfGFP fluorescence (where the effector localizes). <a href="/files/ftp_upload/57719/57719fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/57719/57719fig2.jpg" /> 
Figure 2. Overview of the procedure. The transgenic Arabidopsis or the sfGFP1-10OPT-infiltrated N. benthamiana plant are infected with Pst CUCPB5500 carrying a sfGFP11-tagged effector through syringe infiltration. The fluorescence of the assembled sfGFP can be detected via a confocal microscopy system at the site of the effector localization. <a href="/files/ftp_upload/57719/57719fig2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/57719/57719fig3.jpg" /> 
Figure 3. AvrB-sfGFP detection on N. benthamiana leaves 3 h after infection. The Pst CUCPB5500 harboring a sfGFP11-tagged AvrB gene was infiltrated on the 5th or 6th leaf of N. benthamiana, which was infiltrated by Agrobacterium carrying the sfGFP1-10OPT 2 days previously. The cell wall was stained by propidium iodide. While the cells expressing sfGFP1-10 OPT with AvrB-only do not show any fluorescence signal (A). The GFP signals (yellow arrow) were observed at the infected cells by the Pst containing AvrB-sfGFP11 gene at 3 h post-infiltration (B). This result represents that only AvrB-sfGFP11, not AvrB, is reconstituted with sfGFP1-10OPT at the cytosol and then the assembled sfGFP is translocated to the plasma membrane. Magenta pseudo color represents the cell wall and chlorophyll autofluorescence. Green represents the assembled sfGFP fluorescence. Scale bars = 40 &#181;m. <a href="/files/ftp_upload/57719/57719fig3large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

Watch this Scientific Journal Video about Split Green Fluorescent Protein System to Visualize Effectors Delivered from Bacteria During Infection at JoVE.com

Split Green Fluorescent Protein System to Visualize Effectors Delivered from Bacteria During Infection

Fluorescent protein-based approaches to monitor effectors secreted by bacteria into host cells are challenging. This is due to the incompatibility between fluorescent proteins and the type-III secretion system. Here, an optimized split superfolder GFP system is used for visualization of effectors secreted by bacteria into the host plant cell.

Bacteria, one of the most important causative agents of various plant diseases, secrete a set of effector proteins into the host plant cell to subvert the ...

split-green-fluorescent-protein-system-to-visualize-effectors

Nanyang Technological University

Research

JoVE Journal

Biology

10.4K Views.  Seoul National University. Fluorescerende eiwit gebaseerde benaderingen te controleren effectoren uitgescheiden door bacteriën in de cellen van de gastheer zijn uitdagend. Dit is te wijten aan de incompatibiliteit tussen fluorescente proteïnen en het type III secretie systeem. Hier, wordt een geoptimaliseerde split superfolder GFP-systeem gebruikt voor visualisatie van effectoren uitgescheiden door bacteriën in de fabriek van de gastheercel.

Video: Split Green Fluorescent Protein System to Visualize Effectors Delivered from Bacteria During Infection

A Fluorescence-based Method to Study Bacterial Gene Regulation in Infected Tissues

Bacterial virulence genes are often regulated at the transcriptional level by multiple factors that respond to different environmental signals. Some factors act directly on virulence genes; others control pathogenesis by adjusting the expression of downstream regulators or the accumulation of signals that affect regulator activity. While regulation has been studied extensively during in vitro growth, relatively little is known about how gene expression is adjusted during infection. Such information is important when a particular gene product is a candidate for therapeutic intervention. Transcriptional approaches like quantitative, real-time RT-PCR and RNA-Seq are powerful ways to examine gene expression on a global level but suffer from many technical challenges including low abundance of bacterial RNA compared to host RNA, and sample degradation by RNases. Evaluating regulation using fluorescent reporters is relatively easy and can be multiplexed with fluorescent proteins with unique spectral properties. The method allows for single-cell, spatiotemporal analysis of gene expression in tissues that exhibit complex three-dimensional architecture and physiochemical gradients that affect bacterial regulatory networks. Such information is lost when data are averaged over the bulk population. Herein, we describe a method for quantifying gene expression in bacterial pathogens in situ. The method is based on simple tissue processing and direct observation of fluorescence from reporter proteins. We demonstrate the utility of this system by examining the expression of Staphylococcus aureus thermonuclease (nuc), whose gene product is required for immune evasion and full virulence ex vivo and in vivo. We show that nuc-gfp is strongly expressed in renal abscesses and reveal heterogeneous gene expression due in part to apparent spatial regulation of nuc promoter activity in abscesses fully engaged with the immune response. The method can be applied to any bacterium with a manipulatable genetic system and any infection model, providing valuable information for preclinical studies and drug development.

Described here is a method for analyzing bacterial gene expression in animal tissues at a cellular level. This method provides a resource for studying the phenotypic diversity occurring within a bacterial population in response to the tissue environment during an infection.

Bacterial virulence genes are often regulated at the transcriptional level by multiple factors that respond to different environmental signals. Some ...

Genetics

Application of Fluorescent Nanoparticles to Study Remodeling of the Endo-lysosomal System by Intracellular Bacteria

Fluorescent nanoparticles (NPs) with desirable chemical, optical and mechanical properties are promising tools to label intracellular organelles. Here, we introduce a method using gold-BSA-rhodamine NPs to label the endo-lysosomal system of eukaryotic cells and monitor manipulations of host cellular pathways by the intracellular pathogen Salmonella enterica. The NPs were readily internalized by HeLa cells and localized in late endosomes/lysosomes. Salmonella infection induced rearrangement of the vesicles and accumulation of NPs in Salmonella-induced membrane structures. We deployed the Imaris software package for quantitative analyses of confocal microscopy images. The number of objects and their size distribution in non-infected cells were distinct from the ones in Salmonella-infected cells, indicating extremely remodeling of the endo-lysosomal system by WT Salmonella.

This article describes methods for the synthesis and fluorescent labeling of nanoparticles (NPs). The NPs were applied in pulse-chase experiments to label the endo-lysosomal system of eukaryotic cells. Manipulation of the endo-lysosomal system by activities of the intracellular pathogen Salmonella enterica were followed by live cell imaging and quantified.

Fluorescent nanoparticles (NPs) with desirable chemical, optical and mechanical properties are promising tools to label intracellular organelles. Here, we ...

Immunology and Infection

Electroporation of Functional Bacterial Effectors into Mammalian Cells

The study of protein interactions in the context of living cells can generate critical information about localization, dynamics, and interacting partners. This information is particularly valuable in the context of host-pathogen interactions. Many pathogen proteins function within host cells in a variety of way such as, enabling evasion of the host immune system and survival within the intracellular environment. To study these pathogen-protein host-cell interactions, several approaches are commonly used, including: in vivo infection with a strain expressing a tagged or mutant protein, or introduction of pathogen genes via transfection or transduction. Each of these approaches has advantages and disadvantages. We sought a means to directly introduce exogenous proteins into cells. Electroporation is commonly used to introduce nucleic acids into cells, but has been more rarely applied to proteins although the biophysical basis is exactly the same. A standard electroporator was used to introduce affinity-tagged bacterial effectors into mammalian cells. Human epithelial and mouse macrophage cells were cultured by traditional methods, detached, and placed in 0.4 cm gap electroporation cuvettes with an exogenous bacterial pathogen protein of interest (e.g. Salmonella Typhimurium GtgE). After electroporation (0.3 kV) and a short (4 hr) recovery period, intracellular protein was verified by fluorescently labeling the protein via its affinity tag and examining spatial and temporal distribution by confocal microscopy. The electroporated protein was also shown to be functional inside the cell and capable of correct subcellular trafficking and protein-protein interaction. While the exogenous proteins tended to accumulate on the surface of the cells, the electroporated samples had large increases in intracellular effector concentration relative to incubation alone. The protocol is simple and fast enough to be done in a parallel fashion, allowing for high-throughput characterization of pathogen proteins in host cells including subcellular targeting and function of virulence proteins.

Electroporation was used to insert purified bacterial virulence effector proteins directly into living eukaryotic cells. Protein localization was monitored by confocal immunofluorescence microscopy. This method allows for studies on trafficking, function, and protein-protein interactions using active exogenous proteins, avoiding the need for heterologous expression in eukaryotic cells.

The study of protein interactions in the context of living cells can generate critical information about localization, dynamics, and interacting partners. ...

Analysis of Yersinia enterocolitica Effector Translocation into Host Cells Using Beta-lactamase Effector Fusions

Many gram-negative bacteria including pathogenic Yersinia spp. employ type III secretion systems to translocate effector proteins into eukaryotic target cells. Inside the host cell the effector proteins manipulate cellular functions to the benefit of the bacteria. To better understand the control of type III secretion during host cell interaction, sensitive and accurate assays to measure translocation are required. We here describe the application of an assay based on the fusion of a Yersinia enterocolitica effector protein fragment (Yersinia outer protein; YopE) with TEM-1 beta-lactamase for quantitative analysis of translocation. The assay relies on cleavage of a cell permeant FRET dye (CCF4/AM) by translocated beta-lactamase fusion. After cleavage of the cephalosporin core of CCF4 by the beta-lactamase, FRET from coumarin to fluorescein is disrupted and excitation of the coumarin moiety leads to blue fluorescence emission. Different applications of this method have been described in the literature highlighting its versatility. The method allows for analysis of translocation in vitro and also in in vivo, e.g., in a mouse model. Detection of the fluorescence signals can be performed using plate readers, FACS analysis or fluorescence microscopy. In the setup described here, in vitro translocation of effector fusions into HeLa cells by different Yersinia mutants is monitored by laser scanning microscopy. Recording intracellular conversion of the FRET reporter by the beta-lactamase effector fusion in real-time provides robust quantitative results. We here show exemplary data, demonstrating increased translocation by a Y. enterocolitica YopE mutant compared to the wild type strain.

Analysis of Yersinia enterocolitica Effector Translocation into Host Cells Using Beta-lactamase Effector Fusions

Effector translocation into host cells via a type III secretion system is a common virulence strategy among gram-negative bacteria. A beta-lactamase effector fusion based assay for quantitative analysis of translocation was applied. In Yersinia infected cells, conversion of a FRET reporter by the beta-lactamase is monitored using laser scanning microscopy.

Many gram-negative bacteria including pathogenic Yersinia spp. employ type III secretion systems to translocate effector proteins into eukaryotic target ...

Applying Fluorescence Resonance Energy Transfer (FRET) to Examine Effector Translocation Efficiency by Coxiella burnetii during siRNA Silencing

Coxiella burnetii, the causative agent of Q fever, is an intracellular pathogen that relies on a Type IV Dot/Icm Secretion System to establish a replicative niche. A cohort of effectors are translocated through this system into the host cell to manipulate host processes and allow the establishment of a unique lysosome-derived vacuole for replication. The method presented here involves the combination of two well-established techniques: specific gene silencing using siRNA and measurement of effector translocation using a FRET-based substrate that relies on &#946;-lactamase activity. Applying these two approaches, we can begin to understand the role of host factors in bacterial secretion system function and effector translocation. In this study we examined the role of Rab5A and Rab7A, both important regulators of the endocytic trafficking pathway. We demonstrate that silencing the expression of either protein results in a decrease in effector translocation efficiency. These methods can be easily modified to examine other intracellular and extracellular pathogens that also utilize secretion systems. In this way, a global picture of host factors involved in bacterial effector translocation may be revealed.

Applying Fluorescence Resonance Energy Transfer FRET to Examine Effector Translocation Efficiency by Coxiella burnetii during siRNA Silencing

Investigating the interactions between bacterial pathogens and their hosts is an important area of biological research. Here, we describe the necessary techniques to measure effector translocation by Coxiella burnetii during siRNA gene silencing using BlaM substrate.

Coxiella burnetii, the causative agent of Q fever, is an intracellular pathogen that relies on a Type IV Dot/Icm Secretion System to establish a ...

Using Enhanced Green Fluorescence Protein-expressing Escherichia Coli to Assess Mouse Peritoneal Macrophage Phagocytosis

This manuscript describes a simple and reproducible method to perform a phagocytosis assay. The first part of this method involves building a pET-SUMO-EGFP vector (SUMO = small ubiquitin-like modifier) and expressing enhanced green fluorescence protein (EGFP) in Escherichia coli (BL21DE). EGFP-expressing E. coli is coincubated with macrophages for 1 h at 37 &#176;C; the negative control group is incubated on ice for the same amount of time. Then, the macrophages are ready for assessment. The advantages of this technique include its simple and straightforward steps, and phagocytosis can be measured by both flow cytometer and fluorescence microscope. The EGFP-expressing E. coli are stable and display a strong fluorescence signal even after the macrophages are fixed with paraformaldehyde. This method is not only suitable for the assessment of macrophage cell lines or primary macrophages in vitro but also suitable for the evaluation of granulocyte and monocyte phagocytosis in peripheral blood mononuclear cells. The results show that the phagocytic capability of peritoneal macrophages from young (eight-week-old) mice is higher than that of macrophages from aged (16-month-old) mice. In summary, this method measures macrophage phagocytosis and is suitable for studying the innate immune system function.

Using Enhanced Green Fluorescence Protein-expressing Escherichia Coli to Assess Mouse Peritoneal Macrophage Phagocytosis

Here, we present a protocol to assess mouse peritoneal macrophage phagocytosis using enhanced green fluorescence protein-expressing Escherichia coli.

This manuscript describes a simple and reproducible method to perform a phagocytosis assay. The first part of this method involves building a ...

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

The Dot/Icm secretion system of Legionella pneumophila is a complex type IV secretion system (T4SS) nanomachine that localizes at the bacterial pole and mediates the delivery of protein and DNA substrates to target cells, a process generally requiring direct cell-to-cell contact. We have recently solved the structure of the Dot/Icm apparatus by cryo-electron tomography (cryo-ET) and showed that it forms a cell envelope-spanning channel that connects to a cytoplasmic complex. Applying two complementary approaches that preserve the native structure of the specimen, fluorescent microscopy in living cells and cryo-ET, allows in situ visualization of proteins and assimilation of the stoichiometry and timing of production of each machine component relative to other Dot/Icm subunits. To investigate the requirements for polar positioning and to characterize dynamic features associated with T4SS machine biogenesis, we have fused a gene encoding superfolder green fluorescent protein to Dot/Icm ATPase genes at their native positions on the chromosome. The following method integrates quantitative fluorescence microscopy of living cells and cryo-ET to quantify polar localization, dynamics, and structure of these proteins in intact bacterial cells. Applying these approaches for studying the Legionella pneumophila T4SS is useful for characterizing the function of the Dot/Icm system and can be adapted to study a wide variety of bacterial pathogens that utilize the T4SS or other types of bacterial secretion complexes.

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

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.

The Dot/Icm secretion system of Legionella pneumophila is a complex type IV secretion system (T4SS) nanomachine that localizes at the bacterial pole and ...

Kinetic Visualization of Single-Cell Interspecies Bacterial Interactions

Polymicrobial communities are ubiquitous in nature, yet studying their interactions at the single-cell level is difficult. Thus, a microscopy-based method has been developed for observing interspecies interactions between two bacterial pathogens. The use of this method to interrogate interactions between a motile Gram-negative pathogen, Pseudomonas aeruginosa and a non-motile Gram-positive pathogen, Staphylococcus aureus is demonstrated here. This protocol consists of co-inoculating each species between a coverslip and an agarose pad, which maintains the cells in a single plane and allows for visualization of bacterial behaviors in both space and time.
Furthermore, the time-lapse microscopy demonstrated here is ideal for visualizing the early interactions that take place between two or more bacterial species, including changes in bacterial species motility in monoculture and in coculture with other species. Due to the nature of the limited sample space in the microscopy setup, this protocol is less applicable for studying later interactions between bacterial species once cell populations are too high. However, there are several different applications of the protocol which include the use of staining for imaging live and dead bacterial cells, quantification of gene or protein expression through fluorescent reporters, and tracking bacterial cell movement in both single species and multispecies experiments.

This live-bacterial cell imaging protocol allows for visualization of interactions between multiple bacterial species at the single-cell level over time. Time-lapse imaging allows for observation of each bacterial species in monoculture or coculture to interrogate interspecies interactions in multispecies bacterial communities, including individual cell motility and viability.

Polymicrobial communities are ubiquitous in nature, yet studying their interactions at the single-cell level is difficult. Thus, a microscopy-based method ...

Whole&#45;Organ Pathogen Tracking via Fluorescent Reporter Quantification

High&#45;Resolution Three&#45;Dimensional Whole&#45;Organ Tomography of Microbial Infections

Most infections take place within three-dimensional host tissues with intricate anatomy and locally varying host physiology. The positioning of pathogen cells within this diverse environment significantly affects their stress levels, responses, fate, and contribution to the overall progression of the disease and treatment failure. However, due to the technical difficulties in locating &#181;m-sized pathogen cells within cm-sized host organs, this area of research has been relatively unexplored. Here, we present a method for addressing this challenge. We employ serial two-photon tomography and AI-enhanced image analysis to locate individual Salmonella cells throughout the entire spleen, liver lobes, and whole lymph nodes of infected mice. Using fluorescent reporters and in vivo antibody administration, the replication rate of single Salmonella cells, their local interaction with specific immune cells, and bacterial responses to antibiotics can be determined. These methodologies open avenues for a comprehensive examination of infections, their prevention, and treatment within the three-dimensional tissue context.

Author Spotlight: Unraveling Bacterial Responses to Antibiotics and Immune System in Tissues

Here, we describe a procedure that enables organ-wide detection of pathogenic bacteria during infection and quantification of fluorescent reporter activities.

Most infections take place within three-dimensional host tissues with intricate anatomy and locally varying host physiology. The positioning of pathogen ...

Internalization and Observation of Fluorescent Biomolecules in Living Microorganisms via Electroporation

The ability to study biomolecules in vivo is crucial for understanding their function in a biological context. One powerful approach involves fusing molecules of interest to fluorescent proteins such as GFP to study their expression, localization and function. However, GFP and its derivatives are significantly larger and less photostable than organic fluorophores generally used for in vitro experiments, and this can limit the scope of investigation. 
We recently introduced a straightforward, versatile and high-throughput method based on electroporation, allowing the internalization of biomolecules labeled with organic fluorophores into living microorganisms. Here we describe how to use electroporation to internalize labeled DNA fragments or proteins into Escherichia coli and Saccharomyces cerevisi&#230;, how to quantify the number of internalized molecules using fluorescence microscopy, and how to quantify the viability of electroporated cells. Data can be acquired at the single-cell or single-molecule level using fluorescence or FRET. The possibility of internalizing non-labeled molecules that trigger a physiological observable response in vivo is also presented. Finally, strategies of optimization of the protocol for specific biological systems are discussed.

Studies of biomolecules in vivo are crucial for understanding molecular function in a biological context. Here we describe a novel method allowing the internalization of fluorescent biomolecules, such as DNA or proteins, into living microorganisms. Analysis of in vivo data recorded by fluorescence microscopy is also presented and discussed.

The ability to study biomolecules in vivo is crucial for understanding their function in a biological context. One powerful approach involves fusing ...

Split Green Fluorescent Protein System to Visualize Effectors Delivered from Bacteria During Infection

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