Methods in Molecular Biology 1531 Matthew L Nilles Danielle L Jessen Condry Editors Type Secretion Systems Methods and Protocols METHODS IN MOLECULAR BIOLOGY Series Editor John M Walker School of Life and Medical Sciences University of Hertfordshire Hatfield, Hertfordshire, AL10 9AB, UK For further volumes: http://www.springer.com/series/7651 Type Secretion Systems Methods and Protocols Edited by Matthew L Nilles and Danielle L Jessen Condry Department of Biomedical Sciences, School of Medicine and Health Sciences, University of North Dakota, Grand Forks, ND, USA Editors Matthew L Nilles Department of Biomedical Sciences School of Medicine and Health Sciences University of North Dakota Grand Forks, ND, USA Danielle L Jessen Condry Department of Biomedical Sciences School of Medicine and Health Sciences University of North Dakota Grand Forks, ND, USA ISSN 1064-3745 ISSN 1940-6029 (electronic) Methods in Molecular Biology ISBN 978-1-4939-6647-9 ISBN 978-1-4939-6649-3 (eBook) DOI 10.1007/978-1-4939-6649-3 Library of Congress Control Number: 2016955338 © Springer Science+Business Media New York 2017 This work is subject to copyright All rights are reserved by the Publisher, whether the whole or part of the material is concerned, specifically the rights of translation, reprinting, reuse of illustrations, recitation, broadcasting, reproduction on microfilms or in any other physical way, and transmission or information storage and retrieval, electronic adaptation, computer software, or by similar or dissimilar methodology now known or hereafter developed The use of general descriptive names, registered names, trademarks, service marks, etc in this publication does not imply, even in the absence of a specific statement, that such names are exempt from the relevant protective laws and regulations and therefore free for general use The publisher, the authors and the editors are safe to assume that the advice and information in this book are believed to be true and accurate at the date of publication Neither the publisher nor the authors or the editors give a warranty, express or implied, with respect to the material contained herein or for any errors or omissions that may have been made Cover illustration: Melody N Tooksy, PhD Associations: Harvard TH Chan School of Public Health Printed on acid-free paper This Humana Press imprint is published by Springer Nature The registered company is Springer Science+Business Media LLC The registered company address is: 233 Spring Street, New York, NY 10013, U.S.A Preface The complicated nature of the Type III Secretion System (T3SS) has required many protocols be developed or applied to study this apparatus Variance in the secretion system from bacterial species to bacterial species is heavily influenced by the interacting host, which can vary from mammalian, fungal, protozoan, insect, and plant hosts Subsequently, not every protocol will be useful with every bacterial species that expresses a T3S system Some methods have proven to be useful in every species that contains a T3S system, and other methods may only work in one particular species or family of T3S systems Authors will indicate in their chapters the species that particular protocol has proven successful in and sometimes those species that the protocol has not worked The protocols included in this book have proven to perform well in the indicated species and the results of these protocols published, some many times over Some of these protocols may be modified to work in a different bacterial species than indicated in this book; this is up to you the reader to adapt, try, and of course publish to share with others who study this fascinating system Grand Forks, ND, USA Matthew L Nilles Danielle L Jessen Condry v Contents Preface Contributors v ix Introduction to Type III Secretion Systems Danielle L Jessen Condry and Matthew L Nilles Site-Directed Mutagenesis and Its Application in Studying the Interactions of T3S Components Matthew S Francis, Ayad A.A Amer, Debra L Milton, and Tiago R.D Costa Blue Native Protein Electrophoresis to Study the T3S System Using Yersinia pestis as a Model Thomas A Henderson and Matthew L Nilles In Vivo Photo-Cross-Linking to Study T3S Interactions Demonstrated Using the Yersinia pestis T3S System Thomas A Henderson and Matthew L Nilles Isolation of Type III Secretion System Needle Complexes by Shearing Matthew L Nilles, Danielle L Jessen Condry, and Patrick Osei-Owusu Use of Transcriptional Control to Increase Secretion of Heterologous Proteins in T3S Systems Kevin J Metcalf and Danielle Tullman-Ercek Characterization of Type Three Secretion System Translocator Interactions with Phospholipid Membranes Philip R Adam, Michael L Barta, and Nicholas E Dickenson Analysis of Type III Secretion System Secreted Proteins Danielle L Jessen Condry and Matthew L Nilles Fractionation Techniques to Examine Effector Translocation Rachel M Olson and Deborah M Anderson 10 Measurement of Effector Protein Translocation Using Phosphorylatable Epitope Tags and Phospho-Specific Antibodies Sara Schesser Bartra and Gregory V Plano 11 A TAL-Based Reporter Assay for Monitoring Type III-Dependent Protein Translocation in Xanthomonas Sabine Drehkopf, Jens Hausner, Michael Jordan, Felix Scheibner, Ulla Bonas, and Daniela Büttner 12 Subcellular Localization of Pseudomonas syringae pv tomato Effector Proteins in Plants Kyaw Aung, Xiufang Xin, Christy Mecey, and Sheng Yang He vii 11 33 47 61 71 81 93 101 111 121 141 viii Contents 13 A Method for Characterizing the Type III Secretion System’s Contribution to Pathogenesis: Homologous Recombination to Generate Yersinia pestis Type III Secretion System Mutants Patrick Osei-Owusu, Matthew L Nilles, David S Bradley, and Travis D Alvine 14 Detecting Immune Responses to Type III Secretion Systems Peter L Knopick and David S Bradley 15 Recombinant Expression and Purification of the Shigella Translocator IpaB Michael L Barta, Philip R Adam, and Nicholas E Dickenson 16 Expression and Purification of N-Terminally His-Tagged Recombinant Type III Secretion Proteins Travis D Alvine, Patrick Osei-Owusu, Danielle L Jessen Condry, and Matthew L Nilles 17 Mouse Immunization with Purified Needle Proteins from Type III Secretion Systems and the Characterization of the Immune Response to These Proteins Travis D Alvine, David S Bradley, and Matthew L Nilles 18 Identification of the Targets of Type III Secretion System Inhibitors Danielle L Jessen Condry and Matthew L Nilles 19 Detection of Protein Interactions in T3S Systems Using Yeast Two-Hybrid Analysis Matthew L Nilles Index 155 165 173 183 193 203 213 223 Contributors PHILIP R ADAM • Kansas Department of Health and Environment Laboratories, Topeka, KS, USA TRAVIS D ALVINE • Department of Biomedical Sciences, University of North Dakota, Grand Forks, ND, USA AYAD A.A AMER • Department of Molecular Biology, Umeå University, Umeå, Sweden; Umeå Centre for Microbial Research, Umeå University, Umeå, Sweden; Helmholtz Centre for Infection Research, Braunschweig, Germany DEBORAH M ANDERSON • Department of Veterinary Pathobiology, University of Missouri-Columbia, Columbia, MO, USA KYAW AUNG • Department of Energy Plant Research Laboratory, Michigan State University, East Lansing, MI, USA; Howard Hughes Medical Institute, Michigan State University, East Lansing, MI, USA MICHAEL L BARTA • Higuchi Biosciences Center, University of Kansas, Lawrence, KS, USA SARA SCHESSER BARTRA • Department of Microbiology and Immunology, Miller School of Medicine, University of Miami, Miami, FL, USA ULLA BONAS • Department of Genetics, Institute for Biology, Martin Luther University Halle-Wittenberg, Hale (Saale), Germany DAVID S BRADLEY • Department of Biomedical Sciences, University of North Dakota, Grand Forks, ND, USA DANIELA BÜTTNER • Department of Genetics, Institute for Biology, Martin Luther University Halle-Wittenberg, Halle (Saale), Germany DANIELLE L JESSEN CONDRY • Department of Biomedical Sciences, School of Medicine and Health Sciences, University of North Dakota, Grand Forks, ND, USA TIAGO R.D COSTA • Department of Molecular Biology, Umeå University, Umeå, Sweden; Umeå Centre for Microbial Research, Umeå University, Umeå, Sweden; Institute of Structural and Molecular Biology, University College London and Birkbeck, London, UK NICHOLAS E DICKENSON • Department of Chemistry and Biochemistry, Utah State University, Logan, UT, USA SABINE DREHKOPF • Department of Genetics, Institute for Biology, Martin Luther University Halle-Wittenberg, Halle (Saale), Germany MATTHEW S FRANCIS • Department of Molecular Biology, Umeå University, Umeå, Sweden; Umeå Centre for Microbial Research, Umeå University, Umeå, Sweden JENS HAUSNER • Department of Genetics, Institute for Biology, Martin Luther University Halle-Wittenberg, Halle (Saale), Germany SHENG YANG HE • Department of Energy Plant Research Laboratory, Michigan State University, East Lansing, MI, USA; Department of Plant Biology, Michigan State University, East Lansing, MI, USA; Howard Hughes Medical Institute, Michigan State University, East Lansing, MI, USA THOMAS A HENDERSON • Department of Biomedical Sciences, School of Medicine and Health Sciences, University of North Dakota, Grand Forks, ND, USA ix x Contributors MICHAEL JORDAN • Department of Genetics, Institute for Biology, Martin Luther University Halle-Wittenberg, Halle (Saale), Germany PETER L KNOPICK • Department of Biomedical Sciences, University of North Dakota, Grand Forks, ND, USA CHRISTY MECEY • Department of Energy Plant Research Laboratory, Michigan State University, East Lansing, MI, USA KEVIN J METCALF • Department of Chemical and Biomolecular Engineering, University of California Berkeley, Berkeley, CA, USA DEBRA L MILTON • Department of Molecular Biology, Umeå University, Umeå, Sweden; Umeå Centre for Microbial Research, Umeå University, Umeå, Sweden; Department of Biological and Environmental Sciences, Troy University, Troy, AL, USA MATTHEW L NILLES • Department of Biomedical Sciences, School of Medicine and Health Sciences, University of North Dakota, Grand Forks, ND, USA RACHEL M OLSON • Department of Veterinary Pathobiology, University of Missouri-Columbia, Columbia, MO, USA PATRICK OSEI-OWUSU • Department of Microbiology, University of Chicago, Chicago, IL, USA GREGORY V PLANO • Department of Microbiology and Immunology, Miller School of Medicine, University of Miami, Miami, FL, USA FELIX SCHEIBNER • Department of Genetics, Institute for Biology, Martin Luther University Halle-Wittenberg, Halle (Saale), Germany DANIELLE TULLMAN-ERCEK • Department of Chemical and Biomolecular Engineering, University of California Berkeley, Berkeley, CA, USA XIUFANG XIN • Department of Energy Plant Research Laboratory, Michigan State University, East Lansing, MI, USA Chapter Introduction to Type III Secretion Systems Danielle L Jessen Condry and Matthew L Nilles Abstract Type III secretion (T3S) systems are found in a large number of gram-negative bacteria where they function to manipulate the biology of infected hosts Hosts targeted by T3S systems are widely distributed in nature and are represented by animals and plants T3S systems are found in diverse genera of bacteria and they share a common core structure and function Effector proteins are delivered by T3S systems into targeted host cells without prior secretion of the effectors into the environment Instead, an assembled translocon structure functions to translocate effectors across eukaryotic cell membranes In many cases, T3S systems are essential virulence factors and in some instances they promote symbiotic interactions Key words Type III secretion system, Virulence factor, Injectisomes, Translocon, Effector proteins Type III Secretion Systems In order to manipulate the host, gram-negative bacteria utilize a number of features One of these essential virulence factors is the type III-secretion system (T3SS) T3S systems are important in several known symbiotic relationships, demonstrating a duality of T3S functions ranging from beneficial to detrimental manipulation of eukaryotic cells [1, 2] T3S systems are found in many human pathogenic gram-negative bacteria including pathogenic strains of Escherichia coli, Shigella, Salmonella, Yersinia, and Pseudomonas [3, 4] T3S systems are divided into seven families based on sequence similarities T3S systems from animal pathogens fall into three of those families: Ysc-type injectisomes, SPI-1-type injectisomes, or SPI-2-type injectisomes Although much of the basal structures of these systems are homologous, the secreted effectors and regulation of secretion vary between each family Ysc injectisomes are primarily found in Yersinia species, P aeruginosa, Vibrio, and Bordetella pertussis SPI-1 injectisomes are commonly associated with Shigella and Salmonella SPI-2 injectisomes are associated with enterohemorraghic E coli (EHEC), enteropathogenic E coli (EPEC), and Salmonella [3] The majority of bacteria with T3S Matthew L Nilles and Danielle L Jessen Condry (eds.), Type Secretion Systems: Methods and Protocols, Methods in Molecular Biology, vol 1531, DOI 10.1007/978-1-4939-6649-3_1, © Springer Science+Business Media New York 2017 209 Identification of the Targets of Type III Secretion System Inhibitors Prepare the secondary antibody (Goat Anti-Rabbit IgG (whole molecule)-alkaline phosphatase antibody) in antibody solution diluted 1:20,000 (or as appropriate) Incubate blots for h at °C with constant shaking Wash membrane with wash solution three times for 10 Add 1× AP solution to the blot and incubate for at room temperature with constant shaking 10 Discard 1× AP solution and develop with 5-bromo-4-chloro3-indoylphosphate (BCIP) and nitro blue tetrazolium (NBT) Stop development when bands have reached sufficient intensity by adding cold H2O 11 Blots are dried followed by imaging 3.4 Assessment of Hemolysis Grow overnight cultures of all desired strains to be tested, including wild-type to utilize as control; same as Subheading 3.1, step After 12–16 h, subculture the bacteria, same as Subheading 3.1, step 2, and grow for two generations Spin down bacteria and remove supernatant Resuspend bacteria in 37 °C PBS to a density of 50 A620 per mL Prepare RBCs for hemolysis assay: Centrifuge 1000 × g at RT for 10 Wash twice with cold PBS Determine cell count of RBCs and resuspend to 4×109 cells/ mL PBS Add bacteria to RBCs at 50 μL RBCs l–50 μL bacteria in 96 well plates Add inhibitor at (Subheading 3.1) concentration from growth curves 10 Centrifuge 1000 × g at RT for 10 (to make cells come in contact with bacteria) 11 Incubate for 3–4 h at 37 °C 12 Add 150 μL cold PBS to wells (resuspend cells) 13 Centrifuge 1000 × g at RT for 10 (pellet) 14 100 μL supernatant transferred to a clean microtiter dish 15 Read A570, or evaluate for lysis of RBCs under the microscope and/or measure hemoglobin release via standard kit 3.5 Radioactive Inhibitor Tag Assay Grow overnight cultures of wild-type strain(s) After 12–16 h, subculture and grow strain(s) in secretion inducing conditions with and without the untagged and radioactive tagged inhibitory compound (see Note 3) 210 Danielle L Jessen Condry and Matthew L Nilles Collect supernatant for assessment of secretion to verify experiment (see instructions above for this assessment) May consider assay of secreted proteins Collect whole cell bacteria for assay Divide whole cell into fractions if desired [18] Run protein fractions on SDS-PAGE gel (Subheading 3.1) and/or assess fractions for presence radioactivity Transfer gel to membrane and assess via immunoblot Assess for presence of radioactive inhibitor and correlating antibodies to determine target protein Confirm binding of inhibitor by using purified protein 3.6 Inhibitor Tag Pull-Down Assay Attach tag to inhibitor under the instructions of the tagging kit Verify attachment of tag utilizing inhibitor assay kit Grow overnight cultures of wild-type strain(s) After 12–16 h, subculture and grow strain(s) in secretion inducing conditions with and without the untagged and tagged inhibitory compound (see Note 3) Collect supernatant for assessment of secretion to verify experiment (see Subheading 3.1 above) May consider pull-down assay of secreted proteins too Collect whole cell bacteria for pull-down assay Divide whole cell into fractions if desired [18] Utilize Tag-based pull-down assay kit Elute bound tagged items (see Note 4) 10 Run protein fractions on SDS-PAGE gel (see Note 5) 11 Transfer gel to membrane and assess via immunoblot Utilize antibodies to identify protein target 12 If available: Run mass spectrometry analysis of elution to determine target 13 Confirm binding of inhibitor by using purified protein Notes Inhibitor tag may vary depending on the construct of the inhibitor chosen Induction media varies depending on the species or genera The length of time this takes and conditions to induce secretion may vary by genera or species Elution protocols can vary depending on the interaction of the inhibitor and target protein Sodium dodecyl sulfate-polyacryl- Identification of the Targets of Type III Secretion System Inhibitors 211 amide gel electrophoresis (SDS-PAGE) loading buffer may be sufficient to disrupt inhibitor–protein interaction If not a stepwise gradient of increasing salt concentration or decreasing pH maybe utilized, which allows for selective elution of prey proteins while the bait remains immobilized Many kits come with multiple buffers to aid with this step In the event the inhibitor binds to DNA or RNA instead of protein these methods would not be useful References Keyser P, Elofsson M, Rosell S, Wolf-Watz H (2008) Virulence blockers as alternatives to antibiotics: type III secretion inhibitors against Gramnegative bacteria J Intern Med 264:17–29 Aiello D, Williams JD, Majgier-Baranowska H, Patel I, Peet NP, Huang J, Lory S, Bowlin TL, Moir DT (2010) Discovery and characterization of inhibitors of Pseudomonas aeruginosa type III secretion Antimicrob Agents Chemother 54:1988–1999 Harmon DE, Davis AJ, Castillo C, Mecsas J (2010) Identification and characterization of small-molecule inhibitors of Yop translocation in Yersinia pseudotuberculosis Antimicrob Agents Chemother 54:3241–3254 Kimura K, Iwatsuki M, Nagai T, Matsumoto A, Takahashi Y, Shiomi K, Omura S, Abe A (2011) A small-molecule inhibitor of the bacterial type III secretion system protects against in vivo infection with Citrobacter rodentium J Antibiot 64:197–203 Nordfelth R, Kauppi AM, Norberg HA, WolfWatz H, Elofsson M (2005) Small-molecule inhibitors specifically targeting type III secretion Infect Immun 73:3104–3114 Pan N, Lee C, Goguen J (2007) High throughput screening for small-molecule inhibitors of type III secretion in Yersinia pestis Adv Exp Med Biol 603:367–375 Swietnicki W, Carmany D, Retford M, Guelta M, Dorsey R, Bozue J, Lee MS, Olson MA (2011) Identification of small-molecule inhibitors of Yersinia pestis type III secretion system YscN ATPase PLoS ONE 6:e19716 Bailey L, Gylfe A, Sundin C, Muschiol S, Elofsson M, Nordström P, Henriques-Normark B, Lugert R, Waldenström A, Wolf-Watz H, Bergström S (2007) Small molecule inhibitors of type III secretion in Yersinia block the Chlamydia pneumoniae infection cycle FEBS Lett 581:587–595 Veenendaal AKJ, Sundin C, Blocker AJ (2009) Small-molecule type III secretion system 10 11 12 13 14 15 16 17 18 inhibitors block assembly of the Shigella type III secreton J Bacteriol 191:563–570 Eriksson J, Grundström C, Sauer-Eriksson AE, Sauer UH, Wolf-Watz H, Elofsson M (2012) Small molecule screening for inhibitors of the YopH phosphatase of Yersinia pseudotuberculosis Adv Exp Med Biol 954:357–363 Garrity-Ryan LK, Kim OK, Balada-Llasat J-MM, Bartlett VJ, Verma AK, Fisher ML, Castillo C, Songsungthong W, Tanaka SK, Levy SB, Mecsas J, Alekshun MN (2010) Small molecule inhibitors of LcrF, a Yersinia pseudotuberculosis transcription factor, attenuate virulence and limit infection in a murine pneumonia model Infect Immun 78:4683–4690 Izoré T, Job V, Dessen A (2011) Biogenesis, regulation, and targeting of the type III secretion system Structure 19:603–612 Jessen DL, Bradley DS, Nilles ML (2014) A type III secretion system inhibitor targets YopD while revealing differential regulation of secretion in calcium-blind mutants of Yersinia pestis Antimicrob Agents Chemother 58:839–850 Metcalf WW, Jiang W, Daniels LL, Kim SK, Haldimann A, Wanner BL (1996) Conditionally replicative and conjugative plasmids carrying lacZ alpha for cloning, mutagenesis, and allele replacement in bacteria Plasmid 35:1–13 Lehman MK, Bose JL, Bayles KW (2016) Allelic exchange Methods Mol Biol 1373:89–96 Jakočiūnas T, Jensen MK, Keasling JD (2015) CRISPR/Cas9 advances engineering of microbial cell factories Metab Eng 34:44–59 Osei-Owusu P, Jessen Condry DL, Toosky M, Roughead W, Bradley DS, Nilles ML (2015) The N terminus of type III secretion needle protein YscF from Yersinia pestis functions to modulate innate immune responses Infect Immun 83:1507–1522 Thein M, Sauer G, Paramasivam N, Grin I, Linke D (2010) Efficient subfractionation of gram-negative bacteria for proteomics studies J Proteome Res 9:6135–6147 Chapter 19 Detection of Protein Interactions in T3S Systems Using Yeast Two-Hybrid Analysis Matthew L Nilles Abstract Two-hybrid systems, sometimes termed interaction traps, are genetic systems designed to find and analyze interactions between proteins The most common systems are yeast based (commonly Saccharomyces cerevisae) and rely on the functional reconstitution of the GAL4 transcriptional activator Reporter genes, such as the lacZ gene of Escherichia coli (encodes β-galactosidase), are placed under GAL4-dependent transcriptional control to provide quick and reliable detection of protein interactions In this method the use of a yeast-based two-hybrid system is described to study protein interactions between components of type III secretion systems Key words Type III secretion system, Two-hybrid, Protein–protein interactions Introduction Yeast two-hybrid systems were developed from the observation that some transcriptional activators could be reconstituted by fusing interaction domains of proteins onto separated activation or DNA binding domains of transcriptional activators [1, 2] These observations coupled with reporter gene systems led to experimental approaches where protein interactions could be studied genetically In some cases, the reporter system (e.g the Escherichia coli lacZ encoded β-galactosidase) results in a detectable color change from cleavage of a substrate or the use of simple enzymatic assays In other cases, like the yeast HIS3 gene, activation of the reporter leads to selectable nutritional selections Some yeast-based systems rely on reconstitution of the yeast GAL4 activator, other systems rely on expression of E coli LexA Typical reporters include the E coli lacZ gene and/or yeast HIS3, LEU2 or ADE2 genes Further, two-hybrid systems have also been constructed in E coli Not surprisingly, the flexibility and availability of these diverse systems allows a very broad range of Matthew L Nilles and Danielle L Jessen Condry (eds.), Type Secretion Systems: Methods and Protocols, Methods in Molecular Biology, vol 1531, DOI 10.1007/978-1-4939-6649-3_19, © Springer Science+Business Media New York 2017 213 214 Matthew L Nilles applications In the study of type III secretion (T3S) systems, two-hybrid analysis has been used to map protein interactions of the secretion apparatus, to study the interaction of chaperones and effectors, to find targets of effectors and to study the interactions of regulatory proteins that control T3S systems Some examples from a very small sampling of successful uses of two-hybrid technology on T3S systems include: An early study of Yersinia Ysc proteins encoded by the yscEFGHIKLN and yscQ genes demonstrated interactions between YscN and YscL, YscL and YscQ, and YscQ and YscK, suggesting that YscQ interacted with both YscL and YscK [3] Three-hybrid analysis in yeast suggested that Q, L, and K could form a ternary complex [3], this result was repeated in a later comprehensive screening of Yersinia T3S system interactions [4] Work within the Shigella T3S system was able to demonstrate that 33 C-terminal-amino acids are required for Spa40 interaction with Spa32, demonstrating the ability to map interacting domains [5] Investigators working with the T3S-effector, EspZ, of EPEC used a two-hybrid screen to identify CD98 as an intracellular target of EspZ [6], demonstrating the utility of two-hybrid screens in finding targets for effectors Using yeast two-hybird analysis, networks of interactions have been identified in the T3S system of the obligate intracellular pathogen, Chlamydia [7] In the Bordetella T3S system, two-hybrid analysis was used to demonstrate that the tip complex protein Bsp22 interacted with itself and the translocon protein BopD [8], showing an application to studying the translocon complex Two-hybrid analysis was also applied to study partner switching complexes that regulate T3S in Bordetella, an interaction between BtrW and BtrV was demonstrated that allowed the investigators to examine the phosphorylation steps involved in the BtrW/V regulation of the T3S process [9] A two-hybrid screen was used to demonstrate that the Vibrio parahaemolyticus effector VP1686 interacted with nuclear factor RelA p65/NF-kB to directly influence DNA binding of NF-kB [10] My lab has successfully used two-hybrid analysis coupled with site-directed mutagenesis to identify specific amino acids involved in the interaction between the T3S-regulatory proteins LcrG and LcrV in Y pestis [11–13] We demonstrated amino acids involved in the LcrG/LcrV interaction down to single a residue in LcrG (A16) [12, 13] and down to two amino acids in LcrV (L291 and F308) [11] Clearly, yeast two-hybrid systems have a multitude of uses to analyze T3S systems and their interactions with eukaryotic hosts The method described in this chapter is based on the system used to study the LcrG/LcrV interaction Many different vectors and yeast strains are available from investigators and commercial suppliers The goal of this chapter is to provide a tested system as a starting point, but investigators are encouraged to seek out additional information, other systems may be more suitable for some applications 215 2-Hybrid Analysis of T3S System Proteins Materials 2.1 Saccharomyces cerevisiae Strain 2.2 Plasmids Y187, MATα, ura3-52, his3-200, ade2-101, trp1-901, leu2-3, 112, gal4∆, met, gal80∆, URA3::GAL1UAS-GAL1TATA-lacZ, MEL1 (Clontech, Mountain View, CA) pACT2, GAL4(768–881)AD, LEU2, ampr, HA epitope tag (Clontech) pAS2-1, GAL4(1–147) (Clontech) DNA-BD, TRP1, ampr, CYHs2 Plasmids containing cloned T3S system components in one or both of the above vectors (see Note 1) pACTG: pACT2 + lcrG [12] pASV: pAS2-1 + lcrV [12] pJM15: pASV ∆cyh2 [12] 2.3 Media 40 % glucose (w/v): 40 g/l glucose, brought to l with water, filter sterilize YPD medium (Becton Dickinson, Franklin Lakes, NJ): yeast extract 10 g/l, peptone, 20 g/l, glucose 20 g/l (see Note 3) YPDA medium (Becton Dickinson): YPD medium with 15 g/l agar added Poured into Petri dishes Minimal synthetic dropout (SD) medium (Clontech, see Note 2) Broth (leave out the agar when preparing) or agar can be prepared: 6.7 g/l yeast nitrogen base without amino acids (Becton Dickinson), 20 g/l agar, 850 ml water, 100 ml of appropriate 10× dropout supplement (see below), 50 ml of 40 % glucose, adjust pH to 5.8 and autoclave Cool to 55 °C before adding any supplements 10× dropout (DO) supplements (see Note 3): (a) 200 mg/l L-adenine hemisulfate salt, 200 mg/l L-arginine HCl, 200 mg/l L-histidine HCl monohydrate, 300 mg/l L-isoleucine, 1000 mg/l L-leucine, 300 mg/l L-lysine HCl, 200 mg/l L-methionine, 500 mg/l L-phenylalanine, 2000 mg/l L-threonine, 200 mg/l L-tryptophan, 300 mg/l L-tyrosine, 200 mg/l L-uracil, 1500 mg/l L-valine, dissolve in 1000 ml of water, autoclave and store at °C (b) 10× dropout supplements lacking one or more nutrients are made as above except one or more nutrient is omitted to allow selection for plasmids in yeast For example, for selection of pACT2 or pAS2-1 the following 10× DO 216 Matthew L Nilles solutions are needed For other selections, appropriate 10× DO supplements can be prepared 2.4 Yeast Transformation ● 10× DO-LEU ● 10× DO-TRP ● 10× DO-LEU, -TRP Carrier DNA: 10 mg/ml sonicated high quality DNA, dissolve DNA in water and store at −20 °C, boil for 20 immediately prior to use 40 % polyethylene glycol 4000 (PEG): 40 g PEG 4000 in 100 ml of water, autoclave 10× TE buffer: 0.1 M Tris–HCl, 10 mM EDTA, pH to 7.5, autoclave Diluted 1:10 with sterile water to make 1× TE 10× LiAc: M lithium acetate, pH to 7.5 with dilute acetic acid, autoclave PEG/LiAC solution: for 10 ml of solution, ml of 40 % PEG 4000, ml 10× TE, ml LiAC 100 % DMSO (dimethyl sulfoxide) 1×TE/1× LiAc: Prepare immediately prior to use from the 10× stocks of LiAC and TE 2.5 β-Galactosidase Assays Z buffer (for l): 16.1 g/l Na2HPO4·7H2O, 5.5 g/l NaH2PO4·H2O, 0.75 g/l KCl, 0.246 g/l MgSO4·7H2O, pH to 7.0 and autoclave X-gal stock: 20 mg/ml in dimethyl formamide (DMF) of 5-bromo-4-chloro-3-indolyl-β-D-galactopyranoside (X-gal), store at −20 °C Z buffer/X-gal solution: 100 ml Z buffer, 0.27 ml β-mercaptoethanol, 1.67 ml X-gal stock Z buffer with β-mercaptoethanol: 100 ml Z buffer, 0.27 ml β-mercaptoethanol ONPG/Z buffer: mg/ml o-nitrophenyl β-D-galactopyranoside (ONPG) in Z buffer, pH to 7.0, can be stored at −20 °C or made fresh before use Whatman No or VWR Grade 410 paper filters: 75 and 125 mm filters are available 75 mm filters will work with 100 mm plates, 125 mm filters will work with 150 mm plates Liquid nitrogen Timer M Na2CO3 10 Spectrophotometer 11 Cuvettes 2-Hybrid Analysis of T3S System Proteins 3.1 217 Methods Growth of Yeast Yeast strains can be maintained long-term at 1.5) Subculture 30 ml of the overnight into 300 ml of fresh YPD (in a l flask), the A600 should be 0.2–0.3, more culture may need to be added if the A600 is below that reading, incubate at 30 °C for h with shaking (250–300 rpm), the final A600 should be 0.4–0.6 (if the A600 is not in this range not use the culture) Place cells into 50 ml centrifuge tubes and pellet by centrifugation at 1000 × g for at room temperature Discard the supernatant and resuspend cells in sterile 1×TE and pool all the cells in a final volume of 25–50 ml Centrifuge the pooled cells at 1000 × g for at room temperature, discard the supernatant Resuspend the pelleted cells in 1.5 ml of fresh 1×TE/1× LiAc Add 0.1 μg of plasmid DNA and 0.1 mg of carrier DNA to a 1.5 ml microcentrifuge tube for each transformation and mix thoroughly (see Note 6) Add 100 μl of yeast suspension to each transformation tube and mix by vortexing 218 Matthew L Nilles Add 600 μl of PEG/LiAC solution to each transformation tube, mix by vortexing at high speed for 10 s 10 Incubate the transformation tubes at 30 °C for 30 with shaking at 200 rpm 11 Add 70 μl of DMSO to each transformation tube, mix by inversion, not vortex 12 Heat-shock the yeast cells by incubating for 15 at 42 °C in a water bath 13 Immediately following the heat shock chill the yeast on ice for 1–2 14 After cooling centrifuge the cells at 20,800 × g for s, discard the supernatant and resuspend the yeast in 500 μl of 1×TE 15 Plate 100 μl of each transformation reaction onto appropriate SD/DO plates, also plate 100 μl of 1:10, 1:100, and 1:1000 dilutions of each transformation 16 Incubate plates inverted at 30 °C for 2–4 days until colonies are 2–4 mm in size 17 Pick the largest colonies and streak onto the appropriate SD/ DO plates and incubate them at 30 °C for 2–4 days These will be your master plates for further analysis, they can be stored sealed with Parafilm for ~1 month 3.3 Colony-Lift β-Galactosidase Assay to Evaluate Protein Interactions (See Note 7) Streak appropriate SD/DO plates with the yeast transformants to be screened If screening transformants, colony lifts can be performed on those plates Include vector control strains, notably single transformant strains with vector controls Prepare the Z buffer/X-gal solution as described in Subheading 2.5, item 3 Prepare an empty Petri dish for each transformant plate to be tested, by placing filter paper into the dish and soaking the paper with 2.5–5 ml of Z buffer/X-gal Using forceps place a dry filter paper onto the surface of the plates to be screened Gently use the forceps to push the filter into contact with the agar surface Use a needle to poke holes asymmetrically into the filter and the agar to orient the filter to the plate After the filter has become uniformly wet, lift the filter off the surface of the plate and place colony side up into the cover of a petri dish Carefully pour liquid nitrogen over the filter and allow the filter to freeze for at least 10 s (see Note 8) Remove the filter from the liquid nitrogen and allow the filter to thaw Place the filter, colony side up, onto the filter paper soaked in Z buffer/X-gal, incubate the filters at room temperature for 30 to ~8 h (see Note 9) to allow development of a blue color 2-Hybrid Analysis of T3S System Proteins 3.4 Liquid β-Galactosidase Assay to Perform Relative Quantification of Protein Interactions (See Note 10) 219 Inoculate ml of the appropriate SD/DO medium with one yeast colony and vortex vigorously to disperse the cells Incubate the culture overnight at 30 °C with shaking (250–300 rpm) On the day of the assay, prepare fresh ONPG in Z buffer or thaw ONPG/Z buffer previously stored at −20 °C Subculture the overnight yeast culture(s) by transferring ml (vortex the overnight vigorously) of the overnight into ml of fresh YPD medium Incubate the fresh culture for 3–5 h with shaking (250– 300 rpm) at 30 °C, until the A600 is between 0.5 and 0.8, record the exact A600 reading of each culture prior to harvesting the yeast Add 1.5 ml of each culture into three microcentrifuge tubes (triplicates) and centrifuge at 10,000 × g for 30 s Remove the culture supernatant and resuspend the cells in 1.5 ml of Z buffer Centrifuge the resuspended cells at 10,000 × g for 30 s Resuspend the pelleted cells in 300 μl of Z buffer, achieving a fivefold concentration of the original culture 10 Transfer 100 μl of each concentrated cell pellet to a clean 1.5 ml microcentrifuge tube 11 Place the tubes into liquid nitrogen for 30–60 s to freeze the cells 12 Thaw the frozen cells at 37 °C and repeat steps 11 and 12 two more times (a total of three freeze/thaw cycles) 13 Set up an assay blank with 100 μl of Z buffer in a clean 1.5 ml microcentrifuge tube 14 Transfer 100 μl of each thawed cell lysate to a clean 1.5 ml microcentrifuge tube 15 Add 700 μl of Z buffer with β-mercaptoethanol to the blank and to each assay sample 16 Start your timer and immediately add 160 μl of ONPG/Z buffer to each tube 17 Place tubes at 30 °C to incubate until a yellow color develops to the intensity of a yellow Post It note 18 After the yellow color develops stop the reaction by addition of 400 μl of M Na2CO3, record the elapsed reaction time of each tube in minutes 19 Centrifuge the reaction tubes at 10,000 × g for 10 to remove cells and debris prior to reading absorbance 220 Matthew L Nilles 20 Transfer the supernatants from the reaction tubes to clean tubes, being careful to not carry over any of the pellet 21 Blank the spectrophotometer with the reaction blank at 420 and 550 nm 22 Read the A420 and the A550 of each reaction, the A420 should be between 0.02 and 1.0 to be in the linear range of the assay 23 Calculate the Miller units [14] of β-galactosidase activity for each reaction Miller Units = 1000 × {[A420 − (1.75 × A550)]/(t × V × A600)} A420 is the value read for the reaction A550 is the value read for the reaction t is time in minutes of each reaction V is volume of the culture assayed (in this case, 0.1 ml) × the concentration factor (which is 5) A600 is the A600 reading for ml of culture 24 Analyze your data using an appropriate statistical methodology, recall the assay was performed in triplicate The resulting Miller Unit values are reflective of the relative strength of the protein interactions Notes To test for interacting components one of the interaction partners needs to be cloned into one of the two vectors For example, if you want to test interaction of proteins A and B, you would clone the gene for A into pACT2 and the gene for protein B into pAS2-1 Ideally, you would have potential interaction partners cloned into each vector so that the reciprocal interactions can be assessed Libraries for screening can also be constructed in one of the vectors Commercially obtained YPD medium contains dextrose (glucose), take care to not autoclave at temperatures over 121 °C or for extended periods of time, as the glucose can caramelize As the medium darkens from caramelization YPD becomes a poor medium for yeast growth Some investigators prefer to make YPD (or YPDA) from individual components and add filter-sterilized glucose after autoclaving To add glucose after autoclaving make a 40 % (w/v) glucose solution and filter sterilize After the YPD or YPDA has cooled to 55 °C add glucose to % (50 ml/l of 40 % glucose) Commercially available SD medium and 10× DO supplements are available from various vendors Yeast will not start growing if inoculated in clumps, inoculating a small volume of medium (~500–1000 μl) in a microcentrifuge 2-Hybrid Analysis of T3S System Proteins 221 tube may aid in dispersing the yeast Then move the yeast into the growth flask Yeast competent cells are commonly made using the LiAc method Alternatives to this methodology are commonly used as it can be challenging to make yeast sufficiently competent Commercial kits are also available that can simplify this procedure We have found the Frozen-EZ Yeast Transformation II kit from Zymo Research (Irvine, CA) to be a good alternative as it is quick and allows for the storage of competent yeast at ≤−70 °C If you are transforming two plasmids add 0.1 μg of each plasmid to be transformed Transformation of two plasmids is expected to give lower efficiencies Depending on the efficiency you obtain with the competent cells, transformation of two plasmids may occur If your efficiency is low, you may need to sequentially transform the yeast strain with individual plasmids When transforming two plasmids, one should also set up transformations for each individual plasmid, that will allow a determination of the efficiency and should result in at least obtaining the single transformants The filter lift assay is quick and convenient, it will allow a number of interactions to be tested simultaneously and is useful for screening experiments The method is moderately sensitive and provides qualitative results Be very careful with the liquid nitrogen, safety goggles and protective gloves should be worn Assays for strongly interacting proteins will develop color fairly rapidly (within 30–60 min) Some other interactions may not be detectable until later time points Incubation times over h could lead to false positive results, make sure you compare color development with appropriate negative controls Weak interactions will then need to be confirmed using other assays 10 Liquid assays for β-galactosidase are more sensitive than the filter lift method, and may allow the detection of weaker interactions The liquid assays are less useful for screening The method given is for the use of ONPG as a substrate The ONPG assay is quite robust and easy to perform Other substrates for β-galactosidase are available and they can be more sensitive, allowing the detection of weaker interactions CPRG (Chlorophenol red-β-D-galactopyranoside) is a common alternative substrate CPRG is more sensitive but is also less reproducible for strong interactions A chemiluminescent substrate for β-galactosidase is available (Galacton Star, Thermo Fisher Scientific) and is the most sensitive assay; however, it is significantly more expensive and can suffer from high background 222 Matthew L Nilles References Fields S, Song O (1989) A novel genetic system to detect protein-protein interactions Nature 340(6230):245–246 doi:10.1038/340245a0 Ma J, Ptashne M (1987) Deletion analysis of GAL4 defines two transcriptional activating segments Cell 48(5):847–853 Jackson MW, Plano GV (2000) Interactions between type III secretion apparatus components from Yersinia pestis detected using the yeast two-hybrid system FEMS Microbiol Lett 186(1):85–90 Yang H, Tan Y, Zhang T, Tang L, Wang J, Ke Y, Guo Z, Yang X, Yang R, Du Z (2013) Identification of novel protein-protein interactions of Yersinia pestis type III secretion system by yeast two hybrid system PLoS One 8(1):e54121 doi:10.1371/journal.pone.0054121 Botteaux A, Kayath CA, Page AL, Jouihri N, Sani M, Boekema E, Biskri L, Parsot C, Allaoui A (2010) The 33 carboxyl-terminal residues of Spa40 orchestrate the multi-step assembly process of the type III secretion needle complex in Shigella flexneri Microbiology 156(Pt 9):2807–2817 doi:10.1099/mic.0.039651-0 Shames SR, Deng W, Guttman JA, de Hoog CL, Li Y, Hardwidge PR, Sham HP, Vallance BA, Foster LJ, Finlay BB (2010) The pathogenic E coli type III effector EspZ interacts with host CD98 and facilitates host cell prosurvival signalling Cell Microbiol 12(9):1322– 1339 doi:10.1111/j.1462-5822.2010.01470.x Spaeth KE, Chen YS, Valdivia RH (2009) The Chlamydia type III secretion system C-ring engages a chaperone-effector protein complex PLoS Pathog 5(9):e1000579 doi:10.1371/ journal.ppat.1000579 Medhekar B, Shrivastava R, Mattoo S, Gingery M, Miller JF (2009) Bordetella Bsp22 forms a filamentous type III secretion system tip complex and is immunoprotective in vitro and in vivo Mol Microbiol 71(2):492–504 doi:10.1111/j.1365-2958.2008.06543.x Kozak NA, Mattoo S, Foreman-Wykert AK, Whitelegge JP, Miller JF (2005) Interactions between partner switcher orthologs BtrW and BtrV regulate type III secretion in Bordetella J Bacteriol 187(16):5665–5676 doi:10.1128/ JB.187.16.5665-5676.2005 10 Bhattacharjee RN, Park KS, Kumagai Y, Okada K, Yamamoto M, Uematsu S, Matsui K, Kumar H, Kawai T, Iida T, Honda T, Takeuchi O, Akira S (2006) VP1686, a Vibrio type III secretion protein, induces toll-like receptorindependent apoptosis in macrophage through NF-kappaB inhibition J Biol Chem 281(48):36897–36904 doi:10.1074/jbc M605493200 11 Hamad MA, Nilles ML (2007) Structure-function analysis of the C-terminal domain of LcrV from Yersinia pestis J Bacteriol 189(18):6734–6739 doi:10.1128/JB.00539-07 12 Matson JS, Nilles ML (2001) LcrG-LcrV interaction is required for control of Yops secretion in Yersinia pestis J Bacteriol 183(17):5082–5091 doi:10.1128/jb.183.17.5082-5091.2001 13 Matson JS, Nilles ML (2002) Interaction of the Yersinia pestis type III regulatory proteins LcrG and LcrV occurs at a hydrophobic interface BMC Microbiol 2:16 14 Miller JH (1992) A short course in bacterial genetics: a laboratory manual and handbook for Escherichia coli and related bacteria Cold Spring Harbor Laboratory Press, Plainview, NY Printed on acid-free paperLife Sciences INDEX A F Agrobacterium 34, 137, 142–146, 148, 149, 151 Antibodies 35, 36, 41, 44, 45, 49, 55, 57, 59, 64, 67, 69, 74, 77, 78, 95, 97, 99, 108, 111–117, 128, 131, 133, 136, 158, 161, 170, 171, 194–199, 206–210 Arabidopsis thaliana 141, 143, 144, 146–149 Fractionation 75, 78, 82, 101–109 B Bacterial pathogenesis 12, 111, 155 Bacterial spot 35, 36, 44 Biotechnology 71 Blue native electrophoresis (BNE) 33–35, 39–40, 42–44 Blunt-end PCR cloning 183 Bordetella pertussis 1, 124 C Cell-culture 16, 112–113, 115–116, 157, 160, 163, 166–167, 179, 200 Chaperones 4–6, 8, 12, 17–19, 35, 37, 38, 72, 73, 93, 94, 112, 173, 176, 178, 214 Confocal microscopy 142, 144, 148 Cytokines 4, 7, 8, 62, 164, 165, 167, 169–171, 195, 200 Cytometric bead array (CBA) 167, 169–172 D Detergents 34, 102, 104, 105, 109, 174, 176, 178, 180, 190 Digitonin 34, 102–106 E G Genetic-based screens 11 Golden Gate cloning 124, 128, 129 Gram-negative 1, 6–8, 19, 61, 81, 121, 141, 155, 156, 173, 193 H HEK cells 165–169, 171 Heterologous proteins 13, 71–79, 183 HilA 72, 73, 76–78 His-tag 45, 59, 183–190 Homologous recombination 23, 25, 26, 155–164 Hrp1 Hrp2 2-Hybrid 213–221 Hypersensitive response (HR) 8, 122–124, 133, 134, 136, 137 I Immunization 193–201 Injectisomes 1, 2, 12, 13, 15, 18, 34 IpaA IpaB 81, 82, 86, 87, 89, 90, 173–180 K Knockout mutations 204–207 L Effector proteins 4–6, 93, 101–103, 105–108, 111–117, 121–124, 130, 134, 141–152, 193 Enzyme-linked immunosorbent assay (ELISA) 167, 170–172, 194, 195, 197, 198, 200 Erwinia amylovora Escherichia coli 1, 6, 7, 20, 21, 24–26, 44, 108, 127, 135, 142, 156, 159, 163, 173–177, 179, 180, 183, 187, 189, 213 Esp ETT1 ETT2 Exo LcrG 6, 35, 36, 45, 48–53, 56, 58, 59, 94, 214, 215 LcrV 5, 6, 13, 15, 16, 35, 36, 45, 48–50, 52, 53, 59, 94, 115, 214, 215 Liposomes 81–91, 174 Locus of enterocyte effacement (LEE) Low Calcium Response (LCR) 6, 36, 53, 94, 205 M Membrane proteins .5, 14, 33, 34, 109 Mutant libraries 11 MxiH 3, 7, 155, 183 Matthew L Nilles and Danielle L Jessen Condry (eds.), Type Secretion Systems: Methods and Protocols, Methods in Molecular Biology, vol 1531, DOI 10.1007/978-1-4939-6649-3, © Springer Science+Business Media New York 2017 223 YPE SECRETION SYSTEMS: METHODS AND PROTOCOLS 224 TIndex N Needle proteins 3, 4, 7, 61, 62, 65, 68, 69, 155, 183, 193–201 Needles 2–5, 7, 11–15, 43, 57, 61–69, 83, 85, 90, 103, 106, 121, 144, 151, 155, 158, 173, 193–201, 204, 218 NF-kB 165, 168–169, 214 O Outer proteins 5, 13, 93 P Shigella sonnei 7, 193, 195 Sip Site-directed mutagenesis 11–27, 135, 214 Sop 7, 93 SPI-1-type injectisomes 1, SPI-2-type injectisomes 1, SptP 7, 72 Ssp Subcellular fractionation 103, 104, 106–107 Suicide vector pDM4 13, 21, 24 Syc 6, 19, 94, 112 Synthetic biology 71 Pathogenesis 61, 155–164, 193 pET200 vector 189 Phosphorylatable epitope tags 111–117 Phospho-specific antibodies 111–117 Photo-cross-linking 47–59 Photo-leucine 48, 49, 54, 56, 58, 59 Photo-methionine 48–50, 54, 56, 58, 59 Plant immunity 141 Plant pathogens 8, 123, 124 PopD Protein expression 112, 177, 185, 187–189 Protein-protein interaction assays 47 Protein purification .59, 175, 186, 188–189 Protein secretion 71, 72, 77, 155, 157–160, 207–208 Pseudomonas aeruginosa 1, 5, 7, 19, 94, 96, 112, 193, 194 Pseudomonas syringae 8, 141–152 Pull-down assays 204, 207, 210 Targeted inhibitor identification 204–207 THP-1 62, 165–169, 171 Tobacco .143, 144, 146, 148–150 Transcription activator-like (TAL) effectors 122–124 Translocation 4, 16, 19, 101–109, 111–117, 121–137, 157, 160, 204 Translocators 5, 15, 17, 18, 81–91, 112, 115, 173–180 Translocon 2, 3, 5, 8, 12, 15–17, 121–123, 130, 173, 193, 214 TyeA 6, 18, 19, 94 Type III secretion systems (T3SSs) 1–8, 11–27, 33–45, 47–59, 61–69, 71–79, 81–91, 93–99, 101–105, 108, 111, 112, 114, 115, 121–123, 132, 136, 137, 141, 155–173, 183–190, 193–201, 203–211, 213–221 Type IV secretion system (T4SS) 34, 111, 112 R V Resistance gene 122–123 Rhizobiales Vibrio 1, 19, 214 S Saccharomyces cerevisiae 71, 215 Salmonella Containing Vacuole (SCV) Salmonella enterica serovar Typhimurium 3, 6, 155 Secretion 1, 11–13, 34, 48, 61, 71, 81, 93, 101, 111, 121, 141, 155, 165, 173, 183, 193, 203, 214 Secretion system inhibitors 203–211 Shigella boydii Shigella dysenteriae Shigella flexneri 7, 155, 174, 193, 195 T X Xanthomonas campestris 122–124, 128–134, 136, 137 Y Yersinia enterocolitica 13, 18, 68 Yersinia outer proteins (Yop) 5, 13, 15, 16, 18, 49, 93 Yersinia pestis .4, 5, 13, 18, 33–45, 47–59, 62, 68, 93–94, 96, 102, 112, 115, 117, 155–164, 193, 205, 214 Yersinia pseudotuberculosis 13, 18, 21, 25, 27, 94, 96 Yop secretion (Ysc) 1, 5, 13, 14, 39, 49, 53, 95, 214 Ysc-type injectisomes [...]... After crossing the intestinal barrier the bacteria interacts with macrophages and dendritic cells This interaction causes an increase in pro-inflammatory cytokines and chemokines The increase in inflammation eventually leads to edema, erythema, abscess formation, and mucosal hemorrhages [ 53] The role of the T3S system in Shigella plays out in invasion of epithelial cells and macrophages [ 23] Regulation... is then transformed into chemically competent E coli S17-1λpir strain and stored in a LB broth-DMSO solution at minus 80 °C in readiness for conjugal mating (see Subheading 3. 3) Site-Directed Mutagenesis and Its Application in Studying the Interactions of T3S… 3. 3 Conjugation and the Selection of Allelic Exchange Events Leading to the In Cis Generation of Mutations in the Yersinia Genome 25 1 The... third and last (“late-secreted”) class of protein to be secreted by an assembled T3SS The first are the “early secreted” structural needle components that extend from the bacterial surface, and the second are the pore-forming Matthew L Nilles and Danielle L Jessen Condry (eds.), Type 3 Secretion Systems: Methods and Protocols, Methods in Molecular Biology, vol 1 531 , DOI 10.1007/978-1-4 939 -6649 -3_ 2,... (1998) Type III protein secretion systems in bacterial pathogens of animals and plants Microbiol Mol Biol Rev 62 :37 9– 433 7 Moraes TF, Spreter T, Strynadka NC (2008) Piecing together the type III injectisome of bacterial pathogens Curr Opin Struct Biol 18:258–266 8 Gauthier A, Finlay BB (1998) Protein translocation: delivering virulence into the host cell Curr Biol 8:R768–R770 Introduction to Type III Secretion. .. anti-phagocytic and pro-inflammatory immune suppression properties [33 ] Two translocated Yop effectors contributing to anti-phagocytic function are YopE, a GTPase activating protein (GAP) of RhoA, Rac1, and Cdc42 [34 , 35 ], and YopH, a potent protein tyrosine phosphatase (PTPase) [36 , 37 ] The pDM4mediated site-directed mutagenesis system has played an integral role in understanding the intracellular function... substrates) secretion before effector (late substrates) secretion [3] In Yersinia, a homologue to the InvE-MxiC protein family members is unique in the sense of being a 42 kDa complex of two interacting proteins YopN and TyeA [65–67] Extensive analysis using the pDM4 mutagenesis strategy facilitated an investigation of the role of YopN and TyeA interplay in regulating export of Yop substrates in Y pseudotuberculosis... host cell sensing and translocon insertion Mol Microbiol 63: 1719–1 730 37 Bölin I, Portnoy DA, Wolf-Watz H (1985) Expression of the temperature-inducible outer membrane proteins of yersiniae Infect Immun 48: 234 –240 38 Hamad MA, Nilles ML (2007) Structurefunction analysis of the C-terminal domain of LcrV from Yersinia pestis J Bacteriol 189:6 734 –6 739 39 Matson JS, Nilles ML (2001) LcrG-LcrV interaction... LcrH-interacting domains in YopD—the first being a large N-terminal domain that includes a putative transmembrane domain, and the second being a C-terminal amphipathic α-helix [52] Subsequent site-directed mutagenesis to generate specific point mutations discovered a clear role for hydrophobic residues within this amphipathic α-helix in the interaction with LcrH [52] Interestingly, the amphipathic domain... are largely Fig 1 A representative injectisome: Yersinia Type III secretion system structure [58] (Figure is used unchanged from Frontiers in Cellular and Infection Microbiology under a Creative Commons license http://creativecommons.org/ licenses/by /3. 0/legalcode) Introduction to Type III Secretion Systems 3 conserved between T3S systems, including bacterial flagella [3] On the cytosolic side of the... heterodimer in regulating Yop synthesis and secretion [69–72] Interestingly, Y pestis and Y pseudotuberculosis but not Y enterocolitica were previously shown to produce naturally a singular YopN-TyeA protein [ 73] As this was more in line with the various singular polypeptides making up membership within InvE-MxiC-like protein family, pDM4 site-directed mutagenesis was used in an effort to define the biological ... with T3S Matthew L Nilles and Danielle L Jessen Condry (eds.), Type Secretion Systems: Methods and Protocols, Methods in Molecular Biology, vol 1 531 , DOI 10.1007/978-1-4 939 -6649 -3_ 1, â Springer... surface, and the second are the pore-forming Matthew L Nilles and Danielle L Jessen Condry (eds.), Type Secretion Systems: Methods and Protocols, Methods in Molecular Biology, vol 1 531 , DOI 10.1007/978-1-4 939 -6649 -3_ 2,... clean point mutations and in- frame deletion mutations that have been instrumental in identifying and understanding the molecular interactions between components of the Yersinia type III secretion