Current Topics in Microbiology and Immunology Steffen Backert Elisabeth Grohmann E ditors Type IV Secretion in Gram-Negative and Gram-Positive Bacteria Current Topics in Microbiology and Immunology Volume 413 Series editors RafiAhmed SchoolofMedicine,RollinsResearchCenter,EmoryUniversity,RoomG211,1510CliftonRoad, Atlanta,GA30322,USA KlausAktories MedizinischeFakultät,InstitutfürExperimentelleundKlinischePharmakologieundToxikologie, Abt.I,Albert-Ludwigs-UniversitätFreiburg,Albertstr.25,79104,Freiburg,Germany ArturoCasadevall W.HarryFeinstoneDepartmentofMolecularMicrobiology&Immunology,JohnsHopkins BloombergSchoolofPublicHealth,615N.WolfeStreet,RoomE5132,Baltimore,MD21205, USA RichardW.Compans DepartmentofMicrobiologyandImmunology,EmoryUniversity,1518CliftonRoad,CNR5005, Atlanta,GA30322,USA JorgeE.Galan BoyerCtr.forMolecularMedicine,SchoolofMedicine,YaleUniversity,295CongressAvenue, room343,NewHaven,CT06536-0812,USA AdolfoGarcia-Sastre IcahnSchoolofMedicineatMountSinai,DepartmentofMicrobiology,1468MadisonAve., Box1124,NewYork,NY10029,USA AkikoIwasaki DepartmentofImmunobiology,TACS655,YaleUniversitySchoolofMedicine,POBOX 208011,NewHaven,CT06520-8011,USA BernardMalissen Centred’ImmunologiedeMarseille-Luminy,ParcScientifiquedeLuminy,Case906,13288, MarseilleCedex9,France KlausPalme InstituteofBiologyII/MolecularPlantPhysiology,Albert-Ludwigs-UniversitätFreiburg, Freiburg,79104,Germany RinoRappuoli GSKVaccines,ViaFiorentina1,Siena,53100,Italy Honorary editors MichaelB.A.Oldstone DepartmentofImmunologyandMicrobiology,TheScrippsResearchInstitute,10550North TorreyPinesRoad,LaJolla, CA92037,USA PeterK.Vogt DepartmentofMolecularandExperimentalMedicine,TheScrippsResearchInstitute,10550 NorthTorreyPinesRoad,BCC-239,LaJolla,CA92037,USA More information about this series at http://www.springer.com/series/82 Steffen Backert Elisabeth Grohmann (cid:129) Editors Type IV Secretion in Gram-Negative and Gram-Positive Bacteria Responsible series editor: Klaus Aktories 123 Editors SteffenBackert Elisabeth Grohmann Division of Microbiology, Division of Microbiology, Departmentof Biology Faculty ofLife Sciences Friedrich Alexander University andTechnology Erlangen-Nuremberg BeuthUniversity of Applied Erlangen SciencesBerlin Germany Berlin Germany ISSN 0070-217X ISSN 2196-9965 (electronic) CurrentTopics inMicrobiology andImmunology ISBN978-3-319-75240-2 ISBN978-3-319-75241-9 (eBook) https://doi.org/10.1007/978-3-319-75241-9 LibraryofCongressControlNumber:2018930379 ©SpringerInternationalPublishingAG,partofSpringerNature2017,correctedpublication2018 Thisworkissubjecttocopyright.AllrightsarereservedbythePublisher,whetherthewholeorpart 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 orinformationstorageandretrieval,electronicadaptation,computersoftware,orbysimilarordissimilar methodologynowknownorhereafterdeveloped. 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Printedonacid-freepaper ThisSpringerimprintispublishedbytheregisteredcompanySpringerInternationalPublishingAG partofSpringerNature Theregisteredcompanyaddressis:Gewerbestrasse11,6330Cham,Switzerland Foreword T4SS—Then and Now IwashonoredtobeaskedbySteffenBackertandElisabethGrohmanntoprovidea Foreword for the book “Type IV Secretion in Gram-Negative and Gram-Positive Bacteria,” which they have edited. After having worked in the field of plasmid conjugation for decades, I was excited by the chapter list and impressed with the large group of international experts they had assembled together to contribute. In fact, writing the Foreword provided to me a wonderful excuse to read the entire volume. WhenIwasayounggraduatestudent,Iwasfascinatedbyreadingthepioneering papersofAchtman,Willetts,andClark(1971,1972),whoreportedthefirstgenetic analysis of the F-plasmid transfer region. I was in awe of the complexity of the transfer operon that encoded nine genes and was estimated to be over 30 kb long. Over 20 years later, the complete sequence of the F-plasmid transfer region revealedthepresenceof15genesforF-typepilusassemblyaloneandanother9for DNAtransfer,matingpairstabilization,andsurfaceandentryexclusion(Frostetal. 1994). The 70 amino acid pilin molecule resulted from the removal of a 52 amino acidleaderpeptideandwasacetylatedatitsN-terminus.Atthisstage,wewondered whyitwassocomplicated?Inthesameyear,thegroupofErichLankareportedthe complete sequence of a generic IncP plasmid (Pansegrau et al. 1994). In addition, P-type pilus assembly was shown to require 11 genes plus a peptidase that circu- larizes theP-type pilinafter cleavage attwo sites, amost unusual molecule indeed (Kalkumetal.2004).Asidefromunusualpilinproteins,otherdifferencesincluded anextraATPaseintheP-typesystemsandthepresenceofsixextragenesinF-type systems, some which contain high numbers of conserved cysteine residues (TraN has 22), involved in mating pair stabilization and pilus retraction (Arutyunov and Frost 2013), two traits not seen in P-type systems. At about the same time, the Ti plasmid T-DNA, which is responsible for tumorigenesis in plants, was found to specify a P-type pilus and pilus assembly apparatus capable of delivering the T-DNA into a plant cell. This proved to be a v vi Foreword more tractable system for studying DNA transfer, and rapid progress was made in understandingthatthe11geneproductsdefinedatypeIVsecretionsystem(T4SS), which were duly named VirB1-11 (Christie 1997; Kado 2014). These names are nowuseduniversallyforthecoreT4SSgeneproducts,althoughtheymightnotbe directlyassociatedwithavirulencephenotypeineachcase.MostT4SSgeneshave namesthatarespecifictoaparticularsystemaswellasdesignationasVirBorVirD proteins. A movement is afoot to have VirB1-11 called TivB1-11 to designate membershipintheT4SSfamilyratherthanuse“Vir”attheriskofbeingconfusing (Thomas et al. 2017). In the early days of conjugative plasmid research, the focus was on F and the related R factors within the IncF complex (Incompatibility group F) and the IncI plasmids (Meynell et al. 1968). This early work, using simple reagents and pilus-specific antibodies, showed that the IncF and IncI plasmids specified very different pilus types. Coupled with the work of David Bradley on pilus-specific phages (reviewed in Frost 1993) for a large number of Inc groups, it became clear thatthere weretwopilustypes that definedflexible(F)andrigid(I,P)pilustypes. This work was prescient in that it defined what we now know as the type IVA (T4ASS, F and P-like) and type IVB (T4BSS, I-like) systems (reviewed in Chapter “Biological Diversity and Evolution of Type IV Secretion Systems,” Christie,ValeroandBuchrieser).Thehomologybetween theVirandIncPtransfer regions isstriking, whereasthe homology toF transfer systems ismore difficult to detect,withlowidentityvalues.Initially,thereappearedtobethreetypesofT4SSs, F, P, and I. However, large-scale sequencing projects revealed many chimeric transfersystemsthathadanF-typeT4SS,butexpressedacircularP-typepilin(e.g., theIncHI1plasmidR27,Sherburneetal.2000).Thesearequitecommonlyfound, especiallyinclinicalisolates,suggestingthatF-andP-typeT4SSsaremoresimilar thantheyaredifferent.Consequently,theyarenowconsideredsubtypesofthesame group. Another landmark paper that used thin sectioning of a single layer of mating cells reported on the close contact between the donor and recipient cells over an extensive area of both their surfaces (Dürrenberger et al. 1991). Although the resolutionwasverygood,therewasnoevidenceforamatingapparatusorbridgein theseimages,suggestingthatthematingporeiseitherverysmallorveryscarceor forms transiently in response to an unknown signal. The hunt for this mating pore has been a long-sought goal, and researchers in the T4SS field felt somewhat enviousofthosestudyingtypeIIIsecretionsystems,forinstance,whoseneedle-like structures, related to flagellar basal bodies, could be purified intact (Diepold and Armitage 2015). With the development of high-resolution cryo-electron micro- scopy in the last ten years, the structures of a putative T4SS and the F-type pilus have been determined (see Chapter “Structural and Molecular Biology of Type IV SecretionSystems,”Bergé,WaksmanandTerradot).TheprototypeT4SSisarather large structure,approximately185Åby340Å,anditisamarvel tomethat ithas neverbeenseeninthecellenvelopenorhasitbeenpurifiedintactfromcells.There is something transient and ephemeral about the T4SS. It is as if the pilus is a primitivetouchsystemandthatthereisasignalrelayedfromthetipofthepilusto Foreword vii its base that results in formation of the complete structure, allowing it to transport bothproteinandnucleicacid.Thewholequestionoftheenvironmentalandgenetic signalsthatgoverntheexpression,assembly,andfunctionoftheT4SSisdiscussed in Chapter “Prokaryotic Information Games: How and When to Take Up and Secrete DNA” (Stingl and Koraimann). AlongunansweredquestionhasbeenwhethertheDNAcouldpassthroughthe lumenofthepilus,about20Åindiameter,andwhetherproteinscouldpassthrough it as well. There have been many hints that large complexes can be transported, such as the passage of the RNA-A protein complex of RNA phages during phage eclipseandpenetration(Krahnetal.1972)andtheconjugativetransportofprimase proteins in IncP and IncI systems (Merryweather et al. 1986; Rees and Wilkins 1989), tonametwo.Thefinding thatVirproteinsweretransferredtotheplantcell duringtumorigenesisusinggenetictechniquessuchastheCRAFTassay(Vergunst etal.2000)confirmedwhathadbeenpreviouslyrumoredaboutT4SS.Thisunusual anduniqueabilitytotransportproteinandnucleicacid(eitherRNAorDNAinboth directions, during phage infection, conjugation, or transformation) has provided a target for inhibiting this process as reviewed by Baron and Sharifahmadian (Chapter “Type IV Secretion in Agrobacterium Tumefaciens and Development of Specific Inhibitors”). Conjugation requires an energized membrane, a mating bridge between donor andrecipientcellspresumablyformedbytheT4SS,andtheproteinsresponsiblefor initiating and processing DNA transfer. These latter proteins, in complex with the DNA substrate, form the relaxosome (Chapter “Relaxases and Plasmid transfer in Gram-NegativeBacteria,”ZechneranddelaCruz).Thiscomplexcontains,inmost cases, two ATPases, a relaxase and a coupling protein. Whereas the relaxase is importantforsingle-strandedDNAtransfer,thecouplingprotein,VirD4,isthereal hallmarkofaconjugativesystem.Theessentialroleofthecouplingproteinsinboth ssDNAanddsDNAtransferisreviewedinChapter“CouplingProteinsinTypeIV Secretion” (Llosa and Alkorta). Conjugative transfer in Gram-positive (Gram+) bacteria has long been noted in severalspeciesandhasbeenstudiedindetailinEnterococcusfaecalis(Dunnyand Berntsson2016).ThepresenceinGram+bacteriaofamodifiedT4SS,inwhichtwo ofthesignatureATPases(VirB4andVirD4,thecouplingprotein)andaVirB1-like transglycosylase can easily be discerned, by insilicoanalysis, hasreally expanded our knowledge about the versatility of T4SS. No pilus appears to be required for conjugative DNA transfer in Gram+ species; instead, the T4SS is adapted to their single membrane and robust cell wall that must be breached in both the donor and recipient. Other Gram+ bacteria such as Streptomyces and certain archaea use a coupling protein similar to the partitioning protein FtsK or sporulation protein SpoIIIE that can transfer double-stranded DNA without a T4SS. That plasmid transfer could be whittled down to a single Tra protein and a few inessential accessory proteins was a revelation at the time (Kendall and Cohen 1988). The findings with Gram+ bacteria certainly call into question the role of the pilus. A more detailed discussion of progress in Gram+ conjugation is reviewed in viii Foreword Grohmann, Keller, and Muth (Chapter “Mechanisms of Conjugative Transfer and Type IV Secretion-Mediated Effector Transport in Gram-Positive Bacteria”). Sequencing of bacterial genomes has revealed the presence of T4SS in many − pathogenic Gram-negative (Gram ) bacteria, on plasmids, and within the chro- mosome on pathogenicity islands (PAIs) and integrative conjugative elements (ICEs).IfT4SSscansecreteproteinsduringconjugation,thenanobviousquestion is whether they can transport effector molecules into a host target cell or the subcellular spaces such as vacuoles during the infectious process. Our under- standing of the cagT4SS of Helicobacter pylori has increased greatly in the 22 years since its complete sequence was reported (Backert et al., Chapter “The Helicobacter pylori Type IV Secretion System Encoded by the Cag Pathogenicity Island:Architecture,FunctionandSignaling”).T4ASSsarealsoresponsibleforthe pathogenicity of Bartonella and Brucella species (Chapter “Type IV Effector Secretion and Subversion of Host Functions by Bartonella and Brucella Species,” Dehio and Tsolis) and the tick-borne Rickettsiales members Anaplasma phagocy- tophyllum and Ehrlichia chaffeensis (Chapter “Role and Function of the Type IV Secretion System in Anaplasma and Ehrlichia Species,” Rikihisa). Similarly, the T4BSS, characterized as the icm/dot locus of Legionella pneumophila, has been extensively studied in a few pathogens. This T4SS is responsible for the intravacuolar lifestyle of Legionella (Chapter “Subversion of Host Membrane Dynamics by the Legionella Dot/Icm Type IV Secretion System,” Roy, Hilbi and Nagai) as well as delivery of a vast array of effectors and metaeffectors during infection by Coxiella burnetii (Chapter “Beginning to Understand the Role of the Type IV Secretion System Effector Proteins in Coxiella burnetii Pathogenesis,” Lührmann, Newton and Bonazzi). AbacteriumthatI’vekeptaneyeonformanyyearsisNeisseriagonorrhoeae.It was known to encode a type II secretion system (T2SS) that assembles a type IV pilus,asubjectofstudyformyPh.D.thesis,longbeforeaT4SSwasdiscoveredin its genome sequence. That it uses this T4SS to extrude single-stranded DNA into the media to facilitate transformation and gene conversion, a key factor in Neisseria’s modus vivendi, is truly unique (Chapter “Secretion of Chromosomal DNAbytheNeisseriaGonorrhoeaeTypeIVSecretionSystem,”Callaghanetal.). Thistakesusfullcirclebacktomyyouthwhenwewouldponderwhetherwecould “trick” plasmids into releasing DNA into the medium. TheprogressintheT4SSfield(indeed,secretioningeneral)overthelast10–20 years has been really remarkable (Grohmann et al. 2018). However, there are still many aspects of T4SSs that are puzzling. How does the system know that a recipient or host cell has been found? Is this the sole function of the pilus or is it involvedinmacromoleculartransport?Ifitis,thenwhydoGram+bacterianothave piliandindeed,whyareP-typepilishedfromthecellintothemedium?Howdoes − the nucleic acid or protein getinto thetarget recipient cell? In Gram conjugation, the DNA appears to bypass the periplasm and is transported into the cytoplasm directly.Isthereatubethatextendsthroughthetwocellenvelopessimilartothetail tubes of phages? Would this also be true for the delivery of effector proteins in pathogens? Is it possible to see the T4BSS structure in the cell? Why is there an Foreword ix extra ATPase (VirB11) in P-type systems and what is it doing? There are still so many questions! I am very pleased to see so much progress on these systems. I look forward to new findings in T4SS and congratulate the contributors to this book on their remarkable advancement so far. May it continue. Edmonton, AB, Canada Laura S. 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