Methods in molecular biology vol 1531 type 3 secretion systems methods and protocols

Nilles Key words Type III secretion system , Virulence factor , Injectisomes , Translocon , Effector proteins 1 Type III Secretion Systems In order to manipulate the host, gram-nega

Type 3

Secretion Systems

Matthew L Nilles

Danielle L Jessen Condry Editors

Methods and Protocols

Methods in

Molecular Biology 1531

Series Editor

John M Walker School of Life and Medical Sciences University of Hertfordshire Hatfield, Hertfordshire, AL10 9AB , UK

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Type 3 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

ISSN 1064-3745 ISSN 1940-6029 (electronic)

Methods in Molecular Biology

DOI 10.1007/978-1-4939-6649-3

Library of Congress Control Number: 2016955338

© Springer Science+Business Media New York 2017

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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

The complicated nature of the Type III Secretion System (T3SS) has required many cols be developed or applied to study this apparatus Variance in the secretion system from bacterial species to bacterial species is heavily infl uenced 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 meth-ods 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 modifi ed to work in a different bacterial species than indicated in this book; this is up to you the reader to adapt, try, and

proto-of course publish to share with others who study this fascinating system

Grand Forks, ND, USA Matthew L Nilles Danielle L Jessen Condry

Pref ace

1 Introduction to Type III Secretion Systems 1

Danielle L Jessen Condry and Matthew L Nilles

2 Site-Directed Mutagenesis and Its Application

in Studying the Interactions of T3S Components 11

Matthew S Francis , Ayad A A Amer , Debra L Milton ,

and Tiago R D Costa

3 Blue Native Protein Electrophoresis to Study the T3S System

Using Yersinia pestis as a Model 33

Thomas A Henderson and Matthew L Nilles

4 In Vivo Photo-Cross-Linking to Study T3S Interactions Demonstrated Using

the Yersinia pestis T3S System 47

Thomas A Henderson and Matthew L Nilles

5 Isolation of Type III Secretion System Needle Complexes by Shearing 61

Matthew L Nilles , Danielle L Jessen Condry , and Patrick Osei-Owusu

6 Use of Transcriptional Control to Increase Secretion

of Heterologous Proteins in T3S Systems 71

Kevin J Metcalf and Danielle Tullman-Ercek

7 Characterization of Type Three Secretion System Translocator Interactions

with Phospholipid Membranes 81

Philip R Adam , Michael L Barta , and Nicholas E Dickenson

8 Analysis of Type III Secretion System Secreted Proteins 93

Danielle L Jessen Condry and Matthew L Nilles

9 Fractionation Techniques to Examine Effector Translocation 101

Rachel M Olson and Deborah M Anderson

10 Measurement of Effector Protein Translocation Using Phosphorylatable

Epitope Tags and Phospho-Specific Antibodies 111

Sara Schesser Bartra and Gregory V Plano

11 A TAL-Based Reporter Assay for Monitoring Type III- Dependent

Protein Translocation in Xanthomonas 121

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 141

Kyaw Aung , Xiufang Xin , Christy Mecey , and Sheng Yang He

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 155

Patrick Osei- Owusu , Matthew L Nilles , David S Bradley ,

and Travis D Alvine

14 Detecting Immune Responses to Type III Secretion Systems 165

Peter L Knopick and David S Bradley

15 Recombinant Expression and Purification of the Shigella Translocator IpaB 173

Michael L Barta , Philip R Adam , and Nicholas E Dickenson

16 Expression and Purification of N-Terminally His-Tagged Recombinant

Type III Secretion Proteins 183

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 193

Travis D Alvine , David S Bradley , and Matthew L Nilles

18 Identification of the Targets of Type III Secretion System Inhibitors 203

Danielle L Jessen Condry and Matthew L Nilles

19 Detection of Protein Interactions in T3S Systems Using Yeast

Two-Hybrid Analysis 213

Matthew L Nilles

Index 223

Contents

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

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

Contributors

Matthew L Nilles and Danielle L Jessen Condry (eds.), Type 3 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

Chapter 1

Introduction to Type III Secretion Systems

Danielle L Jessen Condry and Matthew L Nilles

Key words Type III secretion system , Virulence factor , Injectisomes , Translocon , Effector proteins

1 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 func-tions ranging from benefi cial to detrimental manipulation of eukary-otic cells [ 1 , 2 ] T3S systems are found in many human pathogenic

gram-negative bacteria including pathogenic strains of Escherichia

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 regula-tion 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

systems that affect plants fall into two families Hrp1 and Hrp2 and the remaining two families belong exclusively to the phyla of bac-

teria Cylamydiae and the order Rhizobiales It is well known that

one bacteria can express more than one family of T3S systems, as

most notably occurs with the genera Salmonella expressing both a

SPI- 1 and a SPI-2 type of secretion system [ 3 ] The SPI-1 T3S family is also noted for the ability to secrete effectors into multiple

structural proteins of T3S systems are homologous between all these families; those proteins that are not homologous often still have an analogous protein with an equivalent function [ 3 ]

2 Structure

The T3S system is comprised of approximately 25 different proteins that make up the basal body, needle , and translocon [ 3 ] These struc-tural genes are found in a gene cluster in all known species and are thought to be acquired via horizontal gene transfer during evolution These gene clusters could be located on a plasmid or on the main chromosome [ 6 ] The basal body embeds in the inner and outer bac-terial membranes via two ring-like structures connected by a rod structure (Fig 1 ) [ 7 ] The basal structural components 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 )

Danielle L Jessen Condry and Matthew L Nilles

conserved between T3S systems, including bacterial fl agella [ 3 ] On the cytosolic side of the basal structure an ATPase can be found that

is critical for the secretion of proteins [ 3 ] The internal channel of the T3SS is about 2–3 nm, only big enough for unfolded proteins to pass through [ 3 ] The number of needle complexes per bacteria varies, from 10 to 100 complexes, depending on the species [ 8 ]

Extending out from the basal structure is a hollow needle (Fig 1 ) [ 3 ] This portion of the secretion system is made up of repeating subunits of one protein and a cap protein that sits at the tip [ 9 ] The sequence of needle proteins is largely conserved between bacterial species, except the N-terminus X-ray crystallog-raphy and NMR have been utilized to detect structures of some

needle proteins, including MxiH from Shigella [ 10 ], BsaL from

Burkholderia pseudomallei [ 11 ], and PrgI from Salmonella enterica serovar Typhimurium ( S Typhimurium) [ 11 ] The crystal structure

of MxiH was used to generate a model of the T3S needle structure [ 10 , 12 , 13 ] The MxiH-derived model of the needle protein pos-sesses two coiled domains with the N-terminus of the needle pro-tein predicted to line the lumen of the T3S needle [ 10 ] The N-terminus of the needle protein in all these cases was seen to be highly mobile and disordered [ 11 , 14 ] offering little data to defi ne structures of this portion of the protein Sun et al reported the N-terminus in their crystal structure to be largely unorganized and not representative of the protein in its needle conformation [ 15 ] Contrary to previous models, recent work by Loquet et al has

revealed that the N-terminus of the needle protein from Shigella is,

in fact, on the outside surface of the needle, exposing it to host ments, while the conserved carboxy end faces the lumen [ 16 ] How needle length is determined is hypothesized by several models Models suggest a ruler method where a specifi c protein dictates the length of the needle, a cup method where a specifi c number of needle proteins are released to create the needle, or oth-ers suggest a combination of these two models with the proteins that dictate substrate switching also involved in determining needle length [ 3 ] Length of the needle depends on the species of bacteria and studies have shown that this length is critical in the ability of the bacteria to deliver effectors to the host [ 3 ] Length of the needle is correlated with the length of major features on the outer surface of the bacteria such as adhesins [ 17 ] At the tip of the needle is a pro-tein that “caps” the apparatus and interacts with the fi nal portion of the structure that imbeds in the host membrane [ 9 ]

The translocon completes the T3S system (Fig 1 ) This ture is made up of two hydrophobic proteins that insert into the host membrane, thus creating a channel directly from bacteria cytosol into the host cytosol Through this channel unfolded proteins can move from the bacteria into the targeted host cell Some bacterial species show that these proteins make up the cap structure as well; however, this has not been shown true with all T3S systems [ 7 , 9 ]

3 Effectors

Effector molecules can mediate several functions including but not limited to bacterial uptake, alterations of the immune response, or prevention of phagocytosis [ 1 ] There are hundreds of different types of effectors across all T3S systems although some do show homology between different species [ 18 ] Effector proteins can be found within the structural loci or outside that loci, sometimes with regulatory genes [ 6 ] Effector proteins can mimic host cell protein function to irreversibly control specifi c functions of the host cell [ 1 ] The majority of these proteins carry a conserved N-terminal secretion signal [ 19 ] as well as a chaperone -binding domain to allow targeting to the T3S system for export [ 18 ]

4 Regulation

Regulation of this system is crucial for the delivery of effectors at the precise time needed Structural genes are largely regulated by environmental factors such as temperature, osmolality, and pH [ 6 ] Most agree that host cell contact is crucial for activation; however, how this happens and through which proteins is a major debate in this fi eld [ 3 , 7 , 9 , 14 , 20 ] Many proteins function to regulate secretion, though the particular protein and function can vary between different bacterial species and is often located outside the structural gene loci [ 6 ] Overall, however, current theories hypoth-esize the importance of the needle as a regulatory element [ 21 ] In vivo, contact with the host cell membrane is required to initiate translocation of effectors [ 22 ] One hypothesis of regulation via the needle is that the signal is structurally relayed via conforma-tional changes of the needle from the tip to the base Another hypothesis, separate from needle protein structure, involves a pro-tofi lament that once released signals secretion [ 8 ] Several mutants

of needle proteins have been produced that alter the regulatory control of secretion [ 23 – 25 ]; however, an exact mechanism has not been confi rmed by analysis of these mutants

5 Overview of Select Bacteria that Use T3S Systems

Yersinia pestis employs many factors to cause disease; primarily,

these factors are critical for evading detection or suppressing the

immune system of the host More specifi cally, the T3SS in Yersinia

pestis plays a key role in the prevention of phagocytosis, the

manip-ulation of cytokine expression, and killing of immune cells [ 26 ]

In Yersinia pestis the T3S system is encoded by the pCD1

plas-mid Also on this plasmid are effectors, chaperones , and regulatory

5.1 Yersinia

Danielle L Jessen Condry and Matthew L Nilles

proteins that are necessary for expression, construction, and

becomes avirulent and is easily cleared by the host immune system [ 19 ] At 37 °C, the LcrF protein is produced LcrF is responsible for the temperature-dependent activation of genes on pCD1 that

RNA thermosensor, which once shifted to above 30 °C allows for translation to occur [ 27 ]

The base of the T3S system of Yersinia pestis is made up of

proteins termed Ysc ( Yop secretion) (Fig 1 ) [ 28 ] The structure is built in the outer membrane fi rst, made up of YscC, then proceeds

to building the inner ring via YscD and YscJ [ 29 ] YscQ reportedly makes up the C-ring on the cytosolic face of the basal structure [ 29 ] YscQ then interacts with the ATPase, YscN, and subsequently YscN requires YscK and YscL [ 30 ] Also essential are integral mem-brane proteins YscR, YscS, YscT, YscU, and YscV that are thought

to recognize or secrete the Ysc substrates [ 31 ]

Extending out from the base is a hollow needle structure, made up of repeating subunits of YscF Currently, YscF has only been crystallized in complex with its chaperones YscE and YscG [ 32 ] The pore forming structure at the end of the needle is called the translocon [ 9 , 33 , 34 ] This structure is made up of three pro-teins: LcrV , YopB, and YopD [ 9 ] LcrV creates a base on the tip of the YscF proteins that make up the needle [ 3 ] and functions to help insert the hydrophobic translocator proteins, YopB and YopD,

and allow Yops to translocate from the needle apparatus into the host cell [ 9 ] In Yersinia there is no evidence for the order or tim-

ing of secretion to assemble the translocon It is presumed that due

to the hydrophobic nature of YopB and YopD, these proteins are not assembled at the tip prior to cell contact [ 35 ] The translocon

as a whole has yet to be isolated and visualized to confi rm this assumption [ 9 ] This is contrary to the T3S system in Shigella

where the T3S assembles its major hydrophobic translocator before cell contact [ 36 ] In secretion profi les of Yersinia pestis , in vitro, all

three proteins are secreted into the medium

Effector proteins are the toxins of the T3S system These teins, termed Yops (Yersinia outer proteins ), are translocated into the host cell and damage host responses [ 19 ] Yops have an N-terminal secretion signal [ 1 ] and are translocated in an unfolded state [ 19 ] Regulation of the T3S system is a complex process Under

pro-in vivo conditions cell contact is known to trigger secretion pro-in this system [ 10 ] How that signal is relayed to the inside of the bacteria

is not known, although one theory suggests a conformational change occurs in structural proteins that brings the message to appropriate regulatory cytoplasmic molecules [ 14 ] Under in vitro

conditions, the Yersinia pestis T3S and the Pseudomonas aeruginosa

T3S can be triggered by depleting the media of calcium [ 37 ] This

response is known as the Low Calcium Response ( LCR ) Several proteins are involved in the regulation process of secretion from inside the bacteria LcrG blocks secretion that can be alleviated by interaction with LcrV [ 38 – 41 ] YopN and YopN’s chaperones SycB and SycN , along with TyeA , form a complex that also regu-lates secretion of Yops [ 42 , 43 ] YopN regulation is thought to be alleviated by secretion of YopN [ 38 ] Deletion of these regulatory proteins results in an altered ability to secrete Yops Either secre-tion will not occur, such as in the case of deletion of LcrV [ 44 ], these strains are referred to as being calcium independent; or the opposite effect can occur where secretion will occur constitutively resulting in Yops secretion, for example a strain lacking LcrG [ 41 ]

or YopN [ 43 ] These strains are called calcium blind strains An additional factor that occurs in vitro when secretion is triggered is

a twofold event involving a transcriptional increase in Yops sion and an overall growth restriction of the bacteria [ 19 ]

Escherichia coli ( E coli ) is a gram-negative bacterium that can cause enteric diseases in humans Notably, enteropathogenic E coli (EPEC) and enterohemorrhagic E coli (EHEC) are known to utilize the T3S

system to deliver proteins that aid in attachment and effacing of host cells in intestinal epithelial [ 6 , 45 – 47 ] E coli has one confi rmed T3S

system that is called ETT1 This T3S system is encoded on the locus of enterocyte effacement ( LEE ) pathogenicity island [ 47 – 49 ] Another

T3S system is also suspected in E coli , labeled ETT2 The ETT2 gene cluster is highly homologous to the SPI-1 T3S system of Salmonella

tem are referred to as Esp - X Expression of structural ETT1 T3S tem genes is controlled by temperature, as well as, growth phase of the bacteria In vitro activation of secretion can be induced by sodium bicarbonate, calcium, and Fe(NO 3 ) 3 and NH 4 + [ 6 ]

Salmonella enterica is a gram-negative pathogen that causes enteric

disease in humans [ 50 , 51 ] The bacteria are spread by ingestion of contaminated food, and infection causes diseases ranging from

diarrhea to typhoid fever There are several serovars of enterica :

Typhi causes Typhoid fever in humans while Typhimurium causes

a Typhoid like illness in mice [ 33 ] Once Salmonella has reached

the intestine the bacteria attempts to move across the epithelium layer by invading M-cells [ 50 ] This is achieved by the use of one

of Salmonella’s two T3S systems, Salmonella Pathogenicity Island

1 (SPI-1) [ 50 , 51 ] SPI-1 plays multiple roles in infection Initially

in infection SPI-1 effectors cause phagocytosis of the bacteria into epithelial cells and also cause an increase in infl ammatory media-tors and fl uid movement into the intestine [ 51 ] The infl ammation caused by this system loosens tight junctions in the epithelial layer, which can allow more bacteria to pass into the lamina propria [ 50 ] SPI-1 is also capable of causing apoptosis of macrophages [ 51 ]

5.2 Escherichia

5.3 Salmonella

Danielle L Jessen Condry and Matthew L Nilles

macrophages This is accomplished with the other T3S system of

Vacuole ( SCV ) SPI-2 effectors protect the bacteria from reactive oxygen and nitrogen species and orchestrate delivery of materials from the host cell to the SCV to facilitate bacteria growth [ 51 ]

SPI-1 and SPI-2 of Salmonella are found in two separate families

of T3S systems The SPI-1 T3S system is more closely related to the

T3S system found in Shigella , while SPI-2 resembles the E coli T3S

system [ 3 ] Expression of SPI-1 and SPI-2 T3S system structural genes is activated by a combination of low oxygen, high osmolality, and slightly alkaline conditions that vary at different stages of infec-tion [ 6 , 52 ] Effectors of the Salmonella system are referred to by

Sip / Ssp / Sop ; however, many other proteins are able to be secreted

by this secretion system including SptP , AvrA that have been shown

to have homology to secreted effectors in other T3SS [ 6 ]

Shigella is a genus of gram-negative bacteria of the Enterobacteriacae family There are four species: fl exneri , sonnei , dysenteriae , and boy-

dii Shigella fl exneri and sonnei cause endemic forms of dysentery,

while Shigella dysenteriae is associated with epidemics These

bacte-ria are spread by contamination of food or water and only infect

humans Symptoms associated with Shigella range from moderate

to severe diarrhea and in more severe cases fever, abdominal cramps, and bloody mucoid stools Death from this pathogen usually results from septic shock, severe dehydration, or acute renal failure [ 53 ]

Once inside the host Shigella targets the colon and moves past

the epithelial layer via M-cells After crossing the intestinal barrier the bacteria interacts with macrophages and dendritic cells This interaction causes an increase in pro-infl ammatory cytokines and chemokines The increase in infl ammation 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 of the Shigella

T3S structure appears to rely on temperature, osmolality, and pH [ 6 ] Effectors not only mediate uptake into the cell but also begin manipulating the immune response to favor high infl ammation [ 53 ]

Effectors in Shigella include IpaA -D, IpaB-D are known to induce

membrane ruffl ing in epithelial cells via actin rearrangement [ 6 ] IpaA appears to optimize invasion of the host cell [ 54 ] MxiH , which makes up the needle of this T3S system, has been crystal-lized and used to predict the needle structure [ 10 , 12 ] Mutants of MxiH indicate that the needle protein plays a role in “sensing” host cell contact and the triggering of secretion [ 23 ]

Pseudomonas aeruginosa is also a gram-negative pathogen that infects humans This pathogen is associated with several acute dis-ease types ranging from pneumonia to infections of the urinary tract, wounds, burns, and bloodstream Cystic fi brosis patients are

keenly susceptible to Pseudomonas infections as well

5.4 Shigella

5.5 Pseudomonas

Like many gram-negative pathogens Pseudomonas also utilizes

a T3S system to manipulate the host Only four effectors of the

T3S system of Pseudomonas exist: ExoS, ExoT, ExoU, and ExoY

These effectors are capable of preventing phagocytosis, altering cell traffi cking, inhibiting cytokine release, and causing cell death [ 55 ] Ultimately, Pseudomonas ’ goal is to evade innate immunity

[ 24 ] The T3S system of Pseudomonas is closely related to the T3S system of Yersinia and in vitro is also activated by depletion of cal-

cium in the environment [ 3 ] Studies by Broms et al have revealed

the ability of some Yersinia proteins to substitute for homologous Pseudomonas proteins; however, the reverse does not always work YopD specifi cally can function in Pseudomonas ; however, PopD , the Pseudomonas homolog, cannot substitute for YopD, specifi cally

YopD’s regulatory functions This study also revealed the tance of translocon protein chaperones for proper function [ 56 ]

impor-6 Notable Plant Bacteria Species with T3S Systems

T3S systems are conserved in four major plant pathogenic gram-

negative bacteria, as well as involved in symbiotic Rhizobium spp

response defenses and result in resistance to the pathogenic bacteria species [ 6 ] Bacterial genes involved in the T3SS are defi ned as hrp

(hypersensitive response and pathogenicity) [ 57 ] T3S system tors include Avr proteins that function to counteract the resistance

in different plant species Just like in mammalian T3S system tors, the variety of effectors in plant pathogens appears to be specifi c

effec-to the species of plant that bacteria infects but some homology does exist even across effectors that affect mammalian and plant hosts

Regulation of the T3S system in Pseudomonas syringae (bacterial speck) and Erwinia amylovora (Fire Blight) is regulated in vitro by

minimal salts medium, complex nitrogen sources, pH, osmolality, and some carbon sources In vivo regulation is thought to occur by contact and secretion is initiated within hours of inf ection [ 6 ]

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12 Blocker A, Jouihri N, Larquet E, Gounon

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18 Dean P (2011) Functional domains and motifs

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23 Kenjale R, Wilson J, Zenk SF, Saurya S, Picking

WL, Picking WD, Blocker A (2005) The needle component of the type III secreton of Shigella regulates the activity of the secretion apparatus

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24 Sato H, Frank DW (2011) Multi-functional characteristics of the Pseudomonas aeruginosa type III needle-tip protein, PcrV; comparison

to orthologs in other gram-negative bacteria Front Microbiol 2:142

25 Torruellas J, Jackson MW, Pennock JW, Plano

GV (2005) The Yersinia pestis type III tion needle plays a role in the regulation of Yop secretion Mol Microbiol 57:1719–1733

26 Amedei A, Niccolai E, Marino L, D’Elios

MM (2011) Review Article: Role of immune response in Yersinia pestis infection J Infect Dev Ctries 5(9):628–639

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28 Perry RD, Fetherston JD (1997) Yersinia tis—etiologic agent of plague Clin Microbiol Rev 10:35–66

29 Diepold A, Amstutz M, Abel S, Sorg I, Jenal U, Cornelis GR (2010) Deciphering the assembly

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30 Jackson MW, Plano GV (2000) Interactions between type III secretion apparatus compo- nents from Yersinia pestis detected using the yeast two-hybrid system FEMS Microbiol Lett 186:85–90

31 Sorg I, Wagner S, Amstutz M, Müller SA, Broz P, Lussi Y, Engel A, Cornelis GR (2007) YscU recognizes translocators as export sub- strates of the Yersinia injectisome EMBO

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32 Sun W, Curtiss R (2012) Amino acid tutions in LcrV at putative sites of interac- tion with toll-like receptor 2 do not affect the virulence of Yersinia pestis Microb Pathog 53(5–6):198–206

33 Broz P, Mueller CA, Müller SA, Philippsen A, Sorg I, Engel A, Cornelis GR (2007) Function and molecular architecture of the Yersinia injectisome tip complex Mol Microbiol 65:1311–1320

34 Matteï P-JJ, Faudry E, Job V, Izoré T, Attree

I, Dessen A (2011) Membrane targeting and

pore formation by the type III secretion system

translocon FEBS J 278:414–426

35 Mueller CA, Broz P, Müller SA, Ringler P,

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forms a distinct structure at the tip of

injecti-some needles Science 310:674–676

36 Veenendaal AKJ, Hodgkinson JL, Schwarzer

L, Stabat D, Zenk SF, Blocker AJ (2007) The

type III secretion system needle tip complex

mediates host cell sensing and translocon

inser-tion Mol Microbiol 63:1719–1730

37 Bölin I, Portnoy DA, Wolf-Watz H (1985)

Expression of the temperature-inducible outer

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38 Hamad MA, Nilles ML (2007) Structure-

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40 Nilles ML, Williams AW, Skrzypek E, Straley

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Danielle L Jessen Condry and Matthew L Nilles

Matthew L Nilles and Danielle L Jessen Condry (eds.), Type 3 Secretion Systems: Methods and Protocols, Methods in

Molecular Biology, vol 1531, DOI 10.1007/978-1-4939-6649-3_2, © Springer Science+Business Media New York 2017

Chapter 2

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

foremost objective to better understand the fundamental workings of the T3SS, using Yersinia as a model

pathogenic bacterium Examples are given that clearly document how pDM4-mediated site-directed mutagenesis has been used to establish 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 system

Key words Site-directed mutagenesis , Type III secretion systems , Suicide vector pDM4 , Mutant libraries , Genetic-based screens , Protein-protein interaction assays

1 Introduction

Many bacteria evade eukaryotic host immune responses by using type III secretion systems (T3SSs) that inject bacterial effector mol-ecules directly into target host cells (Fig 1 ) [ 1 – 3 ] The T3SS appa-ratus is composed of some 25 proteins, which when completely assembled not only spans the entire bacterial envelope but also pro-trudes outward from the bacterial surface, taking the form of a syringe- needle structure It is through this structure that the effec-tors are directly injected into eukaryotic cells [ 4 ] These injected effectors possess enzymatic activities that subvert host cell signaling for the bacteria’s benefi t They are the third and last (“late-secreted”) class of protein to be secreted by an assembled T3SS The fi rst are the “early secreted” structural needle components that extend from the bacterial surface, and the second are the pore-forming

1.1 Type III Secretion

of Proteins by Bacteria

“middle-secreted” injectisome components that sit at the top of the needle (Fig 1 ) [ 1 – 3 , 5 ] From this position, injectisome compo-nents form pores in infected cell plasma membranes through which the “late” effectors may gain entry into the cell cytosol

The pre-secretory stabilization and effi cient secretion of each pre-secreted substrate generally requires a customized cytoplasmic T3S chaperone ; class I chaperones target “late” effectors, class II target the “middle” pore-formers, and class III target the “early” needle components [ 1 ] Chaperone -substrate complexes are prob-ably recognized by the T3S machinery to act as dedicated substrate secretion signals [ 6 , 7 ] Each substrate has also their own

8 , 9 ] Together, chaperone -dependent and -independent secretion signals could contribute a unique recognition motif allowing the T3SS to demarcate substrates into “early,” “middle,” and “late” secretion events

It is crucial to understand this hierarchal secretion process because it is the basis of T3SS activity, i.e., “early” secreted sub-strates fi rst polymerize needle components at the bacterial surface that then permit secretion of “middle” substrates to form injecti-some pores in the target cell plasma membrane that in turn are needed for the internalization of “late” effector substrates into target cells Research in our laboratory focusing on this issue

employs the model bacterial pathogen Yersinia

Needle components

(early substrates)

Injectisome pore components

(late substrates)

Eukaryotic plasma membrane

OM

CM

Bacterial envelope

Fig 1 The concept of hierarchal substrate by a T3SS In resting state, a T3SS apparatus is capable of secreting

“early substrates” that complete the needle A switching mechanism that senses target cell contact swaps the secretion of earlier cargo for “middle substrates” that form a translocon pore in the eukaryotic cell membrane Once this injectisome assembly is complete, the T3SS is again reprogrammed to prioritize the secretion of

“late substrates” termed effectors that are injected into the host cell through the recently assembled

injecti-some OM bacterial outer membrane, CM bacterial cytoplasmic membrane

Matthew S Francis et al.

Pathogenic Yersinia sp comprise Y pestis , the causative agent

of often fatal bubonic and pneumonic plague, and the enteric Y pseudotuberculosis and Y enterocolitica responsible for self-limiting

food-borne infections [ 2 ] Although the route of infection and ease outcome is different, all three species resist anti-phagocytic host defense mechanisms allowing extracellular replication within lymphoid tissue [ 10 ]—a process mediated by the Ysc- Yop T3SS encoded on a common ~70-kb virulence plasmid [ 2 ] The Yersinia T3SS consists of numerous Ysc ( Yersinia s ecretion) components

dis-that assemble into a functional apparatus specifi cally to secrete at

least three protein classes of Ysc’s and Yop ’s ( Yersinia o uter p

ro-teins ); the “early” needle components (YscF, YscX) and anti-

injected immuno-suppressive enzymes and toxins (YopE, YopH, YopJ, YopM, and YpkA) [ 2 ]

T3SSs are complex biological machines To pry apart the inner

workings of the Y pseudotuberculosis T3SS, we and others have

taken a genetics-based approach reliant on the creation by site- directed mutagenesis of isogenic phenotypic mutants Not only has this provided the basis for understanding the minimal molecu-lar components required for a functional T3SS apparatus, but it has also permitted detailed investigations into the molecular interac-tions among these structural components as well as investigations into the molecular interactions of the secreted cargo, including the intracellular targets of the injected effectors To achieve all of this, genetic studies in our laboratory and in several other Yersinia

research laboratories at Umeå University have relied heavily on the use of a site-directed mutagenesis system based upon the suicide vector pDM4 generated by coauthor Debra Milton [ 11 ] (Fig 2 ) Plasmid pDM4 is sequenced completely, and this sequence has been deposited in the NCBI database with the GenBank accession number KC795686

A T3SS can incorporate some 25 structural components, several regulatory proteins, as well as the numerous examples of secreted cargo Hence, in an effort to understand the inner workings of a vastly complex T3SS, it has been generally convenient to demarcate the many different components into functional categories composed

of a fewer number of components It is in this vein that we and ers have addressed T3SS research, and this section describes a num-ber of studies in which pDM4-based site-directed mutagenesis has been employed to demarcate function of various T3SS components, and in particular to verify the physiological relevance of their homol-ogous and heterologous protein -protein interactions

The T3SS apparatus spans the bacterial envelope and anchors a dle -like appendage that extends out from the bacterial surface In

Yersinia , this apparatus is chiefl y composed of about 20 Ysc proteins,

including the YscF needle Another important protein is YscU, an integral inner- membrane protein absolutely required for T3SS func-tion YscU belongs to a family of proteins that is characterized by auto-cleavage at a highly conserved C-terminal NPTH motif In par-ticular, auto-proteolysis of YscU occurs between the asparagine (N)

1.3.1 Apparatus

Assembly

Fig 2 Schematic diagram of the pDM4 mutagenesis vector Shown are the

salient features that mark pDM4 as a convenient mutagenesis vector including: the chloramphenicol resistant marker (Cm R ), the R6K-derived oriV replicon mak- ing replication dependent on the pir gene, the RP4-derived oriT and associated transfer ( tra ) regions, the counter selectable marker sacBR , and the multiple

cloning site (MCS) harboring various unique restriction enzyme digestion sites See the text for more precise construction details The diagram is drawn to approximate scale only

Matthew S Francis et al.

at position 263 and the proline (P) at position 264 [ 12 ] Critically, when pDM4-mediated site-directed mutagenesis was used to create

a deletion of the NPTH coding sequence, or used to introduce point mutations that affect cleavage effi ciency at the NPTH motif, func-tionality of the T3SS was lost [ 12 – 14 ] Hence, these studies used targeted mutagenesis to identify the importance of YscU auto-cleav-age in the regulation of Yop synthesis and secretion control

Linked to the function of YscU is the protein YscP These teins are thought to cooperate in an assembly checkpoint termed the “substrate specifi city switch.” It has been proposed that this switching machinery identifi es that the apparatus has matured suf-

pro-fi ciently to enable a change in secretion specipro-fi city from the early secretion of needle subunits (e.g., YscF) to the later export of pore-forming and effector Yops Indeed, when pDM4-mediated site-directed mutagenesis was used to create a full-length deletion

of the yscP allele, the resulting mutant was impaired in substrate

remarkably longer needles that were incapable of supporting Yops secretion [ 14 – 16 ] Interestingly, when pDM4-mediated site-directed mutagenesis was used to create the N263A point mutant

in yscU , a similar “long- needle” phenotype was observed [ 14 ] Further site-directed mutagenesis of yscU revealed single point

mutant phenotype to such an extent that Yop secretion was tially restored [ 13 ] This fi nding is consistent with the notion of an interaction between YscP and YscU [ 14 ] Hence, the signifi cant outcome from these genetic approaches is the anticipation that a YscP-YscU interaction is necessary for the regulation of substrate specifi city switching during type III secretion

Upon successful completion of T3SS assembly and in response to eukaryotic host cell contact, a class of pore-forming translocator proteins are secreted via the completed T3SS needle channel The secreted translocators position themselves at the distal end of the needle, where they can oligomerize in the host cell membrane to build up a structure known as the injectisome translocon pore [ 17 ,

18 ] It is assumed that formation of this pore completes the entire T3SS assembly process, with the result being an uninterrupted conduit for the ensuing passage of effector substrates into the host cell, where their activity is responsible for compromising host cell

functions for the benefi t of the bacteria In Yersinia , YopB, YopD,

and LcrV are prominent translocator proteins responsible for tisome formation The two hydrophobic translocators YopB and YopD physically form the pore in the host cell membrane [ 19 – 23 ], and this process is supported by the hydrophilic LcrV translocator that remains capping the distal tip of the YscF needle [ 24 , 25 ] The YopD protein is particularly interesting because it exerts effects on both effector injection into cells as well as on the

injec-1.3.2 Translocon

Assembly

controlled synthesis and secretion of Yops Hence, pDM4 directed mutagenesis has been used to pry apart the various func-tional domains of YopD First, a deletion analysis identifi ed the

Interestingly, this region encompassed predicted structural motifs such as a coiled- coil domain and an amphipathic alpha helix [ 22 ,

27 – 29 ] Follow-up studies in which many point mutations were generated identifi ed key functional residues of YopD In particular, YopD residues localized in the alpha helical amphipathic domain proved to be critical for YopD to establish both self-oligomerization and an interaction with LcrV , and these two properties seemed criti-cal for Yop effector translocation [ 30 ] A similar genetics-based strategy was undertaken to investigate the existence of a short alpha helical stretch that could constitute a coiled-coil domain [ 31 ] Remarkably, disruption of this domain compromised the ability of YopD to integrate with YopB into biological membranes Importantly, one mutant class could still effi ciently translocate Yop effectors in infected cell culture monolayer in vitro systems, but were avirulent in in vivo competitive infection assays in a mouse model Thus, the fall-out from this study is the idea that YopD could also

presence of translocated YopD in the host cell cytosol [ 32 ]

Ysc- Yop T3SS activity in the presence of immune cells contributes both anti-phagocytic and pro-infl ammatory 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

mediated site-directed mutagenesis system has played an integral role in understanding the intracellular function of these two critical virulence determinants For example, the creation of single amino acid substitutions has been used to investigate substrate recogni-tion specifi city by YopE toward RhoA, Rac1, and Cdc42 Being unable to reconcile in vitro and in vivo phenotypes pertinent to YopE function inferred that the true in vivo target of YopE prob-ably remained unknown [ 38 ] Moreover, the identifi cation of a membrane localization domain within YopE that is essential for

Yersinia virulence, but not GAP activity toward known GTPase

targets, further strengthens the notion that alternative intracellular molecular targets of YopE do exist [ 39 ] It was also apparent from these and other genetic studies that an intended consequence of YopE activity inside infected eukaryotic cells was to regulate the

level of Yops expression and translocation by infecting Yersinia

bacteria [ 38 , 40 , 41 ] Hence, pDM4-derived mutagenesis of YopE has revealed novel insight particularly by enabling the discovery that YopE may actually function primarily as a virulence regulator rather than a classical virulence determinant

1.3.3 Molecular Targets

of Translocated Effectors

Matthew S Francis et al.

Regarding YopH, it is the most potent phosphatase known Hence, it is no surprise that several targets of YopH dephosphory-lation have been reported in a variety of cell lines [ 42 ] In particu-lar, the tyrosine kinase FAK and adaptor proteins p130Cas, paxillin, ADAP, and SKAP-HOM associated with focal complexes appear to

be targets of YopH [ 43 – 46 ] In PMNs that are considered a

natu-ral target of Yersinia T3SS activity, the PRAM-1/SKAP-HOM and

SLP-76 signal transduction pathway(s) are targets of YopH activity

(YopHC/A), which is commonly used to trap YopH targets,

ren-ders Yersinia avirulent [ 45 , 48 , 49 ] Furthermore, a focal complex localization signal was identifi ed in the YopH amino acid sequence, and when the codons encoding this stretch of sequence were

deleted, the resulting Yersinia mutant was also severely attenuated

genetic studies to clearly identifi ed YopH as a critical virulence determinant, with a role to disrupt focal complexes via dephos-phorylation of target proteins being a primary function

Secreted type III substrates commonly employ a dedicated small, nonsecreted cytoplasmic chaperone to ensure their effi cient secre-tion [ 1 ] There have been several established ways in which T3S chaperones may impact on the secretion of their cognate substrate cargo They may prime the substrate for unfolding in preparation for entry into the T3SS, they may act as a secretion pilot ensuring substrate secretion through the correct T3SS, or they may even orchestrate a secretion hierarchy ensuring that substrates of differ-ent functional classes are secreted at the appropriate time [ 1 ] In several instances, this is achieved through the coupling of substrate secretion to gene expression [ 1 ]

A particularly interesting T3S chaperone is LcrH This

chaper-one has two principle functions in Yersinia T3S, the fi rst as a

stabi-lizer by preventing premature interactions between the two translocon proteins YopB and YopD [ 51 – 54 ], and the second as a general regulator of type III substrate synthesis and secretion

translocators have been studied in some detail Utilizing in-frame deletion mutagenesis uncovered two LcrH-interacting domains in YopD—the fi rst 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 specifi c point mutations discovered a clear role for hydro-phobic residues within this amphipathic α-helix in the interaction with LcrH [ 52 ] Interestingly, the amphipathic domain is essential for YopD function, and possibly even contributing to YopD oligo-merization [ 19 , 27 , 30 , 32 ], so it is possible that LcrH binding has the purpose to prevent premature YopD oligomerization in the bacterial cytoplasm

1.3.4 Chaperones

and Their Secreted

Substrates

An important feature of LcrH and related chaperones is that their structure is dominated by three tetratricopeptide repeats (TPRs) [ 60 ] Seemingly, these repeats must provide an ideal scaf-fold for poorly characterized interactions with a wide array of addi-tional T3SS components exclusive of the well-established partners

in YopB and YopD [ 57 , 59 , 61 – 63 ] Driven in part by the ery of TPRs in LcrH, extensive pDM4-mediated mutagenesis has since generated a comprehensive collection of LcrH point mutants [ 51 , 64 ] Phenotypic analysis of all of these point mutants leads to the demonstration that the three TPRs actually played signifi cant roles in chaperone stability and dimerization, substrate binding, and substrate secretion [ 51 ] From these data even came the real-ization that only minimal YopB and YopD translocator secretion is necessary for Yop effector delivery into eukaryotic cells [ 64 ], which highlights the impressively tight control LcrH must have over YopB and YopD secreti on

T3SS activity requires the utmost degree of coordination between the different assembly steps to establish an apparatus with optimal functional output Several regulatory proteins are known to assist

in orchestrating injectisome assembly before the injection of tors into the target eukaryotic cell interior can occur [ 1 , 3 , 5 ] The InvE-MxiC family of proteins are a class of regulators known for their role in ensuring translocator (middle substrates) secretion before effector (late substrates) secretion [ 3 ] In Yersinia , a homo-

effec-logue 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 mutagen-esis strategy facilitated an investigation of the role of YopN and

TyeA interplay in regulating export of Yop substrates in Y

pseudo-tuberculosis The analysis of several in cis mutants producing YopN

variants truncated or altered in the C-terminus revealed the

Subsequent use of the pDM4 system was central to the identifi tion of key residues in the YopN C-terminus that were necessary to establish hydrophobic contacts with the N-terminus of TyeA [ 68 ] This study further confi rmed the essential requirement of a

and secretion [ 69 – 72 ]

Interestingly, Y pestis and Y pseudotuberculosis but not Y

enterocolitica were previously shown to produce naturally a

singu-lar YopN- TyeA protein [ 73 ] As this was more in line with the ous singular polypeptides making up membership within InvE-MxiC-like protein family, pDM4 site-directed mutagenesis was used in an effort to defi ne the biological role of this YopN-

TyeA hybrid molecule in the T3SS produced by Y

mutations in yopN that effectively generated translational fusion

1.3.5 Regulatory

Complexes

Matthew S Francis et al.

variants in which the TyeA N-terminus was appended to the YopN C-terminus [ 67 ] These hybrids were effectively synthesized and secreted, and also maintained full T3SS assembly and function

in vitro Yet their ability to polarize YopE translocation upon eukaryotic host cell contact was impaired, and these mutants failed

to compete with parent Yersinia when subjected to competitive

survival assays in orally infected mice [ 67 ] Taken together, these

fi ndings highlighted the signifi cant biological role of YopN and

TyeA in controlling Ysc-Yop T3SS in Yersinia Moreover, it was

quite evident that readouts from in vitro and in vivo assays cannot always be reliably compared, and more emphasis should be placed

on the in vivo analyses of mutant bacteria in animal infection els, and this is especially critical when probing for subtle pheno-typic defects

The pDM4-based site-directed mutagenesis system has proven to

be a powerful and effective method for the manipulation of DNA

in Yersinia The examples described above have focused on the

generation of point mutations or deletions However, the system is equally effective at generating insertion mutations and in the reconstitution of existing mutations with the wild-type allele (i.e.,

in cis complementation) or hybrid variants composed of chimeric

fusions between two or more homologous alleles [ 28 , 57 , 74 ] Moreover, the system results in pure mutations without leaving behind any residual scar in the form of additional nucleotides Finally, although this system was originally designed for use in

Vibrio species [ 11 ], it has since proven to be very effective in a wide

range of Gram-negative bacteria, including Vibrio spp., Yersinia spp., Pseudomonas aeruginosa , Burkholderia spp., and Francisella

spp just to name a few

Despite the success of the pDM4-based mutagenesis system, it would be remiss not to highlight other mutagenesis strategies that

have proven effective in elucidating T3SS function in Yersinia The

works well in Yersinia , and for example has been used to study the

SycN /YscB chaperone and its effects of YopN regulatory function

mutagenesis technique [ 77 ] For example, this technique has been used to verify a collection of YopN residues that constitute an interaction surface for the docking of other regulatory proteins [ 72 ] Other vector-based mutagenesis systems have also been developed

for use in Yersinia The Guy Cornelis laboratory has generated an

effective system [ 78 ] that has been broadly used to study lar interactions between T3SS components [ 19 , 79 , 80 ] The same can be said of a vector system originally developed in the Susan Straley laboratory [ 81 ], with many examples of its utility in creat-

molecu-ing genetic systems to study T3SS function in Yersinia and other

organisms [ 82 – 84 ]

1.4 Epilogue

ing pathogenic Yersinia spp

2 Materials

chloramphenicol- resistant derivative of the pGP704 vector [ 87 ]

that contains the oriV replicon originally from the plasmid R6K

[ 88 ] Plasmid replication is therefore dependent on the pir gene pDM4 is a mobilizable vector and this is conferred by the oriT-tra

region sourced originally from RP4 [ 87 ] Critically, plasmid pDM4

contains the sacBR locus derived from the vector pKNG101 [ 78 ] This locus is normally associated with levansucrase production by

Bacillus subtilis , but is lethal when produced by Yersinia growing

in the presence of 5 % Sucrose The sacBR locus provides a counter selectable marker in Yersinia Additionally, a fi nal inserted syn-

thetic DNA fragment in pDM4 provides the unique restriction

digestion sites of Sal I, Xho I, Spe I, Apa I, Xba I, Bgl II, Sph I, Bst EII, and Sac I [ 11 ] By markedly increasing the available cloning options, this polylinker serves to enhance the utility of pDM4 as a mutagen-esis vector

1 Escherichia coli SY327 λ pir (genotype—Δ( lac pro ), argE (Am), rif , malA , recA56 , LAMpir ) [ 87 ] is routinely used to maintain

pDM4, as replication of the pDM4 plasmid requires the pir gene Since this strain is a lambda lysogen carrying the pir

gene, replication of the pDM4 vector is permitted and the plasmid is stably maintained Compared to other available hosts, these bacteria also allows for large-scale plasmid purifi ca-tion at relatively higher yields

2(Km::Tn7,Tc::Mu-1), pro-82, LAMpir, recA1, endA1, thiE1,

strain in conjugal mating experiments Being a lambda lysogen

carrying the pir gene, the strain can also maintain pDM4

plas-mid replication Moreover, the strain harbors chromosomally integrated conjugal transfer functions to enhance effi cient plas-mid transfer via conjugation

1 Luria-Bertani (LB) broth: 10 g/l tryptone, 5 g/l yeast extract,

10 g/l NaCl E coli bacteria are routinely cultured in LB at

37 °C At least during overnight growth, bacteria are cultured in the presence of appropriate antibiotics to select for maintenance

of pir and mob functions Yersinia bacteria in which mutations

are to be generated are routinely grown in LB broth at 26 °C

2 BD™ Yersinia Selective Agar (Becton Dickinson, Franklin Lakes, New Jersey) To aid in the recovery of Yersinia mutants follow- ing conjugal mating, Yersinia trans-conjugates are most often specifi cally selected for by growth on Yersinia Selective Agar

3 Methods

The generation of a mutated genetic allele employs the standard

sche-matically illustrated in Fig 3 The amplifi ed PCR fragments are then lifted into the pDM4 suicide mutagenesis vector using appro-priate restriction digestion and ligation as described in

introduced into E coli S17-1λ pir , which are then used as the donor

strains in independent conjugal mating experiments with recipient

Y pseudotuberculosis bacteria as presented in Subheading 3.3 Ensuing allelic exchange events are monitored by sensitivity to the presence of 25 μg/ml Chloramphenicol and resistance to the pres-ence 5 % (v/v) Sucrose in the LB broth growth medium as described in Subheading 3.3 and are illustrated schematically in Fig 4 The introduction of mutations introduced in cis on the Y pseudotuberculosis genome is very signifi cant for it ensures that

expression of mutant alleles occurs in the context of native tory elements

1 Design a forward primer that anneals approximately 200 nt upstream (designated primer “a”) and a reverse primer that anneals approximately 200 nt downstream (primer “d”) of the targeted mutagenesis region Note that both “a” and “d” primers must incorporate unique restriction digestion sites at their 5′ terminus that are compatible with the unique restric-tion digestion sites present in the multiple cloning site of the

pDM4 suicide vector ( see Fig 3 and Note 1 )

2 Two additional internal primers (designated reverse primer

“b” and forward primer “c”) are designed to harbor the actual desired mutation in their sequence These two primers must also overlap by containing complementary sequence between

them of about 18 base pairs ( see Fig 3 ) For the design of point mutations, it is usually desirable that both primers “b” and “c” incorporate the necessary nucleotide alterations within the

18 bp overlapping sequence For the design of a deletion, logistics dictates that only one of the primers usually contains the site of deletion Sequence upstream of this site of deletion

is therefore complementary to the other primer, whose 5′ sequence immediately fl anks the deletion site

3.1 Generation

of Mutations

by Overlap-PCR

3 By pairing the forward “a” primer with the reverse “b” primer and the forward “c” primer with the reverse “d” primer, two DNA fragments are amplifi ed by PCR—designated fragments

AB and CD respectively ( see Fig 3 ) For both PCRs, a

Fig 3 Generation of mutated allele by overlap PCR The essence of overlap PCR

is based on four strategically designed primers Internally positioned primers “b” and “c” must contain complementary sequence to each other, and at least one

of them must contain the mutation of choice (indicated by the star shape ) These

primers can be designed to incorporate a point mutation, a deletion of DNA or an insertion of DNA The fl anking primers “a” and “d” would contain a 5′ sequence

for restriction enzyme recognition ( closed circle ) to facilitate cloning of the

ampli-fi ed fragment Two reactions are performed in the ampli-fi rst round of PCR using primer pairs “a” with “b” and “c” with “d.” The resulting amplifi ed fragments AB and CD are annealed While two combinations are possible (not shown), only one of these has the necessary 3′-OH termini for productive end-fi lling by DNA poly-merase It is this template when mixed with the primer pair “a” and “d” that results in amplifi cation of the fi nal AD fragment with the desired mutation in the second round of PCR

Matthew S Francis et al.

Fig 4 Strategy for allelic replacement by a suicide vector Step 1 , the entire plasmid is integrated into the

chromosome by a single-crossover between the homologous genes, producing a chromosomal duplication

Step 2 , the chromosomal duplication is excised by homologous recombination between the fl anking direct repeats Step 3 , the plasmid is cured by exposing the bacteria to 5 % sucrose providing a direct selection for

the loss of the plasmid Ultimately, one copy of the gene is integrated on the targeted region, either the type copy or the mutant copy

reading polymerase enzyme should be used, along with tal bacterial DNA as template (that is often only in the form of boiled total bacterial lysate)

4 Depending on the DNA fragment sizes, run a 0.8–1.5 % rose gel to ensure that specifi c PCR products have been gener-ated Where additional purifi cation is necessary, excise the correct DNA band using any commercial DNA gel extraction

aga-kit ( see Note 2 )

5 Equimolar concentrations (usually 5–10 ng) of purifi ed AB and CD products are combined as a template in a single PCR reaction Due to their terminal sequence complementarity (18 bp) generated by the special design of primers “b” and “c”

(in step 2 ), both products are able to anneal to each other

resulting in hybrid fragments that can be further amplifi ed

using the two external primers “a” and “d” ( see Fig 3 )

6 The newly amplifi ed AD fragment can be subject to analysis

and purifi cation, if needed ( see step 4 )

7 To facilitate automated sequencing of larger DNA fragments generated by overlap PCR, the purifi ed fragment is often

cloned into a commercial T/A cloning system ( see Note 3 )

1 The mutagenized DNA fragment generated by overlap PCR

( see Subheading 3.1 ) is digested with the appropriate tion enzyme combination determined by the sites incorporated into the 5′ terminus of the external fl anking primers “a” and

restric-“d” ( see Note 4 ) In parallel, purifi ed pDM4 plasmid DNA is

similarly restriction digested ( see Note 5 )

2 The DNA fragments can be analyzed and purifi ed as described

in Subheading 3.1 , step 4

3 Ligation of the digested mutagenized insert with the linearized pDM4 plasmid is performed in 10 μl volumes, and can often require up to 10× excess of insert in comparison to the plasmid

( see Note 6 )

4 The ligation reactions are routinely transformed into freshly

prepared chemically competent E coli SY327λ pir ( see Note 7 )

5 Positive transformants containing pDM4 with mutagenized insert are identifi ed by colony PCR using the same primer combination of “a” and “d.” Confi rmation is performed by extracting the pDM4-based plasmid from the PCR-positive transformant(s) using any commercial plasmid isolation kit The purifi ed plasmid is then digested with the same restrictions

enzymes used above ( see step 1 ), before electrophoretic

analy-sis on a 0.8–1.5 % agarose gel

6 A confi rmed pDM4-based mutagenesis plasmid is then

trans-formed into chemically competent E coli S17-1λ pir strain and

stored in a LB broth-DMSO solution at minus 80 °C in

readi-ness for conjugal mati ng ( see Subheading 3.3 )

1 The donor bacteria E coli S17-1λpir containing the suicide

vector are grown without agitation overnight at 37 °C in 5 ml

LB broth with appropriate selection ( see Note 8 ) The ent bacteria Y pseudotuberculosis are grown with agitation

recipi-overnight at 26 °C in 2 ml LB broth with appropriate selection

( see Note 9 )

2 Donor and recipient strains are harvested, then gently washed in

10 ml of fresh antibiotic free LB broth Donor bacteria are centrated 5× by gently resuspending in 1 ml fresh antibiotic free

con-LB broth Recipient bacteria are concentrated 5× by gently resuspending in 400 μl fresh antibiotic free LB broth Equal volumes of 125 μl of donor and recipient are gently combined

on a 0.45 μM HA Nitrocellulose MF™ Membrane Filter (Millipore) overlaid onto a nonselective LB agar plate Filters are left at room temperature undisturbed for a minimum of 4 h

3 Trans-conjugates are recovered by washing the fi lter in 1 ml PBS (0.2 g/l Potassium chloride, 0.2 g/l Potassium dihydrogen phosphate, 8.0 g/l Sodium chloride, 1.15 g/l di-Sodium hydro-gen phosphate), followed by plating appropriate volumes onto BD™ Yersinia Selective Agar with appropriate antibiotic selec-tion (for the mutagenesis plasmid and for the recipient bacteria) Incubate at 26 °C for 48 h or until well-isolated colonies appear

4 Select one or more well-isolated colonies and restreak for

sin-gle colonies on BD™ Yersinia Selective Agar with selection for

the mutagenesis plasmid Incubate at 26 °C for 48 h Following these series of steps, the only way the mutagenesis plasmid can

be maintained by Yersinia is through integration into the

bac-terial genome by a process of homologous recombination (i.e.,

an initial cross-over event) ( see Fig 4 )

5 The next process is designed to encourage a secondary cross- over event that results in allelic exchange of the wild-type copy

for the mutated copy ( see Fig 4 ) To do so, select a lated single colony from the selective agar plate and culture overnight at 26 °C with agitation in 2 ml of LB broth lacking any selection for the mutagenesis plasmid Serially dilute the overnight culture (dilutions of 10 −1 , 10 −2 , 10 −3 usually suffi ce) and spread 100 μl aliquots on LB agar with appropriate antibi-

well-iso-otic selection (for Yersinia only) and supplemented with 5 %

Sucrose Replica patch well-isolated single colonies (less than

50 colonies usually suffi ce, but more can be needed) onto a LB agar plate with selection for the mutagenesis plasmid (i.e., with Chloramphenicol) and a LB agar plate lacking any selection for the mutagenesis plasmid (i.e., without Chloramphenicol)

6 Potential trans-conjugates of interest are those colonies that are chloramphenicol sensitive and sucrose resistant These are further screened by colony PCR to verify the appropriate allelic

exchange event has successfully occurred Moreover, the

ampli-fi ed PCR fragment is cloned into a commercial T/A cloning system ( see Note 3 ) and the fragment sequenced for fi nal confi rmation

7 Confi rmed Yersinia strains harboring the correct mutated

allele are stored in a LB broth-DMSO solution at minus 80 °C

in readiness for phenotypic analysis

4 Notes

1 The size of the regions of DNA fl anking the mutation can be varied, but they should always be of equivalent length It is likely that larger regions can facilitate more effi cient homolo-

gous recombination ( see Fig 4 and Subheading 3.3 ), and this might be necessary in certain situations

2 Often both vector and insert DNA fragments are further

puri-fi ed by phenol-chloroform extraction and then concentrated

by propanol precipitation using glycogen as a DNA carrier

3 To confi rm each mutation by sequence analysis, we clone directly into a sequencing vector such as pTZ57R/T using the InsTAclone PCR cloning strategy of Thermo Scientifi c

4 Note that best results can be achieved by performing a tial digestion by using only one enzyme at a time in its pre-ferred optimal digestion buffer

5 Seldom are the vector termini dephosphorylated before ligation

6 Standard ligation controls of (1) vector alone without the addition of ligase, and (2) vector alone with the addition of ligase are routinely performed to examine the linearity of the digested vector

7 We routinely use the method of Hanahan [ 90 ] for the

prepara-tion of chemically competent E coli bacteria As an alternative,

it should be noted that E coli SY327λ pir can be replaced with the strain E coli DH5αλpir The genotype of this latter strain

is supE 44, Δ lac U 169 (Φ lacZ ΔM15), recA 1, endA 1, hsdR 17, thi -1, gyrA 96, relA 1, λ pir phage lysogen In fact DH5αλpir

has proven to be ideal for the general maintenance of based vectors, and from which high yields of pure plasmid DNA can be obtained that is optimal for subsequent cloning and transformation procedures

8 We grow E coli donor bacteria without agitation in order to

preserve the integrity of the fragile F-pilus required for lization of pDM4 constructs during conjugal mating

mobi-Matthew S Francis et al.

9 Using colony PCR to positively screen for in cis point

muta-tions can be challenging for it is often diffi cult to design a primer pair specifi c for the mutated allele For this reason, we have regularly used as the recipient Y pseudotuberculosis ,

mutant variants that contain a deletion in the region passing the site where the substitution is intended to be posi-tioned To establish the point mutant would simply require reconstitution of a full length allele (containing the point mutation) by crossing- out of the deleted variant via allelic exchange The value of this approach is that a set of screening primers can be designed that are much more discriminatory on the basis of either fragment size and/or the ability to amplify any product at all

Acknowledgments

This work was supported by Swedish Research Council grant 2014–2105 and the Medical Research Foundation of Umeå University to MSF

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