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

Methods in
Molecular Biology 1531

Matthew L. Nilles
Danielle L. Jessen Condry Editors

Type 3
Methods and Protocols




Series Editor
John M. Walker
School of Life and Medical Sciences

University of Hertfordshire
Hatfield, Hertfordshire, AL10 9AB, UK

For further volumes:

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

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
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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


Preface. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Contributors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .


1 Introduction to Type III Secretion Systems. . . . . . . . . . . . . . . . . . . . . . . . . . .
Danielle L. Jessen Condry and Matthew L. Nilles
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
3 Blue Native Protein Electrophoresis to Study the T3S System
Using Yersinia pestis as a Model. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Thomas A. Henderson and Matthew L. Nilles
4 In Vivo Photo-Cross-Linking to Study T3S Interactions Demonstrated Using
the Yersinia pestis T3S System . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Thomas A. Henderson and Matthew L. Nilles
5 Isolation of Type III Secretion System Needle Complexes by Shearing . . . . . .
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 . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Kevin J. Metcalf and Danielle Tullman-Ercek
7 Characterization of Type Three Secretion System Translocator Interactions
with Phospholipid Membranes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Philip R. Adam, Michael L. Barta, and Nicholas E. Dickenson
8 Analysis of Type III Secretion System Secreted Proteins . . . . . . . . . . . . . . . . .
Danielle L. Jessen Condry and Matthew L. Nilles
9 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













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 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .






PHILIP R. ADAM • Kansas Department of Health and Environment Laboratories, Topeka,
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,
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 1
Introduction to Type III Secretion Systems
Danielle L. Jessen Condry and Matthew L. Nilles
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 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



Danielle L. Jessen Condry and Matthew L. Nilles

systems that affect plants fall into two families Hrp1 and Hrp2 and
the remaining two families belong exclusively to the phyla of bacteria 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
kingdoms of organisms, such as plants and animals [5]. Many
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].


The T3S system is comprised of approximately 25 different proteins
that make up the basal body, needle, and translocon [3]. These structural 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 bacterial 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/

Introduction to Type III Secretion Systems


conserved between T3S systems, including bacterial flagella [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 crystallography 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 possesses two coiled domains with the N-terminus of the needle protein 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 define
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 elements, 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 specific protein
dictates the length of the needle, a cup method where a specific
number of needle proteins are released to create the needle, or others 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 protein that “caps” the apparatus and interacts with the final portion of
the structure that imbeds in the host membrane [9].
The translocon completes the T3S system (Fig. 1). This structure 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].



Danielle L. Jessen Condry and Matthew L. Nilles

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 specific 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].


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 field [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 hypothesize 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 conformational changes of the needle from the tip to the base. Another
hypothesis, separate from needle protein structure, involves a protofilament 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 confirmed by analysis of these mutants.


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 specifically, the T3SS in Yersinia
pestis plays a key role in the prevention of phagocytosis, the manipulation of cytokine expression, and killing of immune cells [26].
In Yersinia pestis the T3S system is encoded by the pCD1 plasmid. Also on this plasmid are effectors, chaperones, and regulatory

Introduction to Type III Secretion Systems


proteins that are necessary for expression, construction, and
expression of the T3S. Without the T3S system Yersinia pestis
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
encodes the T3S system [19]. The LcrF transcript has a unique
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 first, 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 membrane 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 proteins: 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,
into the host membrane [9]. YopB and YopD then create a pore
and allow Yops to translocate from the needle apparatus into the
host cell [9]. In Yersinia there is no evidence for the order or timing 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 confirm 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 profiles of Yersinia pestis, in vitro, all
three proteins are secreted into the medium.
Effector proteins are the toxins of the T3S system. These proteins, 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
in vivo conditions cell contact is known to trigger secretion 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


Danielle L. Jessen Condry and Matthew L. Nilles

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 regulates 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 secretion 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 expression 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 confirmed 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
enterica serovar Typhmurium [47]. Effector proteins in the E. coli system are referred to as Esp-X. Expression of structural ETT1 T3S system 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(NO3)3 and NH4+ [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 inflammatory mediators and fluid movement into the intestine [51]. The inflammation
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].
However, it is also possible for Salmonella to survive in

Introduction to Type III Secretion Systems


macrophages. This is accomplished with the other T3S system of
this bacterium SPI-2. Once inside the Salmonella Containing
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 infection [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: flexneri, sonnei, dysenteriae, and boydii. Shigella flexneri and sonnei cause endemic forms of dysentery,
while Shigella dysenteriae is associated with epidemics. These bacteria 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-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 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 inflammation [53].
Effectors in Shigella include IpaA-D, IpaB-D are known to induce
membrane ruffling 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 crystallized 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 disease types ranging from pneumonia to infections of the urinary
tract, wounds, burns, and bloodstream. Cystic fibrosis patients are
keenly susceptible to Pseudomonas infections as well.

Danielle L. Jessen Condry and Matthew L. Nilles


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 trafficking, 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 calcium 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 specifically can function in Pseudomonas; however, PopD,
the Pseudomonas homolog, cannot substitute for YopD, specifically
YopD’s regulatory functions. This study also revealed the importance of translocon protein chaperones for proper function [56].


Notable Plant Bacteria Species with T3S Systems
T3S systems are conserved in four major plant pathogenic gramnegative bacteria, as well as involved in symbiotic Rhizobium spp.
T3S system components are recognized by plant hypersensitive
response defenses and result in resistance to the pathogenic bacteria
species [6]. Bacterial genes involved in the T3SS are defined as hrp
(hypersensitive response and pathogenicity) [57]. T3S system effectors include Avr proteins that function to counteract the resistance
in different plant species. Just like in mammalian T3S system effectors, the variety of effectors in plant pathogens appears to be specific
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 infection [6].

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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
Type III secretion systems are a prolific virulence determinant among Gram-negative bacteria. They are
used to paralyze the host cell, which enables bacterial pathogens to establish often fatal infections—unless
an effective therapeutic intervention is available. However, as a result of a catastrophic rise in infectious
bacteria resistant to conventional antibiotics, these bacteria are again a leading cause of worldwide mortality. Hence, this report describes a pDM4-based site-directed mutagenesis strategy that is assisting in our
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



1.1 Type III Secretion
of Proteins by Bacteria

Many bacteria evade eukaryotic host immune responses by using
type III secretion systems (T3SSs) that inject bacterial effector molecules directly into target host cells (Fig. 1) [1–3]. The T3SS apparatus is composed of some 25 proteins, which when completely
assembled not only spans the entire bacterial envelope but also protrudes outward from the bacterial surface, taking the form of a
syringe-needle structure. It is through this structure that the effectors are directly injected into eukaryotic cells [4]. These injected
effectors possess enzymatic activities that subvert host cell signaling
for the bacteria’s benefit. They are 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. 1531, DOI 10.1007/978-1-4939-6649-3_2, © Springer Science+Business Media New York 2017



Matthew S. Francis et al.

Needle components
(early substrates)

Injectisome pore components
(middle substrates)

Injected effectors
(late substrates)
plasma membrane


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 injectisome. OM bacterial outer membrane, CM bacterial cytoplasmic membrane

“middle-secreted” injectisome components that sit at the top of the
needle (Fig. 1) [1–3, 5]. From this position, injectisome components form pores in infected cell plasma membranes through which
the “late” effectors may gain entry into the cell cytosol.
The pre-secretory stabilization and efficient 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 probably recognized by the T3S machinery to act as dedicated substrate
secretion signals [6, 7]. Each substrate has also their own
chaperone-independent N-terminal secretion signal sequence [2,
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 substrates first polymerize needle components at the bacterial surface
that then permit secretion of “middle” substrates to form injectisome 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.

Site-Directed Mutagenesis and Its Application in Studying the Interactions of T3S…


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 disease 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 secretion) components
that assemble into a functional apparatus specifically to secrete at
least three protein classes of Ysc’s and Yop’s (Yersinia outer proteins); the “early” needle components (YscF, YscX) and antiactivator (LcrQ,), the “middle” injectisome pore-forming
components (YopB, YopD, YopK, and LcrV), and the “late”
injected immuno-suppressive enzymes and toxins (YopE, YopH,
YopJ, YopM, and YpkA) [2].
1.2 Site-Directed
Mutagenesis: Utility
of the pDM4-Based

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 sitedirected mutagenesis of isogenic phenotypic mutants. Not only
has this provided the basis for understanding the minimal molecular components required for a functional T3SS apparatus, but it has
also permitted detailed investigations into the molecular interactions 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.

1.3 Applications
of the pDM4-Based
Mutagenesis in
Yersinia T3SS

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 others have addressed T3SS research, and this section describes a number 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 homologous and heterologous protein-protein interactions.


Matthew S. Francis et al.

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 (CmR), the R6K-derived oriV replicon making 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

1.3.1 Apparatus

The T3SS apparatus spans the bacterial envelope and anchors a needle-like appendage that extends out from the bacterial surface. In
Yersinia, this apparatus is chiefly composed of about 20 Ysc proteins,
including the YscF needle. Another important protein is YscU, an
integral inner-membrane protein absolutely required for T3SS function. YscU belongs to a family of proteins that is characterized by
auto-cleavage at a highly conserved C-terminal NPTH motif. In particular, auto-proteolysis of YscU occurs between the asparagine (N)

Site-Directed Mutagenesis and Its Application in Studying the Interactions of T3S…


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 efficiency at the NPTH motif, functionality of the T3SS was lost [12–14]. Hence, these studies used
targeted mutagenesis to identify the importance of YscU auto-cleavage in the regulation of Yop synthesis and secretion control.
Linked to the function of YscU is the protein YscP. These proteins are thought to cooperate in an assembly checkpoint termed
the “substrate specificity switch.” It has been proposed that this
switching machinery identifies that the apparatus has matured sufficiently to enable a change in secretion specificity 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
switching [14], and this was accompanied by the production of
remarkably longer needles that were incapable of supporting Yops
secretion [14–16]. Interestingly, when pDM4-mediated sitedirected 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
mutations in the C-terminus that could suppress the yscP null
mutant phenotype to such an extent that Yop secretion was partially restored [13]. This finding is consistent with the notion of an
interaction between YscP and YscU [14]. Hence, the significant
outcome from these genetic approaches is the anticipation that a
YscP-YscU interaction is necessary for the regulation of substrate
specificity switching during type III secretion.
1.3.2 Translocon

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 benefit of the bacteria. In Yersinia, YopB, YopD,
and LcrV are prominent translocator proteins responsible for injectisome 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


Matthew S. Francis et al.

controlled synthesis and secretion of Yops. Hence, pDM4 sitedirected mutagenesis has been used to pry apart the various functional domains of YopD. First, a deletion analysis identified the
C-terminus as a region of YopD essential for function [26].
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 identified 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 critical 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 efficiently 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
function beyond translocon formation, which could explain the
presence of translocated YopD in the host cell cytosol [32].
1.3.3 Molecular Targets
of Translocated Effectors

Ysc-Yop T3SS activity in the presence of immune cells contributes
both 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 of these two critical
virulence determinants. For example, the creation of single amino
acid substitutions has been used to investigate substrate recognition specificity 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 probably remained unknown [38]. Moreover, the identification 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.

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