Bacterial Injection Machines*
Annick Gauthier
,
Nikhil A. Thomas
and
B. Brett Finlay ¶
From the
Biotechnology Laboratory and Department of Biochemistry and Molecular
Biology, University of British Columbia, Vancouver, British Columbia V6T 1Z3,
Canada
 |
INTRODUCTION
|
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The delivery of virulence factors directly into host cells is a fascinating
aspect of pathogenesis. For Gram-negative bacteria to translocate virulence
factors into host cells, at least three membranes must be passed (two
bacterial and a host plasma membrane). Bacterial injection machines deliver
virulence factors to a specific cellular location where they intersect and
influence host mechanisms. This minireview focuses on the Gram-negative
bacterial translocation systems that mediate type III and type IV secretion.
Remarkably, although these systems are complex multiprotein structures, there
is significant similarity and analogy in function, and thus a conserved
mechanistic theme in pathogenicity emerges.
Currently there are seven identified types of macromolecular secretion
systems in Gram-negative bacterial pathogens
(1,
2). This minireview focuses on
the two systems that deliver macromolecules directly into eukaryotic cells:
type III secretion system
(T3SS)1 and type IV
secretion system (T4SS). The delivered macromolecules are referred to as
effectors, as they affect and alter the host cellular process. Gram-negative
bacterial effectors cross several biochemically distinct barriers, including
the bacterial inner membrane, peptidoglycan layer, and outer membrane as well
as the host plasma membrane, and even potentially intracellular host
membranes. Plant pathogen effectors have the additional complexity of crossing
the plant cell wall. The biochemistry of these delivery systems will be
discussed, including what is known about how they are assembled and how they
function.
 |
Type III Secretion Systems
|
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In the 1980s and 1990s researchers studying Yersinia, a genus that
causes human diseases ranging from bubonic plague to gastrointestinal disease,
found that the bacteria produced proteins that were thought to be associated
with the outer membrane called Yops. Yops lacked classical signal sequences
and were not secreted via a sec-dependent pathway and thus were
assumed to be delivered by a new type of secretion system, which later became
known as a T3SS, representing its order of discovery in secretion systems. In
the last 10 years T3SS have been identified in more than 20 bacterial
pathogens that infect plants and animals
(Table I). Although there is a
high degree of conservation among the components of the type III apparatus in
different bacterial species, the pathogens often carry a distinct set of
virulence factors with a variety of functions that can be translocated into
either animal or plant cells. The overall theme of these T3SS is the direct
delivery of proteins that alter and in effect "hijack" the
infected host cell for the pathogen (reviewed in Refs.
3 and
4).
 |
Type III Apparatus Components
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Over 20 proteins are proposed to form a functional T3SS
(Fig. 1A)
(4,
5). YscN is thought to energize
the secretion machinery, as it shares homology with the
F0F1-ATPase and has an ATP-binding site. YscN from
Yersinia and its homologue InvC from Salmonella typhimurium
have been shown to have ATPase activity as mutations in the catalytic domain
cause a loss of secretion (6,
7). YscN homologues are
predicted to be located in the cytoplasm where they interact with
membrane-bound components of the type III secretion apparatus, thereby
energizing the system (4). It
has been speculated that the ATPase polymerizes, by itself or with other
components, to form the lower part of the T3SS, but this has not been shown.
Lending support to this model, YscN has been shown to form a complex with
three other cytoplasmic and/or inner membrane-associated Ysc proteins
(8).

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FIG. 1. Models of type III and type IV secretion systems. A,
diagram of the T3SS highlighting apparatus components of EPEC and the direct
nature of translocation to host cells. B, diagram of A.
tumefaciens T4SS highlighting apparatus components and the possibility of
having two (or more) mechanisms of export across the bacterial inner
membrane.
|
|
Many of the proteins involved in forming the T3SS have been localized or
are predicted to be inner membrane proteins with varying numbers of
transmembrane domains. For example the Yersinia YscV (LcrD) contains
eight transmembrane domains and a large cytoplasmic C-terminal domain
(9,
10). YscJ family members carry
sec-dependent signal sequences and are lipoproteins
(4,
11). The Pseudomonas
syringae homologue HrcJ is associated with both inner and outer membranes
(12), suggesting that it spans
the periplasmic space.
Homologues of YscC (e.g. InvG, HrcC) are the only components of
the type III apparatus that are clearly found in the outer membrane
(1215).
YscC belongs to a family of proteins (secretins) that are involved in
transporting large molecules across the outer membrane probably by forming a
channel (4). YscC and its
homologues form a ring-shaped oligomeric complex in the outer membrane with
approximately a 20-nm diameter. Experimentally it has been shown that InvG has
a cleavable signal sequence at residue 25, indicating that secretins are
exported by the sec-dependent pathway
(16). It has been demonstrated
that small outer membrane lipoproteins are required to increase the efficiency
for the correct localization and functioning of the YscC homologues
(13,
14,
1719).
Recently we have shown that correct insertion and function of enteropathogenic
Escherichia coli's (EPEC) EscC secretin in the outer membrane
requires cytoplasmic and inner membrane components of the type III apparatus,
namely EscN and EscV (20).
 |
Syringe and Needle-like Structure
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Electron microscopic analysis has revealed the T3SS to be an organelle that
consists of a base (or syringe) that spans the bacterial membranes and
peptidoglycan layer composed of two pairs of rings that are joined by a
central channel, and a hollow needle-like structure that protrudes outside the
bacteria and in some cases has been observed to contact the host cells
(reviewed in Refs. 21 and
22). There is a remarkable
structural similarity to the bacterial flagellar basal bodies (see
"Origin of Translocation Systems"). Mutants in the needle protein
cannot secrete or translocate effectors, suggesting that either the needle
keeps the pore open or that the needle extends into the "syringe."
Whereas needle proteins exist in all of the animal pathogens, a homologous
protein has not been found in the plant pathogens although the pilus protein
HrpA seems to play a similar functional role
(23).
Elegant immunogold electron microscopy experiments have been conducted with
the plant pathogens Erwinia and P. syringae demonstrating
very clearly that the effectors actually go through the needle/pilus conduit
and can be visualized at the tip of the structure
(24,
25). This indicates that the
type III apparatus and needle are a hollow conduit for protein delivery.
 |
The Translocon: Pore in the Host Cell Membrane
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All of the animal pathogen T3SS have one or more proteins that are thought
to form a pore in the host cell membrane, called the "translocon"
(reviewed in Ref. 26). It is
important to note that although mutations in needle components result in no
secretion or translocation of effectors, mutation of translocon components
yields wild-type levels of secreted but not translocated effectors. Most of
the translocon proteins contain one or two predicted transmembrane domains and
are associated with host cell membranes. Furthermore, in EPEC the needle
sheath protein EspA and the translocon EspB have been shown to interact by a
number of binding assays (27),
suggesting a continuous channel. Additionally, Yersinia and P.
aeruginosa have another type of translocon protein that does not contain
-helical transmembrane domains but is required for pore formation.
Translocon proteins have not been described in plant pathogens, but HrpF from
Xanthomonas is a candidate as it is not needed for secretion but is
required for translocation and forms pores in lipid bilayers
(28).
 |
Recognition of Type III Effectors: mRNA, Protein, and Chaperones
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T3SS effectors do not have an obvious signal sequence. However, there is
evidence for the existence of three different kinds of secretion signals: the
5'-region of the mRNA, the N terminus of the effector, and/or the
ability of a chaperone to bind the effector before secretion (reviewed in Ref.
29). The minimal requirement
for secretion of some effectors is the N-terminal 1015 residues
(30,
31), whereas the minimum
needed for translocation is 5075 N-terminal residues
(30,
31). The so-called mRNA
hypothesis is very controversial. Two groups have shown that certain effectors
have a 5'-mRNA fold that directs translocation
(3235),
but others have refuted these observations with equally convincing experiments
showing that the N-terminal amino acids are the signal
(36).
Many functions have been attributed to T3SS chaperones, but the exact role
or roles of the entire family of chaperones remain to be determined (reviewed
in Refs. 29 and
37). Although most chaperones
have only one cognate effector, there are exceptions with differing numbers of
effectors. Deletion of a chaperone usually results in less of the cognate
effector in the cytoplasm of the bacteria and less secreted/translocated. For
some effectors, chaperone binding prevents degradation, whereas for others it
has been suggested that chaperone binding prevents premature association.
Another model suggests that chaperones escort the effector to the type III
apparatus and play a role in the hierarchy of translocation
(38). Chaperones could
maintain effectors in a secretion-competent state. The needle of the T3SS is
likely too small to allow folded proteins to pass through, but recent data
suggest that effectors are in a partially folded conformation (reviewed in
Ref. 39).
 |
Type IV Secretion Systems
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T4SS are a recent discovery in pathogenic delivery systems (reviewed in
Refs. 40 and
41). These systems deliver
proteins and DNA (often complexed together), but they can also deliver only
proteins. The prototype and most well studied T4SS is involved in the transfer
of oncogenic DNA into plant cells by Agrobacterium tumefaciens
(reviewed in Ref. 42).
Although the function and perhaps the mechanism of the T4SS are similar to the
T3SS, there is little to no conservation in the proteins that comprise the
apparatus. The lack of conservation suggests the mechanism at a molecular and
physical level may differ significantly between the two. Like the T3SS, the
T4SS is considered "promiscuous" in terms of the variety of
substrates and the diversity of target cells
(43)
(Table II). It should be noted
that although T4SS have recently been identified in a number of bacterial
species, their substrates/effectors are often unidentified.
 |
Type IV Apparatus Components
|
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The locus involved in T-DNA transfer in A. tumefaciens contains 11
VirB proteins, 2 of which are ATPases, as well as an additional ATPase, VirD4,
which is involved in the export of DNA
(Fig. 1B) (reviewed in
Ref. 41). It is important to
note that not all of the type IV-containing family of bacteria have homologues
of all of these proteins, and so far a conserved core of 5 proteins exists
(homologues of VirB4, -B7, -B9, -B10, and -B11)
(44). The type IV apparatus
components have been localized using standard biochemical fractionation
techniques like sucrose gradients, alkaline phosphatase insertions, and
protease susceptibility, with most of the studies being done on A.
tumefaciens.
Several proteins are found in the inner membrane and some are integral
membrane proteins like VirB4 and VirB6, whereas others are associated with the
inner side of the inner membrane like VirB11 (reviewed in Ref.
40). VirB7B10
fractionate to both the inner and outer membranes
(45), and it has been
suggested that these proteins act in concert to span both membranes. In fact,
VirB7 is a lipoprotein that is important for structural integrity of many
other components of the T4SS
(46). VirB10 interacts with
VirB9, and results suggest that the outer membrane VirB7-VirB9 complex
interacts with the inner membrane proteins VirB8 and VirB10 to form the
translocation channel
(47).
 |
Energy Requirement
|
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The T4SS has at least two and possibly three ATPases/NTPases. The energy
may be required for the translocation process, to open the channel, as a
coupling factor, or for chaperone functions (reviewed in Refs.
40 and
41). Similar to what has been
observed for the T3SS, studies have demonstrated that NTPase activity is
crucial for export. VirB4 and VirB11 have Walker A nucleotide binding motifs
and have ATPase activity (48).
Interestingly, recent studies support a model whereby there can be
bi-directional DNA transfer aided by the multimeric structure of VirB4 but
that it is the ATP-dependent activity that renders it an export-only system
(49). VirB11 ATPase homologues
from T4SS in Ti-plasmid, RP4, and Helicobacter pylori have been shown
by electron microscopy to form a homohexameric ring structure with a 12-nm
diameter and a 3-nm central channel
(50). The crystal structure of
the H. pylori ATPase HP0525 (homologous to VirB11) has been solved
and suggests that this family of ATPases may function as chaperones similar to
GroEL (51).
 |
Type IV Secretion System Pilus and Translocon
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In A. tumefaciens the T-pilus appears to be 4 nm in diameter and
have a variable length (52),
and it is made up of VirB2, which is a small 7.2-kDa protein
(53,
54), a structure reminiscent
of the T3SS needle/pilus. Recently, it has been suggested that VirB7-VirB9
complexes may link the T-pilus components (VirB2) to the core of the
translocation machinery (55).
VirE2 has been suggested to fulfill the translocon or host-cell pore-forming
function of the T4SS because it forms large anion-selective, voltage-gated
channels and allows the transport of single-stranded DNA
(56).
 |
Chaperone/Coupling Protein
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Chaperones are involved in the export of both DNA and proteins by the T4SS.
There is evidence for the chaperone VirE1 stabilizing VirE2 and inhibiting
aggregation (57), suggesting a
function in preventing premature association, as has been suggested for the
T3SS chaperones. It has been suggested that coupling proteins like the VirD4
family act by specifically recognizing and exporting T4SS substrates (reviewed
in Ref. 43).
 |
Pertussis T4SS: Anomaly or the Norm?
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Pertussis toxin transport seems to be an anomaly in the T4SS substrates
because there are detectable periplasmic intermediates (reviewed in Ref.
40). Pertussis toxin subunits
require the sec-dependent general secretion pathway for export to the
periplasm and then the T4SS for secretion out of the bacterium. Additionally,
the pertussis toxin system is unlike the other T4SS in that it functions
independently of host contact because of the ability of the holotoxin to
autotranslocate itself into the host cell rather than relying on T4SS
delivery.
Although periplasmic intermediates of effectors have not been found in the
T3SS, recent developments in the T4SS field suggest that the pertussis toxin
two-step translocation requirements are not anomalous. Pantoja and colleagues
(58) have demonstrated that
T4SS substrates in A. tumefaciens that lack a signal peptide form a
soluble complex in the periplasm with another protein, VirJ, and then interact
with components of the T4SS. This suggests a two-step model for type IV
secretion in which effectors are exported via different pathways into the
periplasm and then translocated across the outer membrane and host cell
membrane via the T4SS.
 |
Origin of Translocation Systems
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Many but not all of the proteins comprising the T3SS of pathogenic bacteria
have homologues that are known to be involved in flagellar morphogenesis.
Flagella are complex filamentous cell surface organelles that rotate,
resulting in swimming motility. The flagellum includes a long hollow filament
(2025 nm in diameter), a curved hook structure, and a membrane-embedded
basal body. At least 50 gene products are involved in flagellum biogenesis
(for a detailed review see Ref.
59).
Comparatively, T3SS needle complexes are not rotary structures like
flagella, although these respective secretion systems are both involved in
secreting numerous different substrates, which eventually polymerize to form a
cell surface structure. In fact, isolated T3SS needle complexes (from many
bacterial species) resemble flagella basal bodies when viewed by electron
microscopy (16,
60,
61). Proteins that form these
basal bodies are some of the most highly conserved components in all T3SS and
are homologous to specific flagellar proteins. One example is YscJ (and its
homologues in all T3SS), which are lipoproteins that share sequence similarity
with FliF from the flagellar secretion system. This family of proteins is
involved in forming an oligomeric membrane-embedded ring structure.
An interesting question is how have these distinctive secretion systems
evolved with similar components. It is possible that in the evolution of a
bacterial species other proteins somehow became targeted to the flagellar
apparatus and were secreted. This hypothesis is difficult to test, but there
is some evidence to suggest that this is the case. In Yersinia
enterocolitica it has been shown that a small specific number of
virulence substrates are secreted through the flagellar pathway
(62). A complex functional
secretion system for a cell surface organelle has been in place prior to the
appearance of lower eukaryotes, because bacteria have likely been motile cells
for millions of years.
Bacterial pathogens have evolved strategies to infect the diverse
eukaryotic cell types still using the basic T3SS machinery. The extracellular
components of T3SS that directly contact the host cell (i.e. the
needle and translocon components) are not as highly conserved among bacteria
when compared with the basal body components
(21,
63). Moreover, the structural
needle proteins do not appear to share sequence homology with flagellin
proteins. Thus, it is believed that in the flagellar secretion systems and
T3SS of bacteria, the basal body components make up an ancient secretory
apparatus, and the extracellular components have been forged by selective
pressures brought about by sensing the immediate environment (motility) or the
host-pathogen interaction.
 |
Bacterial Conjugation and Type IV Secretion
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Interestingly, some of the components involved in the secretion of
virulence substrates via the T4SS are similar to those required for the
transfer of plasmid DNA by bacterial conjugation. All 12 components of the
plasmid R388 are homologous to components of the A. tumefaciens T4SS,
and 8 components of the plasmid RP4 conjugation system share homology to the
T4SS (44). These include a
family of secretion NTPases and coupling proteins such as TraG and TrwB
(similar to VirD4).
It is believed that bacterial conjugation is a T4SS-like process. In fact,
it has been demonstrated that the T4SS-like DNA transfer pathway can mediate
the DNA-independent translocation of a protein (Sog primase) between E.
coli cells (64).
Similarly, Vergunst et al.
(65) demonstrated that the
agrobacterial system translocates free proteins as well as nucleoprotein
complexes. Moreover, these studies strongly indicate that a system for the
transfer of one kind of substrate (e.g. DNA) could be employed by a
different type of substrate (e.g. effector protein). This paradigm
where an existing translocation system is utilized for a virulence purpose is
also hypothesized to have occurred for the flagellar secretion pathway and
T3SS (62). It has also been
proposed that T4SS were initially for conjugation with other prokaryotes and
later evolved to facilitate virulence relationships with eukaryotes
(66).
 |
Conclusions and Outlook
|
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Both type III and type IV translocation machines are in many ways ingenious
solutions to the dilemma of how to get bacterial proteins across the
Gram-negative envelope as well as into potential target cells. The T3SS field
is advancing rapidly, and it is hoped that soon it will be understood how this
system is assembled and functions to deliver virulent proteins directly into
host cells. The most recent developments in the T4SS suggesting a two-step
transfer with a periplasmic intermediate suggest that more novel findings are
to come. The suggestion of a defined hierarchy of secretion/translocation is
more apparent in the T3SS and likely also occurs in T4SS. Although the
evolution of both these systems is not completely understood, the leap from
flagellar assembly to type III secretion and bacterial conjugation to type IV
secretion may not be such a big jump in the genetically plastic world of
prokaryotes.
 |
FOOTNOTES
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* This minireview will be reprinted in the 2003 Minireview Compendium, which
will be available in January, 2004. Operating grants from Howard Hughes
Medical Institute (HHMI), Canadian Institutes for Health Research (CIHR), and
the Canadian Bacterial Disease Network support work in this laboratory. 
Supported by doctoral research awards from CIHR, Imperial Order of the
Daughters of the Empire, and Michael Smith Foundation for Health Research
(MSFHR). 
Supported by postdoctoral fellowships from Natural Sciences and Engineering
Research Council of Canada and the MSFHR. 
¶
HHMI International Research Scholar, a CIHR Distinguished Investigator, and
the UBC Peter Wall Distinguished Professor. To whom correspondence should be
addressed. Tel.: 604-822-2210; Fax: 604-822-9830; E-mail:
bfinlay{at}interchange.ubc.ca.
1 The abbreviations used are: T3SS, type III secretion system(s); T4SS, type
IV secretion system(s); Ysc, Yersinia type III secretion complex;
EPEC, enteropathogenic E. coli. 
 |
ACKNOWLEDGMENTS
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We thank Fern Ness for artwork for Fig.
1.
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