Structural biology of Type VI secretion systems

CNRS, Laboratoire d'Ingénierie des Systèmes Macromoléculaires, UMR 7255, Institut de Microbiologie de la Méditerranée, Aix-Marseille Université, 31 Chemin Joseph Aiguier, 13402 Marseille Cedex 20, France.
Philosophical Transactions of The Royal Society B Biological Sciences (Impact Factor: 7.06). 04/2012; 367(1592):1102-11. DOI: 10.1098/rstb.2011.0209
Source: PubMed


Type VI secretion systems (T6SSs) are transenvelope complexes specialized in the transport of proteins or domains directly into target cells. These systems are versatile as they can target either eukaryotic host cells and therefore modulate the bacteria-host interaction and pathogenesis or bacterial cells and therefore facilitate access to a specific niche. These molecular machines comprise at least 13 proteins. Although recent years have witnessed advances in the role and function of these secretion systems, little is known about how these complexes assemble in the cell envelope. Interestingly, the current information converges to the idea that T6SSs are composed of two subassemblies, one resembling the contractile bacteriophage tail, whereas the other subunits are embedded in the inner and outer membranes and anchor the bacteriophage-like structure to the cell envelope. In this review, we summarize recent structural information on individual T6SS components emphasizing the fact that T6SSs are composite systems, adapting subunits from various origins.


Available from: Christian Cambillau
Structural biology of type VI secretion
Eric Cascales
and Christian Cambillau
CNRS, Laboratoire d’Inge
nierie des Syste
mes Macromole
culaires, UMR 7255, Institut de Microbiologie de
la Me
e, Aix-Marseille Universite
, 31 Chemin Joseph Aiguier, 13402 Marseille Cedex 20, France
Aix-Marseille Universite
, Architecture et Fonction des Macromole
cules Biologiques, Campus de Luminy,
Case 932, 13288 Marseille Cedex 09, France
CNRS, Architecture et Fonction des Macromole
cules Biologiques, UMR 6098, Campus de Luminy,
Case 932, 13288 Marseille Cedex 09, France
Type VI secretion systems (T6SSs) are transenvelope complexes specialized in the transport of pro-
teins or domains directly into target cells. These systems are versatile as they can target either
eukaryotic host cells and therefore modulate the bacteriahost interaction and pathogenesis or
bacterial cells and therefore facilitate access to a specific niche. These molecular machines comprise
at least 13 proteins. Although recent years have witnessed advances in the role and function of
these secretion systems, little is known about how these complexes assemble in the cell envelope.
Interestingly, the current information converges to the idea that T6SSs are composed of two
subassemblies, one resembling the contractile bacteriophage tail, whereas the other subunits
are embedded in the inner and outer membranes and anchor the bacteriophage-like structure to
the cell envelope. In this review, we summarize recent structural information on individual T6SS
components emphasizing the fact that T6SSs are composite systems, adapting subunits from
various origins.
Keywords: protein transport; protein trafficking; bacteriophage; secretion; Hcp; VgrG
Type VI secretion systems (T6SSs) are the most
recently described specialized secretion systems.
T6SSs are widely distributed in Gram-negative bac-
teria, especially in proteobacteria, where type VI
secretion gene clusters may be found in several
copies on the chromosome [13]. First thought of as
secretion systems dedicated to virulence towards
eukaryotic host cells, recent data have shown unam-
biguously that these systems are regulating bacterial
interactions and competition [410]. T6SSs are
required to kill neighbouring, non-immune bacterial
cells by secreting anti-bacterial proteins directly into
the periplasm of the target cells upon cell-to-cell con-
tact [4,9]. This intense bacterial warfare indirectly
contributes to pathogenesis in plants, fishes, animals
and humans as T6SS facilitates the colonization of
specific niches where pathogens then develop anti-
host defences and toxins. However, a limited number
of T6SSs have been shown to be directly responsible
for pathogenesis, as they deliver toxin modules
interfering with the eukaryotic cytoskeleton [11].
Because T6SSs have only recently been identified, we
have only a limited knowledge on their assembly and
biogenesis, compared with other secretion systems
(see accompanying reviews in this issue). In past
years, a number of studies have identified sub-com-
plexes of this secretion apparatus and have reported
structural information (table 1 and figure 1). This
review will focus on the available structural data and
we also refer the reader to recent reviews on the var-
ious T6SS aspects [1,2,6,1116]. Thirteen subunits,
called core components, are believed to form the mini-
mal apparatus [13]. In several cases, the 13 core
components are supplemented with additional pro-
teins [17]. Although the functions of these proteins
have not been elucidated yet, it has been proposed
that these accessory elements might facilitate or modu-
late T6SS assembly or might confer additional
functions to the secretion machine. Regarding the
core components, several of them share structural
similarities with bacteriophage tail, spike, sheath, hub
or baseplate proteins, whereas a second category
groups proteins embedded within the inner or outer
membrane. However, a number of core components
do not share clear homologies with bacteriophage or
membrane-associated proteins; biochemical and struc-
tural data are therefore required to better understand
their functions. Table 1 summarizes the known and
suggested homologies, as well as the structure infor-
mation currently available. Based on this set of data,
it has been proposed that the T6SS core components
collectively assemble a structure resembling an
upside-down bacteriophage-like structure anchored
* Author for correspondence (
One contribution of 11 to a Theme Issue ‘Bacterial protein secretion
comes of age’.
Phil. Trans. R. Soc. B (2012) 367, 1102–1111
1102 This journal is q 2012 The Royal Society
Page 1
to the bacterial cell envelope [2,16,18]. Figure 1 sum-
marizes the current model for T6SS assembly, as well
as the three-dimensional structures available.
(a) The type VI secretion systems’ needle tail and
The Hcp protein is, together with VgrG, the hallmark
of T6SS secretion. This component of T6SS is essen-
tial to its function and forms a tube of stacked
hexamers in vitro. Three structures of Hcp proteins
have been reported to date: Hcp1 (PDB 1Y12 [19])
and Hcp3 (PDB 3HE1 [20]) from Pseudomonas
aeruginosa as well as EvpC from Edwardsiella tarda
(PDB 3EAA [21]). Additionally, the structure of the
Francisella tularensis IglC protein, an Hcp-like protein,
has also been reported (PDB 2QWU [22,23]). The
three former Hcp proteins exhibit an identical fold of
two antiparallel b-sheets decorated by a b-hairpin
extension (figure 2a), while IglC has a supple-
mental 30-amino acid N-terminal segment [23]. Six
Hcp molecules assemble to form 80 90 A
hexameric rings stabilized by an extension acting as
an inter-subunit belt. The centre of the hexamer has
TagL model (YiaD)
Figure 1. Schematic of type VI secretion systems. Centre: schematic of the current general model of T6SS assembly. Left:
three-dimensional or model structures of the components of T6SS tube and sheath (top) and of the tube hub and the
ATPase (below). Right: three-dimensional or model structures of the components of the T6SS membrane complex. See
text and figures 2 5 for details.
Table 1. Summary of the structural information available on T6SS core components and homologues.
subunit localization PDB homologue/localization PDB
TssA putative cytosolic protein
TssB soluble protein, OM associated bacteriophage sheath 1FOH
TssC soluble protein, OM associated bacteriophage sheath 1FOH
TssD/Hcp soluble, forms hexameric rings,
putative pilus
1Y12, 3HE1,
bacteriophage tail gp19 2K4Q, 2X8K,
TssE putative soluble protein bacteriophage wedge gp25 2IA7
TssF putative soluble protein
TssG putative soluble protein
TssH/ClpV cytosolic protein, AAA
ATPase 3ZRI, 3ZRJ Hsp100/Clp AAA
TssI/VgrG cell puncturing device,
forms trimers
2P5Z bacteriophage tail spike
TssJ/SciN outer membrane lipoprotein 3RX9, 4A1R transthyretin 1SN5
TssK putative cytosolic protein
TssL inner membrane, 1 TM 3U66 T4bSS IcmH/DotU protein
TssM inner membrane, 3 TMs T4bSS IcmF protein
Review. Type VI secretion E. Cascales & C. Cambillau 1103
Phil. Trans. R. Soc. B (2012)
Page 2
a diameter of 35 40 A
, which may therefore accom-
modate a small folded protein or unfolded/partly
folded protein (figure 2b). Although the Hcp proteins
and IglC share structural similarities, the additional
N-terminal segment of IglC interferes with hexamer
formation, and therefore the supramolecular assembly
of IglC remains to be determined [23]. The Hcp
hexamers have been shown to assemble as tubes (red
in figure 2c), either by direct observation of Hcp3
tubes by electron microscopy (EM) [26] or by examin-
ing the crystal packing of Hcp1, Hcp3 and EvpC
(Hcp1 and EvpC hexamers are stacked in a head-
to-tail mode [19,21], whereas Hcp3 hexamers are
packed in a head-to-head mode [20]). This ability
has been used to produce nano-objects, tubes of differ-
ent determined length, by engineering disulphide
bridges at the hexamers’ interfaces [27]. In vivo, Hcp
accumulates in the culture supernatant as well as in
the periplasm, suggesting that it assembles a tubular
structure from the inner membrane which passes
through the outer membrane. However, tubular struc-
tures of Hcp and its packing mode have not been
detected in vivo, leaving open the question on how
its assembly is controlled.
The Hcp tertiary structure is very similar to that of
gpV, the bacteriophage l tail tube protein (PDB 2K4Q
[28]). Hcp structure resembles the N-terminal domains
of Dit proteins from phages SPP1 (PDB 2X8K [29,30])
and p2 (PDB 2WZP [31]), which form hexameric rings
at the distal end of the tails (figure 2a). Finally, the Hcp
proteins share structural resemblance with phage major
tail proteins (MTPs), although the quaternary structure
of phage MTPs has not yet been described. These simi-
larities have been reported before [26,28], and we
recently proposed that they extend to most bacterio-
phages tail proteins, a fact that could be a hallmark of a
common molecular origin [32].
Current models suggest that the tip of the Hcp
tube harbours a trimer of the VgrG protein that
functions as a puncturing device towards the targeted
cells (figure 2d, and orange in figure 2c). As expected
from this model, VgrG proteins are often found in
the culture supernatant of bacteria expressing T6SSs
[33 36]. Interestingly, surface assemblies of Hcp
and VgrG are mutually dependent, as VgrG is not
found in the culture supernatant of hcp
cells and
Hcp is not found in vgrG
cell supernatant [34 36].
While it is easily conceivable that Hcp assembly
pushes out the VgrG trimer located at the tip, the
question why Hcp is not found in the culture super-
natant of vgrG
cells has not yet been experimentally
answered. One may hypothesize that VgrG recruitment
to the apparatus triggers Hcp assembly, with polymeriz-
ation of the Hcp tube then pushing the VgrG protein to
the external medium to puncture target cells [2,18].
The N-terminal domain of the VgrG protein from
Escherichia coli CFT073 (483 residues out of 824) has
been determined by X-ray diffraction (PDB 2P5Z,
[26]) (figure 2d,e). VgrG is composed of four domains,
two side-to-side b-sandwiches, an a/b domain and
T4 gp27
p2 ORF16
p2 ORF15
( f )
Figure 2. Structure of the tail tube proteins. (a) Ribbon views of the three Hcp proteins of known structures and their bacterio-
phage homologues (colours according to secondary structures). (b) Ribbon view of the hexameric ring of Hcp. (c) The tube
surface model formed of stacked Hcp rings (red) and terminated by a VgrG trimer (orange). (d ) Ribbon view of the VgrG
trimer (ribbons, colours per monomer) as determined experimentally by X-ray diffraction and the molecular model of the
needle domain (modelled from the bacteriophage T4 gp5) attached to it. (e) Ribbon view (rainbow colours from blue (Nt)
to red (Ct)) illustrating the VgrG domain topology: the two b-sandwich domains (D1, D2), the a/b domain (D3) and the
OB fold. (f ) Ribbon view of bacteriophage structural homologues of VgrG (colours according to secondary structures).
Figures were drawn with PyMOL [24] or Chimera [25].
1104 E. Cascales & C. Cambillau Review. Type VI secretion
Phil. Trans. R. Soc. B (2012)
Page 3
the oligonucleotide/oligosaccharide-binding (OB) fold
(figure 2e). VgrG architecture is comparable with
that of myophage T4 gp27 and the OB-fold domain
(PDB 1K28 [37]; figure 2f ). By extension, the struc-
tural similarity also applies to p2 ORF16, Mu gp44,
MuSO2 Q8EDP4 and EGD-e gp18, all of which
possess T4 gp27-like topology (figure 2f )[29,31,38].
VgrG forms a trimer in vivo [34,36], as do the similar
phage proteins mentioned above. The two b-sandwich
domains of VgrG share together a highly similar
fold (figure 2e). In the trimer, the three pairs of
b-sandwiches form a pseudo sixfold structure that
establishes the probable interface with the last Hcp
hexamer of the tube (figure 2c). As Hcp resembles the
two b-domains of VgrG proteins, this pseudo sixfold
symmetry probably eases the transition between the
Hcp sixfold and the VgrG threefold symmetries at
the interface.
Sequences and structural comparisons suggest that
full-length VgrG could be described as a T4 punctur-
ing device in which gp27 and gp5 (the needle) are
fused, and the gp5 lysozyme domain removed
[26,34](figure 2d). Although the structure of the
gp5-like domain of the E. coli VgrG has not been
elucidated, it has been modelled and probably folds
as a b-helical prism that will form the puncturing
needle (figure 2d ). In most bacteria, VgrG follows
this scheme, whereas in a few cases, called ‘evolved’
VgrGs, an additional effector domain (most often an
enzyme) is carried at the C-terminus [11,34]. The
C-terminal additional domains of ‘evolved’ VgrG
protein are delivered into the host cell cytosol [39].
Two of these domains have been characterized so far
and induce host cell toxicity through a modification
of the eukaryotic cytoskeleton [34,39,40]. Based on
this observation, it has been proposed that ‘evolved’
VgrGs might target eukaryotic cells, while ‘non-
evolved’ VgrGs would target bacterial cells. In this
latter case, the lack of homologues of the bacterio-
phage gp5 lysozyme domain might be detrimental to
cell wall perforation. It has been shown recently, how-
ever, that the Pseudomonas aeruginosa HSI-1 T6SS
(which carries ‘non-evolved’ VgrGs) can inject the
Tse1 and Tse3 peptidoglycan hydrolases into the peri-
plasm of targeted Gram-negative bacteria, leading to
cell wall destruction and lysis [4,9].
The internal diameter of the Hcp tubes (35 40 A
is sufficient for the canonical T6SS protein substrates
(17 and 43 kDa) to diffuse through it. Importantly, the
VgrG proteins do not possess an open internal chan-
nel, therefore preventing any toxin protein going
through. Obviously, VgrG should disassemble from
the Hcp tube after perforation of the recipient outer
membrane, or should open by a mechanism analogous
to that observed in siphophage p2 [31]. Finally,
although the formation of a needle assembling an
Hcp tube and a puncturing VgrG trimer seem to be
a very likely hypothesis, such assembly has not yet
been structurally documented.
(b) The type VI secretion systems’ needle sheath
In constrast to siphophages, myophages have a
contractile tail sheath enveloping the tail tube through
which DNA is injected. The myophage T4 DNA
injection process involves a cascade of events triggered
by the long fibres attached to the bacterial surface, fol-
lowed by baseplate conformational change, the
attachment of short fibres and contraction of the tail
sheath which pushes the tail tube through the cell
wall breach formed by the actions of the puncturing
device and the lysozyme. We have no structural proof
for a similar mechanism in T6SSs. However, it has
been observed by electron microscopy (EM) that the
Vibrio cholerae TssB and TssC (VipA/VipB) proteins
form tubes hundreds of angstroms long, with an
approximate external diameter of 300 A
and internal
diameter of approximately 100 A
[41](figure 3a). It
seems from the low-resolution pictures that these
tubes obey a 12-fold symmetry. The size of these
tubes is close to that of T4 tail sheath as observed
using single-particle EM maps [26]. After fitting resi-
dues 1 510 of the tail sheath gp18 protein
(approximately three-quarters of the full length; PDB
1FOA) into the cryo-EM map, Rossmann and col-
leagues observed that the tube has an external
diameter of approximately 250 A
and an internal
diameter of approximately 110 A
, allowing a gp19
tail tube to be accommodated (PDB 1FOH [42]).
EM thus showed that the bacteriophage tail sheath
and the TssB TssC tubules share a similar organi-
zation even though TssB and TssC do not have
sequence similarities with gp18. If we consider the
T4 tail sheath as a plausible model for the T6SS
needle sheath, we can attempt to assemble a molecular
model by fitting the Hcp tube structure into that of T4
tail sheath. In this model (figure 3b), the external size
of the Hcp tube (8085 A
) fits well into the internal
size of the tail sheath tube (110 A
). Interaction
between the TssB and TssC proteins has been demon-
strated in several bacteria, including V. cholerae,
Burkholderia cenocepacia, F. tularensis, P. aerug inosa and
Salmonella enterica [43,44]. The TssB and TssC pro-
teins have been shown to localize both in the
cytoplasm as well as at the outer membrane [41,44].
In F. tularensis, the TssB and TssC homologues
IglA and IglB have been proposed to assemble
structures large enough to be pelleted by ultracentrifu-
gation [23]. The TssB and TssC outer membrane
localization might therefore be an artefact due to the
size of the TssBC tubules. The data support the hypoth-
esis that a sheath-like cytoplasmic structure assembles
from the inner membrane. Interestingly, the Hcp-like
IglC protein co-fractionates with IglA and IglB, provid-
ing further support to the tail tube and sheath-like
hypothesis [23]. Based on these similarities, it is thought
that the contraction of the TssBC proteins might
provide the force required for pushing the Hcp tube out-
side the cell [13,26]. This contraction might be
provoked by the ClpV AAA
ATPase that has been
shown to depolymerize TssBC tubules ([41]; see §4).
A number of other subunits of the T6SS apparatus,
e.g. TssA, TssF and TssG, are not predicted to be
anchored to the membrane, but instead are soluble cyto-
plasmic proteins, suggesting that these subunits might
also have structural homologies with bacteriophage
components even though no obvious similarities can
be inferred from the primary sequence or secondary
structure predictions.
Review. Type VI secretion E. Cascales & C. Cambillau 1105
Phil. Trans. R. Soc. B (2012)
Page 4
(c) The type VI secretion systems’ needle hub
A significant (approx. 40%) sequence similarity was
detected between the bacteriophage T4 gp25 component
and the T6SS TssE subunit [26,45]. Although T4
gp25 was first suggested to have a lysozyme-like activity
[46], it is now clear that this protein is a structural com-
ponent of the bacteriophage baseplate [4749]. gp25
contributes to the T4 baseplate structure by interacting
with the (gp27gp5)
complex (at rest) or the tail tube
(in the activated state) [47,50,51]. The structure of a
gp25 homologue has been reported (PDB 2IA7) allowing
modelling of the TssE subunit (figure 3c); the presence
of a homologous protein in the T6SS suggests that it
plays a role either in producing a baseplate-like assembly
interacting with VgrG and Hcp, or may be a constituent
of T6SS hub. Localization studies have shown that
TssE is a cytoplasmic protein in P. aeruginosa suggesting
that if a baseplate-like structure exists in T6SS, it
should be assembled on the cytoplasmic side of the
inner membrane.
The cytosolic ClpV is a member of the Hsp100 (Clp)
family of AAA
proteins, hexameric ATPases involved
in substrate unfolding. The ClpV chaperone, or TssH
in the T6SS nomenclature, has been shown to play a
crucial role in the T6SS assembly mechanism. When
the soluble proteins TssB and TssC are mixed, they
assemble spontaneously in long tubes (see §3b and
figure 3a)[41] that may prevent the translocation of
the monomers into the periplasm to form the putative
tube sheath. The ClpV AAA
chaperone helps solve
this problem by using its non-catalytic N-terminal
domain (residues 1159; PDB 3ZRI) to form a com-
plex with an
-helix from TssC (residues 15 28) and
thus dissociates it from TssB (PDB 3ZRJ [41,52])
(figure 4). The C-terminal ATP-binding domain of
ClpV provides the energy for the dissociation to
occur. This chaperone mechanism has been documen-
ted in other systems and, interestingly, while the
chaperone domains share a structurally conserved a-
helical bundle fold, the helices from the substrates
bind at quite distinct places [52]. The ClpV ATPase
might therefore act at two steps during T6SS assembly
and function by (i) depolymerizing the TssBC tubules
in the cytosol allowing their transport into the peri-
plasm where TssB and TssC will polymerize to form
the sheath-like structure, and then (ii) acting to depo-
lymerize the putative sheath to provide the energy
required for its contraction [13,44].
Three core genes of T6SS gene clusters are predicted to
encode proteins anchored to the cell envelope. Indeed,
240 Å
110 Å
80 Å
Figure 3. Structure of tube sheath and hub proteins. (a) Electron microscopy side-view (left) or cross section (right) of the
TssB/TssC tubes formed in vitro (with permission from Bo¨nemann et al. [41]). The 25 mm scale bar applies to all the electron
microscopy views. (b) Molecular surface model of a cross section of a T6SS tube with Hcp rings in the middle (red) and TssB/
TssC sheath around (blue, modelled from phage T4 tail sheath [1FOH] [42]). (c) Ribbon model (rainbow colours) of the hub
protein TssE, generated from the structure of Geobacter sulfurreducens gp25 (2IA7; Joint Center for Structural Genomics 2006,
unpublished data). Figures were drawn with PyMOL [24] or Chimera [25].
1106 E. Cascales & C. Cambillau Review. Type VI secretion
Phil. Trans. R. Soc. B (2012)
Page 5
TssM and TssL localize at the inner membrane [53],
whereas TssJ is an outer membrane lipoprotein [54].
Binary interactions have been detected: TssM inter-
acts with TssL [35,53] and TssJ [35,55]. TssM
therefore links the inner and outer membrane. Co-
immunoprecipitation studies demonstrated that a
complex of these three subunits, as well as the accessory
TagL protein, assembles in the cell envelope [56].
(a) The inner membrane embedded proteins
and their partners
TssL and TssM are prominent features of T6SS.
These two proteins share similarities with IcmH/
DotU and IcmF, two components associated with
type IVB secretion systems [57,58].
Both TssL and TssM proteins are inserted into the
inner membrane. Sequence analysis and topological
studies have made it possible to determine TssM to-
pology in A. tumefaciens and enteroaggregative E. coli
([53], our unpublished results). The N-terminus of
TssM is composed of a few residues located in the cyto-
plasm, followed by two transmembrane helices
separated by a short loop. A cytoplasmic domain of
approximately 330 residues, predicted as a-helical, is
located between the second and the third TM helices.
Except for a few TssM proteins, this cytoplasmic
domain carries Walker A and B boxes, suggesting that
the TssM proteins have ATP binding and hydrolysing
activities. Mutagenesis studies showed that the Walker
A motif of A. tumefaciens is indeed required for T6SS
assembly [53], while the Walker A mutation has no
effect on E. tarda [35]. Several TssM proteins are
predicted to possess only a single TM, missing the
N-terminal TM hairpin. A large periplasmic domain
of approximately 740 residues follows the last helix
(figure 1). This domain of the entero-aggregative
E. coli TssM protein has been expressed as a soluble
fragment [55]. It has been dissected into two subdo-
mains, an N-terminal one of approximately 540
residues, mainly a-helical (as shown by circular dichro-
ism), and a C-terminal domain of approximately 200
residues, formed essentially by b strands [55](figure 1).
TssL is anchored through a single transmembrane
segment [59]. Most TssL proteins share in their C-
terminal periplasmic domain a peptidoglycan-binding
motif of the OmpA family. When absent, an accessory
protein, TagL, compensates by (i) carrying this motif
and (ii) interacting with TssL [56,60]. Several struc-
tures of peptidoglycan-binding proteins with an
OmpA family fold (PDB 1OAP; 3CYQ; 1R1M,
2K1S) have been solved by allowing a modelling of
the TssL (or TagL) periplasmic domain (figure 5a).
The conserved residues delimitate a groove that
accommodates an N-acetylmuramic acid molecule.
All the residues involved in groove formation and
N-acetylmuramic acid binding are also conserved in
the TssL (or TagL) homologues, and indeed the enter-
oaggregative E. coli TagL protein has been shown to
interact with the peptidoglycan in vivo and in vitro
56]. Besides this periplasmic peptidoglycan-binding
domain, the TssL subunits share an N-terminal
cytosolic domain of approximately 300 residues.
We recently solved the crystal structure of the enteroag-
gregative E. coli TssL cytoplasmic domain (PDB 3U66
[61]; figure 5b). TssL is an eight a-helix protein.
The three first long helices form a tight bundle, and
are followed by a disordered loop of four polar resi-
dues (loop 3 4). Two short helices (h4 and 5) and
loops connect the first helix bundle with the second
helix bundle, formed of three shorter helices (h68).
A long stretch of 10 residues protrudes between helices
7 and 8 (loop 7 8), forming two elongated structures
TssH-NT / TssC-NT
Figure 4. Structure of the ClpV (TssH) ATPase N-terminal domain in complex with the TssC N-terminal helix. Ribbon view
of the N-terminal domain of ClpV (TssH; residues 1158) from Vibrio cholerae (rainbow colours from blue Nt to red Ct) in
complex with the first N-terminal helix of TssC (residues 16 29) (3ZRJ [52]). The critical residues at the ClpVTssC
interface are emphasized in the inset.
Review. Type VI secretion E. Cascales & C. Cambillau 1107
Phil. Trans. R. Soc. B (2012)
Page 6
separated by a loop. The last helix finishes at residue
157, and is followed by an elongated unstructured
segment (residues 158178). The function of the cyto-
plasmic domain of TssL is not clear but a hypothesis is
that it may form a cytosolic hook to recruit the
substrates to be secreted to the T6SS.
(b) The membrane spanning TssM TssJ
The inner membrane protein TssM also interacts with
TssJ [35,55]. Surface plasmon resonance studies
demonstrated that the periplasmic domain of TssM
interacts with TssJ with a 24 mM dissociation con-
stant [55]. TssJ (SciN) is an essential component of
T6SS, approximately 180 residues long, and is
anchored to the outer membrane by the N-terminal
acylated cysteine of the processed form [54]. We
have recently determined the structure of the entero-
aggregative E. coli TssJ protein (PDB 3RX9 [55];
figure 5c). The first 22 residues of the approximately
150 residues mature protein are not visible in the elec-
tron density map, and should therefore form a flexible
linker. TssJ has a b-sandwich fold with two four-
stranded b-sheets (figure 5c). Sheet 1 is composed of
b-strands 4, 1, 7 and 8, and is packed against sheet
2 which contains b-strands 3, 2, 5 and 6. This rep-
resents a common fold, namely transthyretin (PDB
1SN5), shared by several dozens of proteins in the
protein databank. Compared with similar folds, TssJ
owns two additional elements: (i) the external face of
b-sheet 2 is covered in part by three short helices
(h1 3) occurring between b-strands 2 and 3 and
(ii) a loop located between strands 1 and 2 protrudes
from the core of the protein [55]. This fold is
conserved by the Serratia marcescens TssJ protein
loop 3–4
loop 7–8
Figure 5. Structures of the membrane complex proteins. (a) Ribbon view (rainbow colours from blue Nt to red Ct) of the
C-terminal peptidoglycan-binding domain of the enteroaggregative E. coli TagL protein (residues 414557) modelled from
the X-ray structure of E. coli YiaD (2K1S; T. A. Ramelot & M. A. Kennedy 2008, unpublished data). (b) Ribbon view (rain-
bow colours from blue Nt to red Ct) of the N-terminal cytoplasmic domain of the TssL protein from enteroaggregative E. coli
[61]. (c) Ribbon view (rainbow colours from blue Nt to red Ct) of the C-terminal periplasmic domain of the TssJ lipoprotein
from enteroaggregative E. coli [55].
1108 E. Cascales & C. Cambillau Review. Type VI secretion
Phil. Trans. R. Soc. B (2012)
Page 7
(PDB 4A1R [62]). The helical h1 3 domain is
required for stability of TssJ, while the L1 2 loop is
responsible for efficient interaction with TssM
(figure 1)[55]. Interestingly, this fold is also shared
by a number of proteins associated with secretion
systems: the T3SS-associated ExsB lipoprotein (PDB
2YJL [63]), as well as the type IV plus-associated
PilP lipoprotein (PDB 2IVW [64]). This observa-
tion suggests that lipoproteins associated with
distinct secretion systems or trans-envelope complexes
may have evolved from a common ancestor with a
b-sandwich fold.
We have summarized in this review the current know-
ledge on the structural characterization of individual
type VI secretion components. What emerges from
these structures is that T6SSs are patchworks com-
posed of subunits coming from various origins, such
as bacteriophage-like components, type IVb-associated
proteins, lipoproteins with transthyretin fold or
Hsp100/Clp AAA
ATPases. Although the structures
of isolated T6SS subunits provide significant progress
in the field, a comprehensive picture of the overall
structure is still lacking. The crystal structures of the
T6SS-like bacteriophage counterparts provided con-
siderable advances to understanding how T6SSs
assemble and raised exciting hypotheses on how
T6SSs work. The comparison between T6SS biogen-
esis and bacteriophage morphogenesis is therefore
critical to clearly understand T6SS dynamics during
assembly and function. Even though providing new
structures will be important, the next step will be to
obtain high-resolution images of subassemblies of the
T6SS machine, as exemplified for T3SS and T4SS.
In vivo and biochemical data aimed at understanding
the topology of the proteins and the interaction network
will be the foundations for microscopy and structural
studies. If it is reasonable to imagine EM or atomic
definition of several of the T6SS subassemblies in the
future, defining how the different pieces of the jigsaw
are connected is also a fascinating challenge.
We thank the members of the T6SS groups for discussions
and critical reading of the manuscript. We are grateful to
Yves-Michel Cully for figure preparation, Axel Mogk and
Nature Publishing Group for permissions to reproduce
figures, Bernard He
bianca for encouragements, and the
two anonymous reviewers for their corrections. Work in
E.C.s laboratory is supported by the CNRS and funded by
a grant from the Agence National de la Recherche (ANR-
10-JCJC-1303-03). Work in C.C.’s laboratory is supported
by the CNRS, the Universite
Aix-Marseille and by grants
from the Marseille-Nice Ge
nopole, IBiSA and the
Fondation pour la Recherche Medicale (SPF20101221116
and FRM DEQ2011-0421282).
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Phil. Trans. R. Soc. B (2012)
Page 10
  • Source
    • "By motif and profile HMM analysis, we identified presumed functional equivalents of nine of these 13 Proteobacterial core proteins in gut Bacteroidales T6SS loci; however, genes encoding identifiable TssA, TssJ, TssL and TssM proteins were not detected. The function of TssA is currently unknown; however, TssJ, TssL, and TssM likely form a transenvelope complex that anchors the phage tail structure[27,28]. There are five proteins of unknown function encoded within the Bacteroidales T6SS of all three genetic architectures. "
    [Show abstract] [Hide abstract] ABSTRACT: Type VI secretion systems (T6SSs) are contact-dependent antagonistic systems employed by Gram negative bacteria to intoxicate other bacteria or eukaryotic cells. T6SSs were recently discovered in a few Bacteroidetes strains, thereby extending the presence of these systems beyond Proteobacteria. The present study was designed to analyze in a global nature the diversity, abundance, and properties of T6SSs in the Bacteroidales, the most predominant Gram negative bacterial order of the human gut. By performing extensive bioinformatics analyses and creating hidden Markov models for Bacteroidales Tss proteins, we identified 130 T6SS loci in 205 human gut Bacteroidales genomes. Of the 13 core T6SS proteins of Proteobacteria, human gut Bacteroidales T6SS loci encode orthologs of nine, and an additional five other core proteins not present in Proteobacterial T6SSs. The Bacteroidales T6SS loci segregate into three distinct genetic architectures with extensive DNA identity between loci of a given genetic architecture. We found that divergent DNA regions of a genetic architecture encode numerous types of effector and immunity proteins and likely include new classes of these proteins. TheT6SS loci of genetic architecture 1 are contained on highly similar integrative conjugative elements (ICEs), as are the T6SS loci of genetic architecture 2, whereas the T6SS loci of genetic architecture 3 are not and are confined to Bacteroides fragilis. Using collections of co-resident Bacteroidales strains from human subjects, we provide evidence for the transfer of genetic architecture 1 T6SS loci among co-resident Bacteroidales species in the human gut. However, we also found that established ecosystems can harbor strains with distinct T6SS of all genetic architectures. This is the first study to comprehensively analyze of the presence and diversity of T6SS loci within an order of bacteria and to analyze T6SSs of bacteria from a natural community. These studies demonstrate that more than half of our gut Bacteroidales, equivalent to about ¼ of the bacteria of this ecosystem, encode T6SSs. The data reveal several novel properties of these systems and suggest that antagonism between or distributed defense among these abundant intestinal bacteria may be common in established human gut communities.
    Full-text · Article · Dec 2016 · BMC Genomics
  • Source
    • "Over a decade of research led by many laboratories hugely improved our understanding of T6SS. Many comprehensive reviews about different aspects of T6SS were written recently, either with a broad view [11 –13], or with a focus on effectors [14,15] or on structural aspects [1,161718. Here, I will review the progress that has been made towards understanding the molecular mechanism of protein secretion by T6SS and discuss its unique mode of action. "
    [Show abstract] [Hide abstract] ABSTRACT: The type VI secretion systems (T6SS) are present in about a quarter of all Gram-negative bacteria. Several key components of T6SS are evolutionarily related to components of contractile nanomachines such as phages and R-type pyocins. The T6SS assembly is initiated by formation of a membrane complex that binds a phage-like baseplate with a sharp spike, and this is followed by polymerization of a long rigid inner tube and an outer contractile sheath. Effectors are preloaded onto the spike or into the tube during the assembly by various mechanisms. Contraction of the sheath releases an unprecedented amount of energy, which is used to thrust the spike and tube with the associated effectors out of the effector cell and across membranes of both bacterial and eukaryotic target cells. Subunits of the contracted sheath are recycled by T6SS-specific unfoldase to allow for a new round of assembly. Live-cell imaging has shown that the assembly is highly dynamic and its subcellular localization is in certain bacteria regulated with a remarkable precision. Through the action of effectors, T6SS has mainly been shown to contribute to pathogenicity and competition between bacteria. This review summarizes the knowledge that has contributed to our current understanding of T6SS mode of action.
    Full-text · Article · Oct 2015 · Philosophical Transactions of The Royal Society B Biological Sciences
  • Source
    • "The T6SS assembles from 13 conserved components. Architecturally, the T6SS can be seen as a micrometer-long syringe anchored to the cell membrane by a trans-envelope complex3456 . The phage tail-related syringe-like tubular structure is composed of an internal tube tipped by a spike-like complex, wrapped by a contractile sheath and tethered to the membrane through contacts with components of a trans-envelope multiprotein complex [6, 7]. "
    [Show abstract] [Hide abstract] ABSTRACT: The type VI secretion system (T6SS) is a secretion pathway widespread in Gram-negative bacteria that targets toxins in both prokaryotic and eukaryotic cells. Although most T6SSs identified so far are involved in inter-bacterial competition, a few are directly required for full virulence of pathogens. The T6SS comprises 13 core proteins that assemble a large complex structurally and functionally similar to a phage contractile tail structure anchored to the cell envelope by a trans-membrane spanning stator. The central part of this stator, TssM, is a 1129-amino-acid protein anchored in the inner membrane that binds to the TssJ outer membrane lipoprotein. In this study, we have raised camelid antibodies against the purified TssM periplasmic domain. We report the crystal structure of two specific nanobodies that bind to TssM in the nanomolar range. Interestingly, the most potent nanobody, nb25, competes with the TssJ lipoprotein for TssM binding in vitro suggesting that TssJ and the nb25 CDR3 loop share the same TssM binding site or causes a steric hindrance preventing TssM-TssJ complex formation. Indeed, periplasmic production of the nanobodies displacing the TssM-TssJ interaction inhibits the T6SS function in vivo. This study illustrates the power of nanobodies to specifically target and inhibit bacterial secretion systems.
    Full-text · Article · Mar 2015 · PLoS ONE
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