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Type IV pili: e pluribus unum?
Running title: type IV pilus biogenesis
Vladimir Pelicic
Department of Microbiology, Imperial College London, London SW7 2AZ, United Kingdom
For correspondence:
E-mail: [email protected]; Tel: (+44) 20 7594 2080; Fax: (+44) 20 7594 3095
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Summary
The widespread role of pili as colonization factors in pathogens has long been recognized in
Gram-negative bacteria and more recently in Gram-positive bacteria, making the study of
these hair-like filaments a perennial hot topic for research. No other pili are found in as many
or as diverse bacteria as type IV pili. This is likely a consequence of their ancient origin and
unique ability to promote multiple and strikingly different phenotypes such as attachment to
surfaces, aggregation, uptake of DNA during transformation, motility, etc. Two decades of
investigations in several model species have shed some light on the structure of these
filaments and the molecular basis of some of the properties they confer. Moreover, recent
discoveries have led to a better knowledge of the genetic basis and molecular mechanisms
of type IV pili biogenesis. This brings us a few steps closer to understanding how these
filaments are produced, but leaves us wondering whether (as in the famous motto that
inspired the title) out of the many models studied will emerge one unifying mechanism.
Keywords: pili/type IV pili/bundle-forming pili/toxin co-regulated pili/Flp pili/twitching motility
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Type IV pili: bacteria's favourite colonization factors
Attachment to surfaces is a common property of bacteria that permits species-specific
lifestyles by allowing different species to colonize different niches. Attachment is often
promoted by hair-like filaments called pili (also known as fimbriae) that extend from the
bacterial surface and are primarily composed of a single protein generically named pilin (Soto
and Hultgren, 1999). Among the many different types of pili, which were first classified based
on their morphology and more recently based on their mechanisms of assembly, none are as
widespread as type IV pili (Tfp). These filaments have been observed in numerous bacterial
genera (Mattick, 2002) belonging primarily to Proteobacteria but also to other phyla as
diverse as Cyanobacteria (Nostoc punctiforme, Microcystis aeruginosa, Synechocystis sp.),
Deinococcus-Thermus (Deinococcus geothermalis, Thermus thermophilus) or Firmicutes
(Clostridium perfringens, Ruminococcus albus). Tfp are thus the only pili found in both Gramnegative and Gram-positive bacteria, which, together with the fact that this list is far from
exhaustive, suggests that they are the most widespread organs of bacterial attachment.
The unmistakable morphological characteristics that were initially used in electron
microscopy studies to define Tfp as a specific pilus type are still employed in their
identification. Tfp are extremely thin (5-8 nm in width), long (several µm in length) and flexible
filaments that often interact laterally to form characteristic bundles (Fig. 1). There is now
ample molecular evidence confirming the relevance of this original classification, since Tfp
from different species share many sequence and structural properties (Craig et al., 2004).
The pilin subunits, while they are extremely variable in sequence and length (for example, 49
amino acids (aa) for Flp1 in Aggregatibacter actinomycetemcomitans versus 199 aa for TcpA
in Vibrio cholerae), always display a consensus N-terminal motif (Pugsley, 1993). They are
synthesized as precursors (prepilins) with a hydrophilic leader peptide ending with a glycine,
which is cleaved by a unique leader peptidase. Most of the first 20-25 residues of the mature
pilin are hydrophobic, while the fifth residue is almost invariably a glutamate. The lengths of
both the leader peptide and the mature protein define two distinct subtypes of Tfp, which can
co-exist in some species. Type IVa pilins have short leader peptides (less than 10 aa) and a
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characteristic length of about 150-160 aa. Type IVb pilins exhibit long leader peptides (about
15-30 aa) and are either long (180-200 aa) or surprisingly short (only about 40-50 aa) as in
members of the Flp, fimbrial low-molecular-weight protein, family (Kachlany et al., 2001).
Nevertheless, the 3D structures of several type IVa and type IVb pilins (a Flp family pilin has
yet to be crystallized), reveal a conserved architecture consisting of an extended N-terminal
-helix and a globular head (Craig et al., 2004). The atomic models of Tfp based on these
structures have similar helical organizations in which the conserved portions of the Nterminal -helices provide the major assembly interface between pilin subunits and are
buried within the interior of the filament.
As already noted, pili are often involved in promoting bacterial attachment to, and
colonization of, a wide variety of biotic and abiotic surfaces. Tfp facilitate adhesion, directly
and/or indirectly by promoting interbacterial interactions and biofilm formation, on: (1) host
cells in numerous human pathogens such as enteropathogenic Escherichia coli (EPEC),
Neisseria meningitidis and V. cholerae, (2) cellulose in R. albus, (3) stainless steel in
Pseudomonas aeruginosa, (4) papermaking machines in D. geothermalis, etc. However, to
paraphrase George Orwell, compared to other pili Tfp "are more equal" due to their frequent
capacity to promote additional unrelated properties. For example, they permit a form of
locomotion known as twitching motility, as well as DNA uptake during transformation
(Mattick, 2002). These properties depend on the unique capacity of Tfp to retract, which
generates substantial mechanical force (Maier et al., 2002). Moreover, Geobacter
sulfurreducens Tfp are nanowires involved in electron transfer (Reguera et al., 2005). Such
an astonishing and uncommon multi-functionality is a plausible reason for Tfp's success in
the bacterial kingdom.
Biogenesis of simple filaments relies on complex and rather diverse machineries
The first analyses of genes involved in Tfp biogenesis in different species revealed important
commonalities and thus provided definitive genetic evidence that Tfp are a homogeneous
group of pili. In Gram-negative bacteria, Tfp biogenesis machineries comprise a conserved
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core of proteins that includes: (1) one or several proteins, besides the major pilin, with an Nterminal pilin-like motif that might slightly differ from the above consensus, (2) a specific
peptidase that processes the prepilins and prepilin-like proteins, (3) a traffic ATPase that
powers Tfp assembly, (4) an integral inner membrane protein of unclear function and (5) an
integral outer membrane protein, named secretin, necessary for the emergence of Tfp on the
bacterial surface. Hereafter, these conserved proteins will be generically named core
proteins, as opposed to the non-core proteins that are specific to some systems or species.
Unexpectedly, similar proteins were also found in the type II secretion machinery that
mediates the passage of folded proteins through the outer membrane in Gram-negative
bacteria and also, at least in part, in machineries involved in the biogenesis of filamentous
phage and archaeal flagella, or in DNA uptake in Gram-positive bacteria (Peabody et al.,
2003). This suggests that all these systems share a common origin and represent variations
on the theme of macromolecule transport across membranes.
Systematic genetic studies have now defined the complete sets of genes encoding the
proteins specifically dedicated to Tfp biogenesis in several model systems, including both
type IVb pili as the bundle-forming pilus (Bfp) of EPEC (Ramer et al., 2002), the toxin coregulated pilus (Tcp) of V. cholerae (Kirn et al., 2003) and the R64 plasmid thin pilus (R64
Pil) of E. coli (Yoshida et al., 1999), and type IVa pili in P. aeruginosa (Alm and Mattick,
1997) and N. meningitidis (Carbonnelle et al., 2005). All machineries are composed of
between 10 (V. cholerae) and 18 (P. aeruginosa) proteins, and can be subject to complex
regulatory systems, as in P. aeruginosa (Alm and Mattick, 1997). Most importantly, these
studies revealed differences consistent with the subdivision of type IVa and type IVb pili. As
will be put forward in this review, these differences might be more important than previously
thought, and as informative as the commonalities listed above.
Studies in P. aeruginosa and N. meningitidis, which are unrelated  and 
Proteobacteria respectively, showed that Tfp biogenesis requires a large set of extremely
conserved proteins (Alm and Mattick, 1997; Carbonnelle et al., 2005), suggesting that type
IVa pili are a homogeneous group. The rare differences between these bacteria concern the
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different genomic organization of few pil genes, notably pilin and pilC (Fig. 2). Unless
otherwise stated, the N. meningitidis nomenclature is used in this review. Moreover, a N.
meningitidis pilZ mutant is piliated but affected for Tfp-linked properties (Carbonnelle et al.,
2005), while the corresponding P. aeruginosa mutant is non-piliated (Alm et al., 1996a), and
the pilY2 gene apparently plays a role in Tfp biogenesis only in P. aeruginosa (Alm et al.,
1996b), since it is not found in any other bacterial genus sequenced to date. In bacteria
producing type IVa pili, Tfp biogenesis genes are always scattered throughout the genome
(the only notable exception coming from Gram-positive bacteria in which they are apparently
clustered), but the same genes or gene clusters (e.g., the pilMNOPQ cluster, the pilW gene
and the pilFDG cluster encoding the traffic ATPase, the prepilin peptidase gene and the
conserved inner membrane protein) are almost invariably flanked by the same, mainly
housekeeping, genes (Fig. 2). In addition, the pilin-like genes essential for Tfp biogenesis are
also clustered (Fig. 2). However, not all of the proteins cleaved by PilD are required for pilus
biogenesis, as shown for FimT in P. aeruginosa (Alm and Mattick, 1996) and PilX, ComP and
PilV in N. meningitidis (Carbonnelle et al., 2005). As suggested by the recent
structure/function analysis of PilX (Helaine et al., 2007), at least some of these latter proteins
might be minor pilins with important modulatory roles in Tfp biology.
A systematic search using the MaGe microbial annotation system (Vallenet et al.,
2006), which allows easy exploration of gene context by highlighting conserved synteny,
showed that the above gene clusters are conserved in all the species in which type IVa pili
have been characterized experimentally or that are known to exhibit twitching motility
(Mattick, 2002), even if they are phylogenetically distant (Fig. 2). Strikingly, virtually entire
sets of homologous genes could also be found in the same genomic locations in more than
150 sequenced species in various phyla, but primarily Proteobacteria where they are found
in genera belonging to all orders of the ,  and  classes. This includes many well-studied
species of Enterobacteria such as E. coli, Klebsiella pneumoniae, Salmonella enterica
serovar Typhimurium, Shigella flexneri and Yersinia pestis, in which type IVa filaments or
their linked phenotypes have not been observed. However, although the corresponding
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genes in laboratory workhorse E. coli K12 are poorly expressed and conditions leading to the
biogenesis of Tfp could not be found (Sauvonnet et al., 2000), type IVa pili are produced in
enterohemorrhagic E. coli O157:H7 (EHEC) upon growth on minimal casein medium and in
vivo during EHEC infections (Xicohtencatl-Cortes et al., 2007). Therefore, together with our
analysis, this leaves open the possibility that type IVa pili are far more widespread than
usually thought. These findings suggest that pil genes, which are not present on obvious
pathogenicity islands, are of ancient origin and were already present in a common ancestor
to many bacterial phyla, in which they encoded a machinery whose exact role can only be
speculated upon, although it certainly represented a variation on the theme of
macromolecule export.
In comparison, the picture in bacteria producing type IVb pili, which emerged from the
systematic studies of Bfp, Tcp and R64 Pil, is dramatically different. Type IVb pilus
biogenesis genes are less numerous, encoding smaller sets of 10-12 proteins (Yoshida et
al., 1999; Ramer et al., 2002; Kirn et al., 2003), and are always clustered. Moreover, when
the different type IVb systems are compared, the striking difference with type IVa pili is that
there is no significant conservation in the order of the genes, nor in the sequence of the
corresponding proteins except for the universally conserved core proteins (Fig. 2). This
indicates that type IVb pili are far less homogeneous than type IVa pili. One notable
exception to this is the recently described Flp family of type IVb pili (Kachlany et al., 2001), in
which flp biogenesis genes (whose exact number remains to be determined) are conserved
"en bloc" (Fig. 2), with a similar organization in A. actinomycetemcomitans and Caulobacter
crescentus (Tomich et al., 2007). Although such a gene organization has made genetic
studies more difficult because of the possibility of polar effects, it has allowed the transfer of
these genes and synthesis of pili in surrogate hosts (Sohel et al., 1996; Stone et al., 1996),
unambiguously confirming that they encoded complete sets of Tfp biogenesis proteins. This
has not been achieved with type IVa pili. Such an organization and the role of Bfp and Tcp in
pathogenesis suggested that type IVb pili-encoding genes could be parts of pathogenicity
islands, which is indeed the case for the tcp cluster in V. cholerae. Moreover, although this is
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not universally accepted (Faruque et al., 2003), this pathogenicity island might be a
filamentous bacteriophage (Karaolis et al., 1999). This phage could be transferred between
V. cholerae strains, endowing the recipient with the ability to assemble Tcp. However, this
potential for mobility has not led to a wide distribution of type IVb pili, which, unlike type IVa
pili, are found in a small subset of genera. BLASTP analyses at the NCBI using default
parameters reveal that complete sets of homologous non-core biogenesis proteins are found
only in EPEC and Yersinia mollaretti ATCC 43969 for the Bfp, in species of the genus Vibrio
for the Tcp, and in a few genera of  and  Proteobacteria for the R64 Pil. Again, Flp are an
exception, since non-core flp genes are found in distant phyla such as Chlorobi and
Actinobacteria (Tomich et al., 2007), although often as incomplete subsets.
Taken together, these observations suggest that the two subtypes of Tfp, although
sharing a common origin, separated long ago and have since evolved in parallel. The
obvious lack of conservation of Tfp biogenesis proteins other than the universally conserved
core proteins is particularly striking. When bacteria producing type IVa and IVb pili are
compared, none of the non-core Pil proteins (PilC, PilM, PilN, PilO, PilP and PilW), which
account for up to 40% of the proteins essential for Tfp biogenesis in N. meningitidis, are
found in type IVb systems, and vice versa. Moreover, as noted above, except for Flp, none of
the non-core proteins is shared among the species that produce type IVb pili. Again, this is
very significant because these proteins account for 33% of the Bfp proteins (BfpG, BfpC,
BfpU and BfpL), 40% of the Tcp proteins (TcpQ, TcpR, TcpD and TcpS) and as many as
45% of the R64 Pil proteins (PilK, PilL, PilM, PilO and PilP). This indicates that Tfp
biogenesis is less uniform than is widely accepted, with possible consequences for the
underlying molecular mechanisms of pilus assembly. Either the different pilus biogenesis
systems have evolved convergently, so that unrelated proteins perform similar functions, or
they have evolved divergently with unrelated proteins performing different functions, which
would suggest that there is more than one way to assemble Tfp. Although this problem
remains to be solved, it has important practical consequences, since it cautions against hasty
extrapolations of results obtained in one Tfp subtype to the other, or even in the case of type
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IVb pili to the other members of the same subtype.
Molecular mechanism(s) of Tfp biogenesis
How are Tfp assembled? Although some aspects of Tfp biogenesis remain foggy, partly as a
consequence of the differences outlined above, much progress has been made recently and
several key issues have been solved. The data are generally consistent with the following
molecular mechanism(s) for Tfp biogenesis.
Prepilin transport and processing
A shown in the related type II secretion system, prepilins are co-translationally targeted by
the signal recognition particle to the Sec machinery, which is solely responsible for
translocating them across the inner membrane (Arts et al., 2007; Francetic et al., 2007). Due
to their hydrophobic N-terminal -helix, the prepilins remain in the membrane as bitopic
proteins, with the charged leader peptide in the cytoplasm and the C-terminal domain in the
periplasm (Strom and Lory, 1987). This topology is required for correct recognition and
processing of prepilins by the prepilin peptidase, a polytopic inner membrane enzyme.
Systematic mutagenesis in P. aeruginosa showed that the only residue in prepilins key for
efficient processing was the last glycine residue of the leader peptide, while the conserved
Glu5 was dispensable for processing but important for subsequent pilus assembly (Strom and
Lory, 1991). A somehow more complex picture emerged when such a study was performed
with the R64 Pil, in which in addition to the last glycine residue of the leader peptide other
residues distributed throughout the prepilin sequence were also important for efficient
processing (Horiuchi and Komano, 1998). Although the molecular mechanism of processing
remains to be understood, it is now clear that two conserved aspartate residues in a Cterminal cytoplasmic loop of prepilin peptidases are critical (LaPointe and Taylor, 2000). This
finding indicated that type IV prepilin peptidases represent a novel family of aspartate
proteases and that, unlike originally suggested, the N-terminal cytoplasmic domain, which is
missing in the Flp family enzymes, is not crucial for function.
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Filament assembly
How Tfp are actually assembled is the main unsolved mystery in Tfp biogenesis.
Nevertheless, some aspects are now better understood. Filament assembly requires energy,
which is provided by a conserved traffic ATPase, and it relies on an inner membrane multiprotein machinery.
Early genetic studies in P. aeruginosa (Turner et al., 1993), later reproduced in different
piliated species and other related systems, indicated that Tfp assembly requires a traffic
ATPase belonging to the core set of proteins. The corresponding proteins in the R64 Pil
(PilQ) and Bfp (BfpD), which were purified and characterized in vitro (Sakai et al., 2001;
Crowther et al., 2005), form multimers and exhibit ATPase activity. The importance of several
conserved motifs was confirmed both in vivo and in vitro by showing that purified proteins
containing aa substitutions displayed reduced levels of ATPase activity (Sakai et al., 2001).
The recent solving of the 3D structure of several traffic ATPases revealed that these are
dynamic hexamers that can undergo large domain rearrangements upon ATP binding and
hydrolysis (Satyshur et al., 2007; Savvides, 2007). These rearrangements are probably
coupled to the generation of mechanical leverage through a "push and pull" mode of action
that powers extrusion of the pilin subunits from the inner membrane.
How this conversion of chemical energy into mechanical energy might occur is best
illustrated in a series of studies done in EPEC. A wide array of experimental approaches first
identified multiple interactions between the different Bfp proteins, leading to an emerging
picture of the topography of the Bfp assembly machinery (Ramer et al., 2002; Hwang et al.,
2003; Crowther et al., 2004). This provided evidence for the existence of a subcomplex in the
inner membrane (Crowther et al., 2004), composed of the traffic ATPase BfpD, the
conserved polytopic inner membrane protein BfpE (both core proteins) and the bitopic
cytoplasmic membrane protein BfpC, which is specific to EPEC. BfpC and BfpE interact with
each other and recruit BfpD to the inner membrane. The in vitro ATPase activity of BfpD was
increased by more than 1200-fold in the presence of BfpE and BfpC (Crowther et al., 2005),
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showing that this protein acts within a multiprotein machinery. The finding that BfpD binds to
two different adjacent sites in the N-terminus of BfpE, depending on the phosphorylation
status of the bound nucleotide, led to an attractive model explaining how BfpD might
transduce mechanical energy to the Bfp biogenesis machinery (Crowther et al., 2005). In
brief, BfpD-ATP is first recruited to the inner membrane by binding to BfpC and to a first
binding site in BfpE (residues 77-114), which dramatically stimulates its ATPase activity. ATP
hydrolysis is likely to lead to the noted large domain rearrangements (Savvides, 2007) within
BfpD, which promote the binding of Bfp-ADP to an adjacent binding site in BfpE (residues
39-76), thereby pushing the BfpE N-terminus through the cytoplasmic membrane and
energizing this protein to act as a piston pushing pilin subunits out from the inner membrane.
While this model is very elegant, it is not clear how relevant it is to other Tfp biogenesis
systems since BfpC, which serves as a scaffold protein anchoring the core proteins BfpD and
BfpE in place, is unique to EPEC. However, as noted above, unrelated bitopic inner
membrane proteins might substitute for BfpC in other systems. In support of this possibility, a
recent study in V. cholerae demonstrated that the traffic ATPase TcpT is recruited to the
inner membrane by the non-core bitopic inner membrane protein TcpR (Tripathi and Taylor,
2007). Nevertheless, it remains to be seen whether the core polytopic inner membrane
protein TcpE is part of this complex and whether the above model still holds.
What happens after extrusion of the pilin subunits from the inner membrane? Recent
findings in N. meningitidis obtained upon introduction of a pilT mutation into each of the 15
non-piliated pil mutants (Carbonnelle et al., 2006) provide some clues and suggest an
intriguing possibility. Namely, that Tfp might be assembled by a different mechanism in type
IVa and type IVb systems. This systematic study, which was inspired by a seminal report in
N. gonorrhoeae showing that the lack of piliation in a pilC mutant could be suppressed by a
second mutation in the gene encoding the PilT traffic ATPase powering pilus retraction
(Wolfgang et al., 1998), showed that piliation could be restored in the absence of most of the
Pil proteins when retraction is abolished (Carbonnelle et al., 2006). These proteins (PilC,
PilG, PilH, PilI, PilJ, PilK, PilQ and PilW) act after pilus assembly to promote emergence of
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the filaments on the surface and/or to antagonize PilT-mediated pilus retraction. The number
of proteins actually required for pilus assembly per se is surprisingly low and apparently
consists of PilD, PilE, PilF, PilM, PilN, PilO and PilP. Strikingly, although the pilMNOPQ gene
cluster is one of the most conserved in bacteria producing type IVa pili (Fig. 2), it encodes
four non-core proteins absent in species producing type IVb pili. As the roles of PilD (prepilin
peptidase), PilE (pilin) and PilF (traffic ATPase) are clear, the four non-core proteins PilM,
PilN, PilO and PilP could constitute the essence of the pilus assembly subcomplex. This
subcomplex is not only expected to assemble Tfp, but also to convert the chemical energy
provided by PilF into mechanical leverage, leading to the extrusion of pilin subunits from the
inner membrane. This later idea comes from the perhaps unexpected finding that the core
protein PilG (the equivalent of BfpE) is dispensable for pilus assembly since a pilG/T mutant
is piliated (Carbonnelle et al., 2006). This apparently rules out that PilG might be the piston
that pushes the pilin out of the inner membrane in N. meningitidis and conflicts with the
above described EPEC model, but this remains to be formally demonstrated. Another finding
supporting the possibility that different mechanisms exist for filament assembly is the
observation that while species producing type IVa pili can process and assemble type IVa
pilins from other species into filaments, as shown in P. aeruginosa and N. gonorrhoeae
(Elleman et al., 1986; Winther-Larsen et al., 2007), such gene interchange has so far not
been possible in species producing type IVb pili (McNamara and Donnenberg, 2000).
Tfp emergence on the bacterial surface
A particularly well-characterized core protein is the secretin that forms very stable multimers
in the outer membrane through which pili are thought to emerge on the bacterial surface.
These multimers in different species and related systems have a similar basic structure, as
determined by electron microscopy. They appear as ring-like cylindrical structures with large
gated central cavities (Opalka et al., 2003; Collins et al., 2004; Chami et al., 2005) that can
form channels in planar lipid bilayers in accordance with their role (Nouwen et al., 1999).
Biochemical and structural studies in N. meningitidis further showed that Tfp interact with the
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PilQ secretin, filling the large central cavity and inducing significant structural changes
(Collins et al., 2005). The strongest evidence that Tfp pass through this cavity comes from
the finding that piliation could be restored in N. gonorrhoeae and N. meningitidis secretin
mutants in the absence of pilus retraction but that the filaments remain trapped within the
periplasm (Wolfgang et al., 2000; Carbonnelle et al., 2005). These studies, which captured
Tfp in an intermediate intraperiplasmic state, provided several important clues for Tfp
biogenesis in general and the role of secretins in particular. First, they provided evidence that
Tfp assembly occurs before these filaments emerge on the bacterial surface. Second, pilus
translocation seems to be the major function of secretins, since these proteins are
dispensable for Tfp assembly. Third, they showed that secretins are the one and only route
to the surface for Tfp.
There is now growing evidence for the existence of a subcomplex of Tfp biogenesis
proteins in the outer membrane that is centred on the secretin and contains other proteins
required for secretin multimerization and/or stability. Unfortunately, these proteins are often
erroneously considered as pilot proteins or pilotins, even though most of them have not been
shown to pilot the secretin to its final destination. Moreover, targeting to the outer membrane
and multimerization are likely to be independent events (Guilvout et al., 2006). In the model
type IVb systems, the BfpB, PilN and TcpC secretins clearly do not need pilotins to reach the
outer membrane, since they are lipoproteins (Ramer et al., 1996; Sakai and Komano, 2000;
Bose and Taylor, 2005) that are probably transported to the outer membrane by the Lol
lipoprotein sorting pathway (Narita and Tokuda, 2006). However, the formation and/or
stability of these secretin multimers is still dependent on their interaction with non-core outer
membrane proteins such as BfpG and TcpQ (Schmidt et al., 2001; Bose and Taylor, 2005).
In the type IVa systems, where secretins are not lipoproteins, there is more confusion about
the need for pilot proteins. This is mainly due to common belief that the lipoprotein PilP might
be the PilQ pilotin in N. gonorrhoeae, which is based on the slight decrease in PilQ multimers
observed in a pilP mutant (Drake et al., 1997). However, no such effect was seen in N.
meningitidis (Carbonnelle et al., 2005; Carbonnelle et al., 2006) nor in M. xanthus (Nudleman
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et al., 2006), and it now seems that the observed decrease in N. gonorrhoeae PilQ multimers
might have been due to a polar effect on pilQ transcription of the mutation in the upstream
pilP gene (Balasingham et al., 2007). Finally, although PilP and PilQ interact, the finding that
PilP is an inner membrane lipoprotein argues against a role as a pilotin (Balasingham et al.,
2007). Several lines of evidence now indicate that type IVa secretins might not need pilotins
for proper targeting to the outer membrane. First, in the systematic study of pil mutants in the
absence of pilus retraction mentioned above, intraperiplasmic fibres, which could result either
from the absence or the mistargeting of the secretin, were seen exclusively in the pilQ/T
mutant (Carbonnelle et al., 2006). Second, PilQ insertion in the outer membrane in N.
meningitidis is dependent on Omp85, a protein ubiquitous in Gram-negative bacteria
(Voulhoux et al., 2003), involved in the membrane insertion and/or multimerization of many
outer membrane proteins. Nevertheless, the stability of type IVa secretin multimers is still
dependent on a partner non-core protein, such as PilW in N. meningitidis (Carbonnelle et al.,
2005) and its Tgl ortholog in M. xanthus (Nudleman et al., 2006). However, it remains to be
determined whether these proteins actually play an active part in the assembly of secretin
multimers or whether they merely contribute to their exceptional stability. The latter
possibility, which implies that PilQ multimers too unstable to be detected exist in the absence
PilW, is supported by the observation that the pili assembled by a pilW/T mutant are exposed
on the surface (Carbonnelle et al., 2005).
Future prospects and challenges
As reviewed here, our understanding of Tfp biogenesis has improved but remains
fragmentary. This might soon change, since the constantly increasing distribution of these
filaments and their key role in colonization in important pathogens should further boost efforts
in the field. However, the significant differences between the various systems, purposely
underscored in this review, suggest that a global understanding of Tfp biogenesis could only
come from parallel studies in several model organisms expressing various subtypes (type
IVa and type IVb) and families (Flp) of Tfp. It is only once the molecular mechanims of Tfp
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biogenesis are better understood in each class that overall common themes will be
discernible. Essential next steps involve completing the biochemical characterization of each
Tfp biogenesis protein (expression, localization, topology and protein-protein interactions).
This is particularly important for type IVa pili that are lagging behind compared with Bfp and
Tcp. Similarly to what has been done in EPEC and V. cholerae, interactions between the
type IVa system Pil proteins could be unravelled by two-hybrid studies (Crowther et al.,
2004), pull-down assays (Tripathi and Taylor, 2007), and by determining the stability and/or
localization of every protein in bacteria containing mutations in each of the other pil genes
(Ramer et al., 2002; Tripathi and Taylor, 2007), etc. Complementary approaches such as
surface plasmon resonance, used to study interactions between components of the
chaperone-usher pilus assembly pathway (Saulino et al., 1998), could also be explored.
Another appealing prospect would be to determine the influence, if any, of pilus retraction on
Tfp biogenesis in a species producing type IVb pili, as done in N. meningitidis (Carbonnelle
et al., 2006). The obvious candidate is EPEC, which might be the only model species
producing retractile type IVb pili, based on the fact that it is the only one with a gene
homologous to pilT (BfpF) whose inactivation leads to increased piliation and aggregation
(Bieber et al., 1998). In parallel, type IVb pilus retraction in EPEC or another suitable species
could be tested on a single-molecule level, as done in N. gonorrhoeae (Maier et al., 2002).
This could be instrumental in determining whether type IVb pili are actually non-retractile
filaments and could offer some clues as to why different species might have evolved different
ways to assemble Tfp.
Although the results that will be generated during such studies are the sine qua non for
understanding the molecular mechanisms of Tfp biogenesis, they are unlikely to be sufficient,
and new avenues of research will be required. What could be the new technical or
conceptual approaches that will open up the field? Although the answer to this question is
obviously subjective, two possibilities are particularly attractive. Due to the high intrinsic
value of atomic level 3D structures in understanding protein function, as illustrated in the field
by the example of pilins leading to atomic models of Tfp (Craig et al., 2004), one possibility
15
would be to start a concerted program to determine the structure of as many Tfp biogenesis
proteins as possible by nuclear magnetic resonance and/or X-ray crystallography. Although
this would require tremendous effort, the immense impact that atomic level structures of a
prepilin peptidase or a secretin, for example, would inevitably have suggests that it is
worthwhile. Difficulties will arise from the fact that many of these proteins are membrane
proteins whose structural characterization is notoriously difficult. Moreover, beside pilin
subunits, only few atomic level structures are currently available for proteins such as traffic
ATPases (Satyshur et al., 2007; Savvides, 2007), the N. meningitidis PilX minor pilin
(Helaine et al., 2007), PilP from N. meningitidis (Golovanov et al., 2006) and the two
orthologs PilF in P. aeruginosa (Kim et al., 2006) and PilW in N. meningitidis (our
unpublished data).
The second novel research avenue could be to use a reductionist approach in which
smaller pieces of the Tfp biogenesis machinery would be studied because every reduction in
complexity is likely to yield a biological system that is easier to manipulate and ultimately
understand. Two recent findings pave the way for this approach. First, some Gram-positive
bacteria such as C. perfringens (Varga et al., 2006) and R. albus (Rakotoarivonina et al.,
2002) produce Tfp. Studying Tfp biogenesis in a Gram-positive bacterium, which is inherently
"simpler" because of the absence of outer membrane, is an attractive possibility, since pilus
assembly is expected to require fewer dedicated proteins (Fig. 2). However, a suitable Grampositive species producing Tfp remains to be identified, since both C. perfringens and R.
albus present severe limitations, above all poor genetic tractability. Second, another way to
reduce complexity could be to create a minimal system capable of Tfp biogenesis by building
on the finding that Tfp biogenesis in N. meningitidis occurs in four distinct steps relying on
distinct subsets of proteins and that, in the absence of pilus retraction, the number of proteins
required for pilus assembly consists of PilD, PilE, PilF, PilM, PilN, PilO and PilP (Carbonnelle
et al., 2006). The unambiguous confirmation that these proteins are sufficient for pilus
assembly could be achieved either by expressing the genes encoding these proteins in a
surrogate non-piliated organism, similar to what was done for type IVb pili (Sohel et al., 1996;
16
Stone et al., 1996), or by introducing mutations in every pil gene that is not involved in pilus
assembly per se in a N. meningitidis pilT mutant.
In conclusion, unravelling the molecular basis for Tfp biogenesis is likely to have a
significant impact both on a fundamental level, by contributing to a better understanding of a
widespread colonization factor and related transport systems, and practically, by providing
clues for the rational design of new drugs that could inhibit Tfp assembly, as done for the pili
assembled by the chaperone-usher pathway (Pinkner et al., 2006). In turn, this could have
far-reaching consequences for public health due to the key role of these filaments in many
human pathogens.
17
Acknowledgements
I would like to thank present and former members of my group, especially Etienne
Carbonnelle and Sophie Helaine, for their invaluable contributions. I am deeply indebted to
Katrina Forest (University of Wisconsin-Madison, USA) and Andrea Dessen (lnstitut de
Biologie Structurale Jean-Pierre Ebel, France) for our fruitful structural collaborations. I wish
to express my gratitude to the members of the Department of Microbiology (Imperial College
London, UK) for their everyday support and useful comments on this manuscript. Work in my
group is supported by funding from the Agence Nationale de la Recherche (ANR) and the
Biotechnology and Biological Sciences Research Council (BBSRC).
18
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25
Legends to Figures
Fig. 1. Typical morphology of Tfp, as illustrated by a transmission electron microscopy
picture of Neisseria meningitidis filaments.
Fig. 2. Distribution and organization of the genes coding for Tfp biogenesis proteins
throughout the bacterial kingdom: common features and peculiarities.
All the genes are drawn to scale, with the scale bar representing 1kb. Genes of the same
colour encode proteins of similar function, as determined by sequence similarity and/or
conserved synteny. The core Tfp biogenesis genes, found in all Gram-negative bacteria,
encode: a pilin (
), one or several pilin-like proteins (
a traffic ATPase (
), an inner membrane protein (
), a prepilin peptidase (
) and a secretin (
),
). Genes
marked with an asterisk are dispensable for Tfp biogenesis and include, in order to
emphasize the conserved gene clustering, some mainly housekeeping genes unrelated to
Tfp biogenesis such as yggS (
ponA (
), aroK (
), yacG (
) and aroB (
), coaE (
), ispG (
), yfgB (
),
). For the sake of clarity, the type IVa Tfp
biogenesis gene clusters, within which the order of genes has been conserved, have all been
artificially organized as in the genome of N. meningitidis 8013 displayed on top (our
unpublished data). Within the phyla in which essentially complete sets of Tfp biogenesis
genes are found (Proteobacteria, Cyanobacteria, Deinococcus-Thermus, Acidobacteria and
Firmicutes),
species
have
been
chosen
to
cover
most
taxonomic
ranks
(class>order>family>genus) in order to perform a comparison among bacteria as
phylogenetically distant as possible, therefore including species in which Tfp production is
yet to be demonstrated. The strains that are displayed are: N. meningitidis 8013, R.
solanacearum GMI1000 (it should be noted that this strain contains another set of Tfp
biogenesis genes not represented here, which is present on a megaplasmid), M. flagellatus
KT, Azoarcus sp. BH72, P. aeruginosa PA01, S. putrefaciens CN-32, D. nodosus
VCS11703A, L. pneumophila Paris, M. capsulatus Bath, F. tularensis Schu4, V. cholerae El
26
Tor, X. campestris ATCC33913, B. bacteriovorus HD100, G. uraniumreducens Rf4, M.
xanthus DK 1622, Nostoc sp. PCC 7120, D. geothermalis DSM11300, T. thermophilus HB27,
A. bacterium Ellin 345, C. perfringens SM101 and S. sanguinis SK36. Enterobacteria stands
for well-studied species of enterobacteria in which the gene organization is virtually identical,
such as E. coli, K. pneumoniae, S. enterica serovar Typhimurium, S. flexneri and Y. pestis.
27