Molecular Microbiology (2006) 62(2), 320–330
x
MicroReview
Pili with strong attachments: Gram-positive bacteria do
it differently
June R. Scott* and Dorothea Zähner
Department of Microbiology and Immunology, Emory
University School of Medicine, Atlanta, GA 30322, USA.
Summary
Bacteria attach to their appropriate environmental
niche by using adhesins. To maximize their contact
with the environment, adhesins are often present on
the ends of long hairlike structures called pili.
Recently, attention has focused on pili of Gram-
positive bacteria because they may be vaccine can-
didates in important human pathogens. These pili
differ from the well-studied pili of Gram-negative bac-
teria because their subunits are covalently linked,
they do not require specific chaperones for assembly,
and the tip protein (likely to be the adhesin) is not
required to initiate formation of the pilus structure.
In Gram-positive bacteria, the genes for pili occur
in clusters, which may constitute mobile genetic
elements. These clusters include the transpepti-
dase(s) of the sortase family that is/are required for
polymerization of the subunit proteins. However, effi-
cient covalent attachment of the completed pilus
structure to the cell wall is accomplished, in cases
where this has been studied, by the ‘housekeeping’
sortase, which is responsible for attachment to the
peptidoglycan of most surface proteins containing
cell wall sorting signals. This enzyme is encoded
elsewhere on the genome. Because pili of Gram-
positive bacteria have not been extensively investi-
gated yet, we hope that this MicroReview will help to
pinpoint the areas most in need of further study.
Introduction
For many years, bacteriologists have been aware that pili,
multisubunit hairlike extensions from the bacterial cell
surface, are important for attaching Gram-negative bac-
teria to their appropriate environmental niche. Pili (some-
times called fimbriae) serve as the primary colonization
Accepted 8 June, 2006. *For correspondence. E-mail scott@
microbio.emory.edu; Tel. (+1) 404 727 0402; Fax (+1) 404 727 8999.
© 2006 The Authors
Journal compilation © 2006 Blackwell Publishing Ltd
doi:10.1111/j.1365-2958.2006.05279.
First published online 15 September 2006
factors for many Gram-negative pathogens and have
been used successfully as vaccines. Although pili on
Gram-positive bacteria were first described in 1968 in
Corynebacteria (Yanagawa et al., 1968) and shortly there-
after in Streptococci that inhabit the oral cavity (reviewed
by Wu and Fives-Taylor, 2001), they have only been
studied in these and other Gram-positive bacteria by a
few groups and have only recently been discovered in
many additional Gram-positive human pathogens. The
finding that newly discovered pili might be excellent
vaccine candidates in important Gram-positive human
pathogens (Malone et al., 2005; Mora et al., 2005) has
generated much excitement. This MicroReview will sum-
marize current knowledge about Gram-positive pili, focus-
ing on their assembly and structure, and will emphasize
similarities to and differences from Gram-negative pili.
Pili in Gram-negative bacteria
In Gram-negative bacteria, pili consist of multiple subunits
that are non-covalently attached to each other and
enzymes are not required for their assembly. Pili can be
removed from the bacterial surface by shearing, or by
dissociating the non-covalent bonds holding the subunits
together, for example by boiling in SDS. An adhesin
protein is located at the pilus tip. It is generally believed
that the pilus shaft is required to distance this adhesin far
enough from the bacterial surface (which may be encap-
sulated) to permit unimpeded contact with its receptor.
The best-understood pili of Gram-negative bacteria are
those in the family that is assembled by the ‘chaperone-
usher’ pathway, including Pap and type 1 pili of Escheri-
chia coli (Soto and Hultgren, 1999). The genes required
for assembly of this type of pilus are usually clustered
together in an operon. In this pathway, all of the pilus
proteins have N-terminal Sec signal peptides for transport
through the cytoplasmic membrane. The proteins found in
the final structure are unstable as monomers in the peri-
plasm unless they associate with a specific chaperone,
which is also encoded in the pilus operon. A large protein
in the outer membrane (the usher) serves to transport the
pilin subunits out of the cell. The pilus shaft is composed
of multiple copies of a single subunit, and the tip contains

Outer C C C C
C D
Membrane D
A A
B B
D
A A
B B B BB
several different proteins capped by the adhesin. The
most distal tip protein also serves as the initiating nuclea-
tor for production of a new pilus because these pili grow
by adding subunits to the base. The stochastic incorpora-
tion of a special protein at the base of the shaft of an
assembled pilus is thought to determine the length of the
structure by preventing incorporation of further subunits.
Non-covalent association with the outer membrane usher
presumably anchors the pili.
A simpler variant of this assembly pathway, dubbed the
‘alternate chaperone-usher’ pathway, is used for pili pre-
dominantly found in enterotoxigenic E. coli (e.g. CS1,
CFA/I and relatives; Sakellaris and Scott, 1998). Only four
proteins are required to make these pili (Fig. 1). In addi-
tion to the large outer membrane usher, there is only one
tip protein, which is needed for adherence, one shaft
protein, and a pilus-specific chaperone. It is not clear if the
length of these pili is controlled. The number of pili per
bacterial cell seems to be determined by the availability of
tip protein, because this serves as the initiator for forma-
tion of a new pilus structure.
Type 4 pili, a family unrelated to those described above,
are found on many different Gram-negative organisms
and even some Gram-positive bacteria, and their assem-
bly requires many more genes. Type 4 pili will not be
discussed here, but are reviewed by Mattick (2002).
Pili on Gram-positive bacteria
In contrast to the above, pili identified on Gram-positive
bacteria are composed of subunits that are covalently
attached to each other and cannot be dissociated by hot
SDS. The assay generally used to determine whether a
protein is polymerized into a pilus structure is to subject
the cell wall fraction (extracted with mutanolysin with or
without addition of lysozyme, or with a phage lysin specific
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Gram-positive pill 321
Fig. 1. Model of CS1 pilus assembly in
D enterotoxigenic E. coli (based on Starks et al.,
2006). Letters specify: B = CooB, the
A
D periplasmic chaperone; D = CooD, the
A nucleating tip protein; C = CooC, the outer
A membrane usher; A = CooA, the major pilin
subunit. Pilus formation is initiated when a
A CooD molecule, escorted by CooB, reaches
A the outer membrane protein CooC. Growth
continues by addition of CooA subunits,
A chaperoned by CooB, to the growing pilus
A C structure.
B
B
for the organism) to boiling in SDS followed by SDS-
PAGE. (It should be noted that simple presence in the cell
wall fraction isolated following lysin extraction does not
indicate covalent linkage to the cell wall.) A protein that is
part of a pilus will appear as a high molecular weight
(HMW) ladder in an immunoblot. The other method used
to detect pili is visualization by negative staining, or, more
specifically, by immunogold electron microscopy (EM),
although this may be less reliable because the pili may be
very short. However, immunogold EM can reveal the
localization of a protein within the pilus structure.
Like the simple CS1 pili of ETEC, Gram-positive pili are
composed of multiple copies of a single shaft pilin. They
may also have a different protein, which may be an
adhesin, at the tip. In pili of some Gram-positive bacteria,
additional proteins are associated in an unknown way with
the shaft, but these are not required for the integrity or
synthesis of the pilus.
As in the case of pili in Gram-negative bacteria, pilins of
Gram-positive bacteria are secreted through the cell
membrane by the Sec machinery. Polymerization of the
subunits of these pili is catalysed by an enzyme with
homology to sortases, which ‘sort’ proteins to the cell wall
fraction and attach them covalently to the peptidoglycan.
Sortases (reviewed recently by Marraffini et al., 2006) are
membrane-associated transpeptidases that recognize
surface protein precursors containing C-terminal cell wall
sorting signals (CWSSs) composed of a recognition pen-
tapeptide followed by a hydrophobic region of 30–40 resi-
dues and a charged tail. The hydrophobic domain and
charged tail retains the protein in the cell membrane,
where the sortase is also located. The recognition peptide
(usually LPXTG) is cleaved by the sortase at the T to form
an acyl-enzyme intermediate. The enzyme is released
from the protein by nucleophilic attack of an amino group,
which is provided by a peptidoglycan precursor for cell

322 J. R. Scott and D. Zähner
wall-anchored proteins. The final result is cleavage and
removal of the C-terminal segment of the cell wall-
anchored protein and covalent linkage of the protein to the
peptidoglycan. The ‘housekeeping’ sortase, usually des-
ignated SrtA, is responsible for anchoring most of the
cell wall proteins. Many Gram-positive bacteria encode
sortase homologues and there is a growing realization
that a subgroup of these transpeptidases is required for
pilus polymerization.
The prototype: Corynebacterium diphtheriae
SpaA pili
The Gram-positive bacterial pili whose mechanism of
assembly is best understood are found on C. diphtheriae.
First observed by EM in the 1970s (Kumazawa and Yana-
gawa, 1972; Yanagawa and Honda, 1976), the assembly
of these structures has been studied extensively most
recently by Schneewind and colleagues (reviewed by
Marraffini et al., 2006). Three different types of pili are
present in one strain of C. diphtheriae and the genes for
each are in loci containing different transpeptidases. The
pili are named according to their major subunit, Spa
(sortase-mediated pilin assembly) A, D or H (see Table 1
for summary).
Biogenesis of SpaA pili requires four adjacent genes
(Fig. 2). The three Spa proteins of this locus have N
terminal Sec signal peptides and C terminal CWSSs,
although their recognition sequences differ (Table 1). On
the basis of immunogold EM, it appears that SpaA con-
stitutes the pilus shaft while SpaC is at the tip and SpaB
is associated in an unknown way along the pilus length
(Ton-That and Schneewind, 2003). These conclusions
were supported by experiments in which each spa gene
was deleted in turn, and the ability of the other pilus
proteins to form the HMW ladder characteristic of pili was
evaluated. As expected, the absence of spaA, encoding
the major pilus shaft protein, resulted in failure to detect
SpaA protein by immunoblotting, but HMW forms of SpaC
were also absent, and HMW SpaB was greatly reduced.
On the other hand, deletion of spaB or spaC did not
prevent pilus formation by SpaA and the other minor pilin,
as assayed by the presence of their HMW forms. Further-
more, affinity chromatography showed that HMW SpaB
and HMW SpaC co-eluted with his-tagged SpaA, support-
ing the idea that all three are part of the same structure.
For a pilin protein to cross-link not just to the cell wall
but also to another pilin subunit, two different motifs rec-
ognized by the transpeptidase that polymerizes the
protein are required. Ton-That and Schneewind (2003)
identified a ‘pilin’ motif centrally located in pilins of
C. diphtheriae as well as in the pilins of Actinomyces
naeslundii and several streptococci (Table 1). They sug-
gested that the conserved lysine in the pilin motif may be
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critical for subunit–subunit cross-linking because it could
provide a free amino group to initiate the nucleophilic
attack, which would release the previous subunit from its
acyl linkage to the sortase. In support of this, HMW SpaA
was not formed when lys490 in this motif of SpaA was
replaced by ala. Further, they demonstrated the generality
of this process by inserting the pilin motif into a non-pilin
cell wall-anchored protein of Staphylococcus aureus, sta-
phylococcal enterotoxin B or SEB. When this gene was
expressed in C. diphtheriae in the absence of SpaA,
HMW SEB polymers were produced (Ton-That et al.,
2004).
The location of SpaC at the pilus tip is supported by
several lines of evidence (Ton-That and Schneewind,
2003). In strains overexpressing SpaA, anti-SpaC labels
the tips of the long pilus structures. In addition, deletion of
spaA prevents formation of HMW SpaC, and the SpaC
protein lacks the pilin motif, suggesting that it can only be
attached by one site to another pilin molecule. However,
deletion of spaC does not interfere with production of pili
or of HMW SpaA and SpaB, showing that SpaC is not
required for formation of these structures. Nevertheless, it
seems likely that SpaC is added first during pilus forma-
tion because the transpeptidase required for its linkage to
SpaA is membrane-located (Fig. 3). Thus, these pili must
grow by a mechanism that differs fundamentally from that
of pili of Gram-negative bacteria, because the tip protein
of the latter is required for initiation of pilus formation. A
new pilus may be initiated when the concentration of free
SpaA shaft protein reaches a threshold because overpro-
duction of the SpaA protein (encoded on a plasmid)
increases the number of pili per cell. In addition, these pili
are much longer, suggesting that the amount of the major
pilin shaft protein is limiting for pilus length.
The mechanism of incorporation of SpaB into SpaA pili
and its location in the final structure are not yet clear.
Anti-SpaB antiserum stains pili, but not evenly, suggesting
that SpaB is intermittently associated with the pilus shaft
along its length. SpaB is co-eluted with his-tagged SpaA,
demonstrating a close association (Ton-That and Schnee-
wind, 2003). Deletion of spaB does not affect pilus forma-
tion, and does not prevent formation of HMW SpaC or A.
Furthermore, SpaB does not have a pilin motif and the
pentapeptide in the CWSS of SpaB differs in sequence
from that of SpaA and SpaC (Table 1).
Ton-That et al. (2004) identified an additional motif in
the proteins that constitute the shafts of the known pili of
Gram-positive bacteria and named it the ‘E box’ (Table 1).
The conserved glutamate residue in the E box in SpaA is
needed for incorporation of SpaB into SpaA pili. Changing
the glu to ala in this E box motif in SpaA reduces incor-
poration of SpaC into HMW forms, but does not affect
SpaA polymerization. No further evidence about the role
of the E box in pilus assembly is currently available. The
© 2006 The Authors
 © 2006 The Authors
Table 1. Conserved motifs in pilin proteins.

Major pilin protein Minor pilin proteins

Strain Name CWSSa Pilin motif E box Name CWSSa Role References

C. diphtheriae NCTC13129 SpaA LPLTGG WLQDVHVYPK FCLVETATASGY SpaC LPLTGS Tip Ton-That et al. (2004)
SpaB LAFTGA
C. diphtheriae NCTC13129 SpaD LPMTGG WNYNVVAYPK FCLKETKAPAGY SpaF LPKTGG Tip Ton-That et al. (2004)
SpaE LALTGV
C. diphtheriae NCTC13129 SpaH LPLTGG WLYDVNVFPK YVLVETEAPTGF SpaG LPLTGG Tip Ton-That et al. (2004)
SpaI LGNTGA
S. agalactiae COH1 GBS80 IPKTGE LSEDKVIYPK YVLKEIETQSGY GBS52 IPNTGG Lauer et al. (2005)
GBS104 FPKTGG
S. pneumoniae TIGR4 RrgB IPQTGG DVVDAHVYPK YYLEETKQPAGY RrgC VPDTGE Tip Barocchi et al. (2006)
RrgA YPRTGG
S. pyogenes SF370 Spy0128 EVPTGV – – Cpa VVPTGV Adhesin Mora et al. (2005)
Spy0130 LPSTGE
S. pyogenes MGAS315 Orf100 QVPTGV – – Cpa VPPTGL Adhesin Mora et al. (2005),
Orf102 LPLAGE Barnett et al. (2004)
S. pyogenes MAS10394 T6 LPSTGS – – FctX LPSSGG Mora et al. (2005)
A. naeslundii T14V, type 1 FimP LPLTGA WNYNVHVYPK YCLVETKAPEGY Orf1 LPLSGG Adhesin Yeung and Ragsdale (1997)
A. naeslundii T14V, type 2 FimA LPLTGA WIYDVNVYPK YVLVETKAPAGY Orf977 LPLTGG Adhesin Yeung et al. (1998),

Journal compilation © 2006 Blackwell Publishing Ltd, Molecular Microbiology, 62, 320–330
Hoflack and Yeung (2001)
Consensus sequencesb LPxTG WxxxVxVYPK YxLxETxAPxGY LPxTG

a. CWSS, cell wall sorting signal motif preceding the hydrophobic stretch and charged tail.
b. Consensus sequences according to Schneewind et al. (1992) (LPxTG motif), Ton-That and Schneewind (2003) (pilin motif), and Ton-That et al. (2004) (E box motif). Highly conserved residues
are indicated (bold).
Gram-positive pill 323

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324 J. R. Scott and D. Zähner

C. diphtheriae NCTC13139
spaA srtA spaB spaC

C. diphtheriae NCTC13139 srtC spaE

srtB spaD spaF

C. diphtheriae NCTC13139 spaI

spaG spaH srtD srtE

S. agalactiae COH1 gbs80 gbs52 srt647 srt648 gbs104

S. pneumoniae TIGR4 IS1167 rlrA rrgA rrgB rrgC srtB srtC srtD IS1167

S. pyogenes SF370 (M1)

cpa sipA Spy0128 srtC1 Spy0130

S. pyogenes 10394 (M6) fctX tee6 srtB

A. naeslundii T14V type 1 orf3 orf2 orf1 fimP orf4

A. naeslundii T14V type 2

orf977 fimA orf365

Fig. 2. Organization of gene clusters encoding described pili. Genes encoding proteins involved in pilus biogenesis are shown as arrows with
black outlines. These encode: enzymes homologous to sortases (blue arrows), and major (green arrows) and minor (orange arrows) pilin
proteins containing CWSSs. Other genes in the cluster are shown by white arrows. Arrows outlined in grey indicate the genes flanking the
cluster. For references, see Table 1.

mechanism of incorporation of the accessory protein
SpaB awaits further experimentation.
As expected, SrtA, which is encoded in the locus that
includes the SpaA pilus genes (Fig. 2), is required for
assembly of SpaA pili. Ton-That and Schneewind (2003)
demonstrated that deletion of srtA prevents formation of
pili that are detectable by immunogold EM and also pre-
vents formation of HMW SpaA and SpaC. Thus, it appears
that SrtA is responsible for SpaA pilus subunit poly-
merization. However, C. diphtheriae has genes for six
sortase homologues, five of which are in loci that include
predicted surface proteins likely to be pilins. Because the
srtF gene is not linked to presumptive pilin genes, it is the
‘housekeeping’ sortase required for anchoring of most
non-pilin LPXTG-containing proteins to the C. diphtheriae
cell wall. Mutants deleted for srtB through F still produce
HMW SpaA, indicating that they are not required for SpaA
polymerization. However, in a mutant deleted for srtF,
boiling the cells in SDS solubilizes significant amounts of
HMW SpaA, B and C (Ton-That and Schneewind, 2003;
Ton-That et al., 2004). This shows that SrtF, the presumed
housekeeping sortase, is required for correct covalent
attachment of SpaA pili to the cell wall. As the transpep-
tidase encoded in the SpaA pilin gene cluster, called SrtA,
cannot perform this reaction efficiently, this enzyme could
be considered a pilin polymerase rather than a sortase.
The mechanism of cell wall attachment of pili has not yet
been investigated in further detail. In Fig. 3, we have
expanded the current model, proposed by Schneewind
and coworkers, in which the growing SpaA pilus remains
attached to the pilin polymerase, SrtA, in the membrane
(Marraffini et al., 2006), to include transfer of the pilus to
the membrane-associated sortase, SrtF, which links it
covalently to the peptidoglycan. It remains possible that
additional chromosomal genes are needed for pilus
production.
General principals resulting from the work of the
Schneewind group on SpaA pilus assembly include: (i)

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Gram-positive pill 325

Fig. 3. Model for pilus assembly in Gram-positive bacteria (modified from Marraffini et al., 2006). The pilin subunit that becomes the tip is
probably the first subunit incorporated into the new pilus. Following transport through the cell membrane (by the Sec system), the pilin subunit
remains associated with the membrane by the hydrophobic tail of its CWSS. The sortase-related pilin polymerase (Pip), which is also
membrane-located, presumably forms an acyl-enzyme intermediate with the tip protein and a cleavage process releases the tip protein from
the cell membrane. To add the shaft protein, the free amino group in the lysine residue of the pilin motif of the shaft protein presumably
attacks the threonine of the CWSS of the tip protein, forming a peptide bond. The CWSS of the shaft protein remains linked to the pilin
polymerase until the next shaft protein is added by the same type of enzymatic cleavage reaction. After a series of similar polymerization
reactions, the growing pilus is probably transferred to the housekeeping sortase (SrtA), which attaches it to the cell wall precursor, resulting in
its covalent linkage into the growing peptidoglycan chain of the cell wall.

genes for all pilin proteins and the transpeptidase that
polymerizes them (hereafter called pilin polymerase) are
genetically linked, (ii) the sortase homologue genetically
linked to the pilin gene cluster is needed for pilus poly-
merization, (iii) the presumed housekeeping sortase is
needed for efficient covalent attachment of the pili to the
cell wall, (iv) the shaft protein contains a pilin motif that
is required for polymerization, (v) the shaft protein is the
only one required for polymerization of the others and its
quantity determines pilus length, (vi) additional pilus-
associated proteins, one of which localizes to the tip, also
contain LPXTG that is needed for polymerization, (vii) an
additional motif in the shaft protein is needed for associa-
tion of the accessory protein, and (viii) specific chaper-
ones do not appear to be required. In the discussion to
follow, we will try to illustrate the extent to which these
principals apply to other Gram-positive pili.
SpaD pili of C. diphtheriae
The other type of C. diphtheriae pilus that has been inves-
tigated is called SpaD (Gaspar and Ton-That, 2006).
Unlike the spaA locus, the spaD gene cluster includes
genes for two putative transpeptidases, srtC and srtB
(Fig. 2). As far as is currently known, the role of each
protein in SpaD pilus formation is similar to the roles of the
analogous proteins for SpaA pilus formation. The two
proteins with LPXTG motifs form the pilus, while SpaE,
which contains LALTG, is an accessory protein that is
incorporated in an unknown way along the pilus shaft
(Table 1).
Immunogold EM identifies SpaD on the surface of cells,
but this does not seem to form fibres unless the spaD
gene is overexpressed. Thus, as in the case of SpaA, the
amount of the major pilin protein appears to limit the
length of the structure. SpaE appears to be located irregu-
larly along the pilus shaft (like the accessory protein SpaB
in SpaA pili), and SpaF cannot be detected clearly, but
may be present at the pilus tip (like SpaC in SpaA pili). In
addition, formation of HMW species of minor pilins
requires the major pilin, SpaD, but neither minor pilin is
required for HMW SpaD formation.
Deletion mutants were used to show that either of the
two transpeptidase genes in the spaD gene cluster, srtB

© 2006 The Authors

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326 J. R. Scott and D. Zähner
or srtC, can allow polymerization of HMW SpaD and the
minor pilins, although srtB may be more efficient (or its
product may be more abundant) (Gaspar and Ton-That,
2006). As for SpaA pili, the pilin motif of SpaD is required
for pilin polymerization because a K107A substitution in
the pilin motif of SpaD prevents formation of HMW SpaD,
SpaE or SpaF (Gaspar and Ton-That, 2006). A difference
between SpaD and SpaA pili is found in the behaviour of
the accessory protein. The E box of the major pilin, which
is essential for incorporation of the accessory protein of
the SpaA pili, is not required for polymerization of the
accessory protein of SpaD pili.
In summary, assembly of SpaD pili seems to be very
similar to that of SpaA pili, except that either of the two
transpeptidases encoded in the gene cluster is capable of
pilus polymerization and the E box of the major pilin is not
needed for polymerization of the accessory protein. The
role of SrtF, the housekeeping sortase, in attaching the
SpaD pilus to the cell wall has not been investigated.
Pili of group B streptococci
Lauer et al. (2005) discovered pili on Streptococcus aga-
lactiae (group B streptococci) by analysing the genome
sequence for clustered genes encoding proteins with
LPXTG-related motifs (Fig. 2 and Table 1). Two of the
genes (gbs80 and gbs104) encode surface-exposed anti-
gens that mediate complement-dependent opsonophago-
cytic killing of virulent bacteria and confer passive
protection against challenge in a mouse maternal immu-
nization model (Malone et al., 2005). The gene cluster
encodes two predicted transpeptidases and three pro-
teins, which have an N-terminal Sec signal and a CWSS.
The products of the first and last genes in the cluster
(gbs80 and gbs104 respectively) form HMW ladders in
cell wall extract fractions and anti-Gbs80 labels pili detect-
able by immunogold EM (Lauer et al., 2005). As for SpaA
and SpaD pili of C. diphtheriae, overexpression of the
gene for the presumed pilus shaft, gbs80, results in longer
pili. Antibody to the product of the last gene in this cluster
(gbs104) was reported to stain the pili more weakly, but as
the data were not shown, it is not clear whether this
staining is intermittent along the whole shaft, as expected
for an accessory pilin protein. Both sortases in the gene
cluster are reportedly needed for correct pilus assembly.
Pili of Streptococcus pneumoniae
Pili are visible on some, but not on all cells in populations
of some strains of S. pneumoniae (Barocchi et al., 2006).
Pili may not be detectable by immunogold EM even when
HMW pilin is detected by immunoblots, indicating that
detection of pilin multimers by SDS-PAGE and immunob-
lotting may be more reliable than immunogold EM. The
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genes required for pilus production are located in the rlr
pathogenicity islet (Fig. 2). This islet is flanked by IS1167
elements, suggesting that it may be a mobile genetic
element. In agreement with this, it is present only in some
strains. This islet is a virulence factor in a murine lung
infection model (Hava and Camilli, 2002) and is important
for adherence to lung epithelial cells (Barocchi et al.,
2006).
In the rlr islet (Fig. 3), rlrA encodes a RofA-like positive
regulator of the gene cluster, the rrg genes have homol-
ogy to the genes for pili of group B streptococci, and there
are three transpeptidase gene homologues. RrgB con-
tains an N terminal Sec signal, a CWSS, a pilin motif and
an E box, while RrgC lacks the pilin motif (Table 1). Anti-
RrgB labels the entire pilus (Barocchi et al., 2006;
LeMieux et al., 2006) and anti-RrgC labels the tip (Baroc-
chi et al., 2006). Deletion of rrgB and rrgC prevents pilus
formation, while deletion of rrgC alone does not (LeMieux
et al., 2006), indicating that RrgB is the shaft protein, as
expected from the fact that it has all the described pilus-
related motifs (Table 1). RrgA and B form HMW ladders.
Anti-RrgA was reported to label the bacterial surface
(Barocchi et al., 2006), consistent with the possibility that
RrgA is located at the pilus base, while LeMieux et al.
(2006) found anti-RrgA to label ‘patches’ spaced along the
pilus fibres, which are present in the absence of RrgC but
require RrgB for formation. RrgA may be present in both
locations. As RrgA has a motif likely to be recognized by
the housekeeping sortase, it should be covalently linked
to the cell wall as a monomer while it may, in addition,
become attached to the pili.
SrtD, also encoded in the pilin gene cluster, is required
for formation of HMW RrgA (the accessory pilin; LeMieux
et al., 2006). Changing the LPXTG-related motif of RrgA
to that of RrgB or C or to the canonical LPXTG prevented
the formation of HMW RrgA, consistent with the interpre-
tation that the wild-type CWSS of the accessory protein is
required for its incorporation into pili by SrtD.
In summary, it appears that these S. pneumoniae pili
are assembled much like those of C. diphtheriae.
T antigen pili of group A streptococci
The T (trypsin resistant) antigen of Streptococcus pyo-
genes (the group A streptococcus or GAS) was used by
Lancefield and coworkers along with the M protein more
than 50 years ago to classify GAS isolates into serotypes.
Recently, Mora et al. (2005) found this protein to be the
shaft of a pilus in the four GAS strains of different sero-
types that they studied. The T antigen is encoded in a
region named FCT by Bessen and Kalia (2002) because
it encodes proteins that bind fibronectin or collagen, as
well as the T antigen (Fig. 2). The FCT region is always
located between the same two genes, but, for each GAS
© 2006 The Authors

strain examined, it contains a unique combination of
partially conserved genes (Bessen and Kalia, 2002).
Encoded in the FCT region are a sortase homologue and
several proteins with an N-terminal Sec signal and a
CWSS. The sequence of these predicted surface proteins
is strain-specific, like the T antigen. In some strains, the
first and largest gene in the pilin cluster encodes a known
adhesin (collagen binding protein encoded by cpa: Fig. 2
or a fibronectin binding protein).
The T antigen in the cell wall fraction appears as HMW
forms (Barnett et al., 2004), as does the protein encoded
by the first gene, the presumed adhesin (Mora et al.,
2005). Furthermore, immunogold EM shows that anti-T
antiserum stains the entire length of long trypsin-resistant
pili, while antibody to the protein encoded by the pre-
sumed adhesin does not stain these (Mora et al., 2005).
Thus, the T antigen is the pilus shaft. The T antigen
protein is able to polymerize in the absence of any of the
other CWSS proteins encoded by the gene cluster, but
requires the transpeptidase encoded in the FCT region
(Barnett et al., 2004; Mora et al., 2005). Mora et al. (2005)
also found that the transpeptidase in the FCT region is
needed for HMW formation by all the FCT pilins tested, as
well as by the adhesin. This indicates that the adhesin is
part of the pilus. We have recently found that, although the
shaft protein T6 in the serotype M6 strain contains the
canonical LPXTG motif and this protein is polymerized by
the sortase homologue in the FCT region (SrtB), the
housekeeping sortase, SrtA, is required for cell wall
attachment of the pili (D. Zähner, T.N. Jones and J.R.
Scott, in preparation).
In contrast to the T6 antigen protein that has a canoni-
cal LPXTG motif, the T antigen shaft proteins of the other
GAS strains have QVPTG or EVPTG in its place
(Table 1). All T antigens lack the canonical pilin motif and
E box (Table 1), suggesting that the pilin polymerase
encoded in these FCT regions recognizes different
sequences from those present in other Gram-positive pili.
Furthermore, the QVPTG in the CWSS of the T antigen
protein is required for HMW laddering, because no HMW
forms were seen when QVPTG was changed to the
canonical LPXTG (Barnett et al., 2004). In summary, GAS
pili seem to be assembled similarly to the prototype SpaA
pili of C. diphtheriae. However, in some strains of GAS,
the sequences involved in peptide bond formation
between subunits are different from the canonical
sequences used by the other pili described above.
Pili of A. naeslundii
Two antigenically different types of pili (types 1 and 2)
have been identified on the dental pathogen A. naeslundii
(reviewed by Yeung, 2000). Type 1 is required for adher-
ence to saliva-coated hydroxyapatite and type 2 for
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Gram-positive pill 327
co-aggregation with viridans streptococci and interactions
with mammalian cells. These structures resist chemical
treatments designed to dissociate them into subunits.
Type 1 pili
Type 1 pili are much shorter than type 2 (reviewed by
Yeung, 2000). The major subunit of type 1 pili is the FimP
protein, which includes a CWSS with an LPXTG motif, a
pilin motif and an E box (Fig. 2, Table 1). Antibody against
FimP does not inhibit the adherence of bacteria, suggest-
ing that it may not be the adhesin. Immediately down-
stream of fimP lies orf4, encoding a member of the
sortase-like enzyme family, whose presence is needed to
form pili.
The gene immediately upstream of fimP encodes a
protein that has a CWSS with a canonical LPXTG (Li
et al., 2001; Fig. 2). Deletion mutants in this gene and
additional mutants in the two genes encoded upstream,
orf2 and orf3, produce no pili and are unable to adhere
(Yeung and Ragsdale, 1997). All three open reading
frames show homology to a major part of a single open
reading frame of the pilus gene cluster of the related
organism Actinomyces viscosus (Li et al., 2001). Further
molecular evidence is needed to decide whether three
genes are present in this region or whether sequencing
errors disguise a large single surface protein that might be
the adhesin.
Type 2 pili
The gene cluster encoding type 2 pili of some strains of
A. naeslundii (Fig. 2) is one of the smallest yet described:
it contains only three genes and they are flanked by the
gene encoding Ef-Tu and ribosomal genes. The product
of the first gene in the cluster, orf977, shares 36%
sequence identity in the last 300 amino acids with SpaG
of the SpaH pilus cluster of C. diphtheriae. The second
gene encodes the major subunit of type 2 pili, FimA, which
contains a CWSS including LPXTG (Table 1). The last
gene in the cluster, orf365, encodes the transpeptidase
required for assembly of these type 2 structures. Further-
more, Yeung et al. (1998) recognized that the surface-
attached form of FimA lacks the C terminal cell wall
anchor, consistent with its attachment to the peptidogly-
can by a transpeptidation reaction.
In summary, although little is known about assembly of
A. naeslundii pili, biogenesis of both types 1 and 2 pili
requires a transpeptidase encoded in the same gene
cluster as the shaft protein. Both types of pili are also
required for adherence. The type 2 gene cluster appears
to be the simplest possible for Gram-positive pili, suggest-
ing that further study of the assembly of these pili would
be rewarding.
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328 J. R. Scott and D. Zähner

‘Pili’ of Streptococcus parasanguis teria are predicted to encode pili. The only genes in the
clusters with high sequence homology between clusters
The surface of the dental pathogen S. parasanguis is
are the sortase homologues, which have been classified
covered with hairlike intertwined structures that look like,
into families using different criteria (Comfort and Clubb,
and were called, fimbriae or pili. These are involved
2004; Dramsi et al., 2005). However, the SrtC pilin poly-
in adherence to saliva-coated hydroxyapatite, co-
merase of some strains of GAS does not belong to a
aggregation with other oral pathogens and in biofilm for-
family containing other pilin polymerases in either classi-
mation (reviewed by Wu and Fives-Taylor, 2001). These
fication scheme and thus would not be recognized by
structures, which are not solubilized by boiling in SDS, are
these criteria. Additional bioinformatic analyses based on
composed of Fap1. Deletion of the CWSS in Fap1, which
clustered genes encoding putative surface proteins and
includes LPXTG, results in the presence of large amounts
additional supporting experimental evidence can be
of monomeric Fap1 in the medium. Fap1 is, however, very
expected to lead to the identification of many more pili in
different from Gram-positive pilins. It is a large protein,
Gram-positive organisms.
predicted from its gene size to be 260 kDa, and is com-
posed of two regions of dipeptide repeats that comprise
80% of the protein. In addition, Fap1 is glycosilated and Conclusions
for secretion through the cell membrane it requires
Although pili are used for adherence by both Gram-
SecA2, encoded by a gene located close to fap1, which is
negative and Gram-positive bacteria, the structures them-
different from the canonical secA (Chen et al., 2004).
selves are substantially different and therefore their
Fap1 resembles a large group of structurally related
morphogenesis is fundamentally different. Gram-negative
adhesins, all of which are glycosilated and in the cases
bacterial pili are composed of subunits that are not
investigated, require a non-canonical secA gene for
covalently linked, and initiation of the structure requires
export. These include GspB of Streptococcus gordonii,
the presence of a protein that ends up on the tip. The
SrpA of Streptococcus parasanguis and SraP of Staphy-
prepilins are assembled on the outer membrane usher
lococcus aureus (Bensing et al., 2005). FimA, an adhesin
protein and require a specific chaperone for stability. Pili of
highly homologous to the metal binding protein PsaA of
Gram-positive bacteria, in contrast, are composed of sub-
S. pneumoniae, was identified as forming the tip of the
units that are covalently linked by a sortase family
Fap1 structures on the basis of immunogold EM
transpeptidase, encoded in the gene cluster containing
observations. However, FimA lacks a CWSS and is
the pilin genes. It is not clear yet what initiates the forma-
encoded in a locus with genes for an ATP-binding cas-
tion of a new pilus structure. The housekeeping sortase,
sette transport system. Furthermore, FimA is a lipoprotein,
encoded elsewhere in the chromosome, appears to
so although it is an adhesin, it does not appear to be
perform the final transpeptidation reaction that attaches
related to the ‘pili’.
the structure to the peptidoglycan.
The gene downstream of fap1 is not required for forma-
The model of C. diphtheriae SpaA pili developed by
tion of the structures, and fap1 is transcribed as a mono-
Schneewind’s group (Marraffini et al., 2006) is an excel-
cistronic message (Wu and Fives-Taylor, 1999). It seems
lent starting point for understanding morphogenesis of the
likely that the Fap1 structures on the S. parasanguis
other pili of Gram-positive bacteria (Fig. 3). However,
surface, which are attached covalently to the peptidogly-
further investigation is needed to provide greater detail
can, are composed of single Fap1 molecules decorated
and to indicate which aspects of the model are conserved
with sugars. This is supported by the recent finding of Wu
in assembly of other pili. Questions that remain include:
et al. (H. Wu, P. Fives-Taylor, T. Ruiz, pers. comm.) that a
what is/are the functional role(s) of the pilus and its sub-
mutant in the glucosyltransferase gene of the fap1 locus,
units in cases where adherence has not been demon-
which initiates Fap1 glycosylation, produces no visible
strated? How is the growing pilus transferred from the pilin
surface structures. Therefore, these Fap1 structures do
polymerase to the sortase responsible for cell wall attach-
not seem to be pili, because they probably are not com-
ment? Is this process stochastic, suggesting that pilus
posed of subunits.
length is determined by the ratios of the pilin shaft protein
and the two transpeptidases? In cases where a minor pilin
protein is found at the tip, how is it localized there? Can
Perspectives
transpeptidase recognition be provided by different
At this time, bioinformatic identification of putative pilus sequence motifs in different pili? How are the accessory
gene clusters relies on the presence of several genes proteins associated with the pilus and is there an order to
encoding proteins with predicted N-terminal Sec signal the process? What determines whether a protein will be
peptides and CWSSs linked to a sortase homologue. polymerized into pili by the pilin polymerase or anchored
Using these criteria, many additional Gram-positive bac- to the cell wall by the sortase, or is this process stochas-

© 2006 The Authors

Journal compilation © 2006 Blackwell Publishing Ltd, Molecular Microbiology, 62, 320–330

tic? In addition, very little is currently known about regu-
lation of synthesis of the proteins in the pilin gene clusters
and this might be important for determining their relative
stoichiometry. In summary, this is an exciting time in the
investigation of pilus assembly in Gram-positive bacteria,
both because the molecular mechanisms of their morpho-
genesis are beginning to be determined and also because
they appear to be promising candidates for vaccine devel-
opment in important human pathogens.
Acknowledgement
The work in our lab on this subject was supported in part
by Grant AI055605 from the National Institutes of Health to
J. R. S.
References
Barnett, T., Patel, A.R., and Scott, J.R. (2004) A novel
sortase, SrtC2, from Streptococcus pyogenes anchors a
surface protein containing a QVPTGV motif to the cell wall.
J Bacteriol 186: 5865–5875.
Barocchi, M.A., Ries, J., Zogaj, X., Hemsley, C., Albiger, B.,
Kanth, A., et al. (2006) A pneumococcal pilus influences
virulence and host inflammatory responses. Proc Natl Acad
Sci USA 103: 2857–2862.
Bensing, B.A., Takamatsu, D., and Sullam, P.M. (2005)
Determinants of the streptococcal surface glycoprotein
GspB that facilitate export by the accessory Sec system.
Mol Microbiol 58: 1468–1481.
Bessen, D.E., and Kalia, A. (2002) Genomic localization of a
T serotype locus to a recombinatorial zone encoding
extracellular matrix-binding proteins in Streptococcus
pyogenes. Infect Immun 70: 1159–1167.
Chen, Q., Wu, H., and Fives-Taylor, P.M. (2004) Investigating
the role of secA2 in secretion and glycosylation of a fimbrial
adhesin in Streptococcus parasanguis FW213. Mol Micro-
biol 53: 843–856.
Comfort, D., and Clubb, R.T. (2004) A comparative genome
analysis identifies distinct sorting pathways in gram-
positive bacteria. Infect Immun 72: 2710–2722.
Dramsi, S., Trieu-Cuot, P., and Bierne, H. (2005) Sorting
sortases: a nomenclature proposal for the various sortases
of Gram-positive bacteria. Res Microbiol 156: 289–297.
Gaspar, A.H., and Ton-That, H. (2006) Assembly of distinct
pilus structures on the surface of Corynebacterium
diphtheriae. J Bacteriol 188: 1526–1533.
Hava, D.L., and Camilli, A. (2002) Large-scale identification
of serotype 4 Streptococcus pneumoniae virulence factors.
Mol Microbiol 45: 1389–1406.
Hoflack, L., and Yeung, M.K. (2001) Actinomyces naeslundii
fimbrial protein Orf977 shows similarity to a streptococcal
adhesin. Oral Microbiol Immunol 16: 319–320.
Kumazawa, N., and Yanagawa, R. (1972) Chemical proper-
ties of the pili of Corynebacterium renale. Infect Immun 5:
27–30.
Lauer, P., Rinaudo, C.D., Soriani, M., Margarit, I., Maione, D.,
Rosini, R., et al. (2005) Genome analysis reveals pili in
Group B Streptococcus. Science 309: 105.
© 2006 The Authors
Journal compilation © 2006 Blackwell Publishing Ltd, Molecular Microbiology, 62, 320–330
13652958, 2006, 2, Downloaded from https://onlinelibrary.wiley.com/doi/10.1111/j.1365-2958.2006.05279.x by Cochrane France, Wiley Online Library on [03/05/2023]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License
Gram-positive pill 329
LeMieux, J., Hava, D.L., Basset, A., and Camilli, A. (2006)
RrgA and RrgB are components of a multisubunit pilus
encoded by the Streptococcus pneumoniae rlrA pathoge-
nicity islet. Infect Immun 74: 2453–2456.
Li, T., Khah, M.K., Slavnic, S., Johansson, I., and Strom-
berg, N. (2001) Different type 1 fimbrial genes and
tropisms of commensal and potentially pathogenic Acti-
nomyces spp. with different salivary acidic proline-rich
protein and statherin ligand specificities. Infect Immun 69:
7224–7233.
Malone, D., Margarit, I., Rinaudo, C.D., Masignani, V., Mora,
M., Scarselli, M., et al. (2005) Identification of a universal
Group B streptococcus vaccine by multiple genome
screen. Science 309: 148–150.
Marraffini, L.A., Dedent, A.C., and Schneewind, O. (2006)
Sortases and the art of anchoring proteins to the envelopes
of gram-positive bacteria. Microbiol Mol Biol Rev 70: 192–
221.
Mattick, J.S. (2002) Type IV pili and twitching motility. Annu
Rev Microbiol 56: 289–314.
Mora, M., Bensi, G., Capo, S., Falugi, F., Zingaretti, C.,
Manetti, A.G., et al. (2005) Group A Streptococcus produce
pilus-like structures containing protective antigens and
Lancefield T antigens. Proc Natl Acad Sci USA 102:
15641–15646.
Sakellaris, H., and Scott, J.R. (1998) New tools in an old trade:
CS1 pilus morphogenesis. Mol Microbiol 30: 681–687.
Schneewind, O., Model, P., and Fischetti, V.A. (1992) Sorting
of protein A to the staphylococcal cell wall. Cell 70: 267–
281.
Soto, G.E., and Hultgren, S.J. (1999) Bacterial adhesins:
common themes and variations in architecture and
assembly. J Bacteriol 181: 1059–1071.
Starks, A.M., Froehlich, B.J., Jones, T.N., and Scott, J.R.
(2006) Assembly of CS1 pili: the role of specific residues of
the major pilin, CooA. J Bacteriol 188: 231–239.
Ton-That, H., and Schneewind, O. (2003) Assembly of pili on
the surface of Corynebacterium diphtheriae. Mol Microbiol
50: 1429–1438.
Ton-That, H., Marraffini, L.A., and Schneewind, O. (2004)
Sortases and pilin elements involved in pilus assembly of
Corynebacterium diphtheriae. Mol Microbiol 53: 251–
261.
Wu, H., and Fives-Taylor, P.M. (1999) Identification of dipep-
tide repeats and a cell wall sorting signal in the fimbriae-
associated adhesin, Fap1, of Streptococcus parasanguis.
Mol Microbiol 34: 1070–1081.
Wu, H., and Fives-Taylor, P.M. (2001) Molecular strategies
for fimbrial expression and assembly. Crit Rev Oral Biol
Medical 12: 101–115.
Yanagawa, R., and Honda, E. (1976) Presence of pili in
species of human and animal parasites and pathogens of
the genuscorynebacterium. Infect Immun 13: 1293–1295.
Yanagawa, R., Otsuki, K., and Tokui, T. (1968) Electron
microscopy of fine structure of Corynebacterium renale
with special reference to pili. Jpn J Vet Res 16: 31–37.
Yeung, M.K. (2000) Actinomyces: surface macromole-
cules and bacteria–host interactions. In Gram-Positive
Pathogens. Ferretti, J.J., Novick, R.P., Fischetti, V.A.,
Portnoy, D.A., and Rood, J.I. (eds). Washington, DC:
American Society for Microbiology Press, pp. 583–593.

330 J. R. Scott and D. Zähner
Yeung, M.K., and Ragsdale, P.A. (1997) Synthesis and
function of Actinomyces naeslundii T14V type 1 fimbriae
require the expression of additional fimbria-associated
genes. Infect Immun 65: 2629–2639.
13652958, 2006, 2, Downloaded from https://onlinelibrary.wiley.com/doi/10.1111/j.1365-2958.2006.05279.x by Cochrane France, Wiley Online Library on [03/05/2023]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License
Yeung, M.K., Donkersloot, J.A., Cisar, J.O., and Ragsdale,
P.A. (1998) Identification of a gene involved in assembly of
Actinomyces naeslundii T14V type 2 fimbriae. Infect
Immun 66: 1482–1491.

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Journal compilation © 2006 Blackwell Publishing Ltd, Molecular Microbiology, 62, 320–330