Molecular Microbiology (1998) 30(4), 681–687
MicroReview
New tools in an old trade: CS1 pilus morphogenesis
Harry Sakellaris and June R. Scott
Department of Microbiology and Immunology, Emory
University, School of Medicine, Atlanta, GA 30322, USA.
Summary
CS1 pili serve as the prototype for a large class of sero-
logically distinct pili associated with enterotoxigenic
Escherichia coli that cause diarrhoea in humans. The
four genes essential for CS1 pilus morphogenesis,
cooB , A , C and D , are arranged in an operon and encode
structural and assembly proteins unlike those of other
pilus systems commonly associated with Gram-nega-
tive bacteria. CS1 pili are composed primarily of the
major pilin subunit, CooA, which determines the sero-
logical type of the pilus. The major pilin subunit is
assembled into pili by the proteins CooB, CooC and
CooD. CooD is both a minor component found at the
pilus tip and an essential assembly protein, whereas
CooC is an outer membrane protein thought to be
involved in pilin transport. CooB is a novel periplas-
mic chaperone-like protein that forms intermolecular
complexes with and stabilizes the major and minor
pilins. Unlike other pilin chaperones, CooB also stabil-
izes the outer membrane component of the assembly
system, CooC. The proteins of CS1 pili have no signifi-
cant homology to those of the well-characterized Pap
(pyelonephritis-associated) pili and related systems,
although most of the features of pilus morphogenesis
are similar. Therefore, these appear to be among the
rare cases of convergent evolution. Thus, for CS1 pili,
enterotoxigenic E. coli use new protein ‘tools’ in the
old ‘trade’ of forming functional pili.
Introduction
Enterotoxigenic Escherichia coli (ETEC) are important
bacterial pathogens that cause diarrhoeal disease in millions
of people every year (Black, 1993). In infants, this disease
is often fatal, so development of a protective vaccine is an
important priority. Once they are established in the small
intestine, ETEC secrete the heat-stable and/or heat-labile
Received 28 May, 1998; revised 27 July, 1998; accepted 29 July,
1998. *For correspondence. E-mail scott@microbio.emory.edu; Tel.
(404) 727 0402; Fax (404) 727 3659.
Q 1998 Blackwell Science Ltd
enterotoxins that cause diarrhoea. Colonization of the intes-
tine, the first step in the disease process, is mediated by
factors on the bacterial cell surface that recognize and
bind to host cell receptors. Most ETEC colonization factors,
called pili or fimbriae, are rigid, rod-like organelles of 5–
7 nm in diameter consisting of thousands of identical pro-
tein subunits (pilins) (Gaastra and Svennerholm, 1996).
Owing to their critical role in colonization and the success
of pilus-based vaccines in animals (Rutter et al ., 1976;
Morgan et al ., 1978; Nagy, 1980), ETEC pili are attractive
targets for the development of vaccines against human
diarrhoeal disease (Levine, 1987; Evans et al ., 1988;
Ahren et al ., 1993; Rudin et al ., 1994; Rudin and Svenner-
holm, 1994; 1996).
Human ETEC strains display a variety of serologically
distinct pili on their cell surfaces (Gaastra and Svenner-
holm, 1996). Among these, CS1 pili represent a distinct
family of ETEC pili and therefore serve as a model system
for the study of structure, function and morphogenesis in
this pilus family. Most of our current knowledge of the mor-
phogenesis of pili comes from investigation of E. coli Pap
pili and their relatives, including type I pili found on many
E. coli strains (Hultgren et al ., 1991) and the K88 and
K99 pili of animal ETEC strains (Bakker et al ., 1991). How-
ever, CS1 pili bear no resemblance to Pap-related pili and
other pilus types. As the proteins needed for the morpho-
genesis of CS1 pili are unrelated to those of other pilus
types (Perez-Casal et al ., 1990; Scott et al ., 1992; Froeh-
lich et al ., 1994; Sakellaris et al ., 1996; Voegele et al .,
1997), analysis of CS1 pilus morphogenesis should reveal
new mechanisms by which proteins interact to assemble
macromolecular structures of biological importance.
Definition of the CS1 pilus family
There are over 20 serologically and genetically distinct
colonization factors found on human ETEC strains (Gaastra
and Svennerholm, 1996). The major subunits of CFA/I,
CS2, CS4, CS14, CS17 and CS19 pili share extensive
amino acid sequence similarity (H. Sakellaris, unpublished)
and therefore define a distinct pilus family. CS1, which is
the best-characterized member of this group, serves as
a prototype for its study.
The CS1 major pilin shares extensive sequence similar-
ity with the pilins of several human ETEC strains including

682 H. Sakellaris and J. R. Scott
CFA/I, CS2, CS4, CS14, CS17 and CS19, (H. Sakellaris,
unpublished). The only other CS1 pilus family member
identified so far is the Cable type II pilus found on the cystic
fibrosis-associated pathogen Burkholderia cepacia (Sajjan
et al ., 1995). Owing to the high level of sequence similar-
ity, many of the CS1-related pilins on human ETEC strains
share cross-reactive epitopes that are exposed upon
denaturation of the pilus (Rudin and Svennerholm, 1994;
1996). However, in their native state, the members of this
group elicit specific serological responses used to define
the distinct types of pili. This antigenic diversity may be
the result of selective pressures imposed by the human
immune system as for pili in other families (Hanley et al .,
1985; Boslego et al ., 1991).
The structure and assembly of CS1 pili is simpler than
those of many other types of pili because only four struc-
tural and assembly genes from the ETEC strain are
required to produce functional pili in an E. coli K-12 back-
ground (Froehlich et al ., 1994). If additional genes are
involved in pilus biogenesis, they are not ETEC-specific.
In contrast, Pap pili require nine structural and assembly
genes (Hultgren et al ., 1991), although as many as 14
genes are required for the assembly of the type IV Tcp
pili in Vibrio cholerae (Ogierman et al ., 1993) and BFP pili
in enteropathogenic E. coli (Sohel et al ., 1996; Stone et
al ., 1996).
The structure of the CS1 major pilin, CooA, also differs
significantly from both the type IV pilins and the Pap-related
pilins. CooA does not have the conserved N-terminal
and C-terminal sequences, including a highly conserved
glycine residue and a penultimate tyrosine residue, found
in several Pap-related pilins (Lindberg et al ., 1986).
Furthermore, both Pap-related (van Die et al ., 1987) and
type IV pilins (Strom and Lory, 1993; Zhang and Donnen-
berg, 1996) contain conserved cysteine residues that form
disulphide bridges, whereas CooA lacks cysteine residues
13652958, 1998, 4, Downloaded from https://onlinelibrary.wiley.com/doi/10.1046/j.1365-2958.1998.01088.x by Cochrane Mexico, Wiley Online Library on [05/12/2023]. 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
(Perez-Casal et al ., 1990). Type IV pilins also contain con-
served leader peptides that differ from Sec-dependant
signal sequences (Strom and Lory, 1993) and mature
type IV pilins have characteristic hydrophobic N-terminal
sequences (Strom and Lory, 1993), whereas CooA has a
normal Sec-dependent signal sequence and its N-terminal
sequence differs from those of Type IV pilins.
Thus, CS1 pili and their relatives constitute a new pilus
family because of the high sequence homology they share,
because of the lack of sequence homology with type IV
and Pap-related pilins, and because of the lack of specific
primary structural elements found in type IV and Pap-
related pilins.
Genes encoding CS1 pili
The genes encoding the structural and assembly proteins
required for CS1 pilus production are contiguous on a
large plasmid in the wild-type ETEC strain from which
they were cloned (Fig. 1 and Perez-Casal et al ., 1990;
Scott et al ., 1992; Froehlich et al ., 1994; Marron and
Smyth, 1995). These four genes, cooB , A , C and D , are
transcribed in the same direction and appear to constitute
an operon dependent on the AraC-related activator Rns
for expression. Mutations in either cooB , cooC or cooD
abolish the assembly of the major pilin subunit, CooA,
into functional CS1 pili on the cell surface (Scott et al .,
1992; Froehlich et al ., 1994). Therefore, cooB , cooC and
cooD may be regarded as assembly genes.
The four linked coo genes are flanked by remnants of
IS elements and, with the exception of cooA , they have
a significantly lower GþC content than the rest of the E.
coli genome (Scott and Froehlich, 1994). This suggests
that they have been introduced relatively recently from a
foreign genetic background and may be considered a
small pathogenicity island.
Fig. 1. Physical maps of the homologous operons
encoding CS1 (Froehlich et al ., 1994), CFA/I (Jordi
et al ., 1992) and CS2 (Froehlich et al ., 1995) pili of
enterotoxigenic E. coli . Genes and their direction of
transcription are indicated by arrows. cfaD 8 is an
inactive copy of the cfaD regulator gene carrying a
premature stop codon indicated by the break in the
arrow (Jordi et al ., 1992). Remnants of IS elements
and sequences homologous to IS elements are
indicated by boxes. White boxes indicate homology
to IS 150; grey boxes, homology to IS 629; spotted,
IS 2; diagonal stripes, IS 3; narrow black box (CS2
gene cluster), IS1.
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The proteins encoded by the CFA/I cluster (CfaA, B, C
and E; Jordi et al ., 1992) and CS2 cluster (CotB, A, C and
D; Froehlich et al ., 1995) are highly homologous to the
Coo proteins (50–65% identity) and all three gene clusters
have the same gene order (Fig. 1). In several cases when
the experiment has been performed, the genes from one
pilus type are usually so closely related to those of another
type that they can functionally substitute for each other.
For example, cooB and cooA can complement cotC and
cotD to produce pili and vice versa (Froehlich et al ., 1995).
The serological type of the pili produced in these experi-
ments is determined by the major pilin subunit being
expressed. Similarly, the CFA/I major pilin gene, cfaB ,
can complement a cooA deletion mutation in the CS1
gene cluster to produce pili (Marron and Smyth, 1995).
However, in the converse complementation, Marron and
Smyth did not see pili when the CS1 major pilin gene
cooA was used to complement a cfaB deletion mutation
in the CFA/I gene cluster. This suggests that although
the CS1 and CFA/I genes are closely related, the major
pilins may have diverged sufficiently so that they are not
entirely functionally interchangeable.
Roles of the Coo proteins in pilus morphogenesis
CooA
CooA is the major pilin subunit of CS1 pili (Perez-Casal et
al ., 1990). Like all of the other Coo proteins, its sequence
suggests that CooA relies on the E. coli Sec system for its
secretion across the cytoplasmic membrane (Perez-Casal
et al ., 1990). The 16 kDa cytoplasmic form of CooA is pro-
teolytically processed at a canonical signal peptidase I
recognition site to yield the mature 15.2 kDa form which
polymerizes into pili on the cell surface (Perez-Casal et
al ., 1990).
Periplasmic CooA occurs mostly as intermolecular com-
plexes with the CooB assembly protein, but a small pool of
CooA multimers of unknown significance is also present
(Sakellaris et al ., 1996). When they are synthesized
from a strain that does not produce the other Coo proteins,
CooA multimers are not capable of interacting with CooB
in vitro (H. Sakellaris, unpublished). This may be either
because the multimers are composed of misfolded pro-
teins or because, in the polymerized form of CooA, the
sites for interaction with CooB are not exposed. CooA
also forms periplasmic complexes with the minor pilin
subunit, CooD (Sakellaris et al ., 1996). As for the CooA
multimers, the CooA–CooD complexes may be natural
assembly intermediates or dead end products with no
function in assembly.
CooC
CooC is a 94 kDa protein that has several properties in
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CS1 pilus morphogenesis 683
common with outer membrane proteins, including a high
b-sheet and low a-helix content, a high content of charged
residues and no extensive hydrophobic regions (Froehlich
et al ., 1995). Although all of the other Coo proteins are
periplasmic, CooC is an integral outer membrane protein
(Sakellaris et al ., 1996). This makes it likely that CooC is
involved in the transport of the Coo pilins across the
outer membrane. Although CooC has no sequence simi-
larity to the proteins of other pilus families, it may have a
function like that of a class of outer membrane proteins
of similar size associated with Pap and its relatives.
These proteins, termed ‘ushers’, are presumed to trans-
port pilins from the periplasm across the outer membrane.
CooD
CooD is a 38 kDa minor pilin associated with the tip of the
CS1 pili (Sakellaris et al ., 1996). In CS1 pili, the molar ratio
of CooD/CooA is about 1:1800, suggesting that there may
be only one CooD subunit per pilus (Sakellaris et al .,
1996). It is not surprising that no minor proteins were
found in Coomassie blue-stained gels of purified CFA/I
pili (Evans et al ., 1979; Buhler et al ., 1991) and CS2 pili
(Sjoberg et al ., 1988), because the sensitivity of this tech-
nique is inadequate to detect one molecule in about 2000.
However, using immunoblotting, we have recently found
that the CooD homologue, CotD, is present in purified CS2
pili (H. Sakellaris, unpublished). Thus, in contrast to Pap,
which has a complex tip structure consisting of four distinct
minor pilin subunits (Jacob-Dubuisson et al ., 1993), the tip
of the CS1 pilus appears to be simple.
CooD has only 13% sequence identity to CooA (H.
Sakellaris, unpublished). This degree of homology is similar
to that of the minor tip-associated pilins, PapG and FimH,
with their cognate major pilins in Pap and type I pili,
15% and 20%, respectively (Girardeau and Bertin, 1995).
However, the CS1 pilin proteins share a conserved amino
acid sequence motif (A–G–x–Y–x–G–(x)6 –T, where ‘x’
denotes a non-identical amino acid residue), which is
found very close to their C-termini. This motif is conserved
in all major and minor pilins of the CS1 family whose
sequence is currently available, including those of CS1,
CS2, CFA/I, CS4, CS14, CS17 and CS19, (Sakellaris et
al ., 1996; H. Sakellaris, unpublished). In addition, five of
the six absolutely conserved residues in the C-terminal
motif are also conserved in the Cable type II major pilin
of B. cepacia . This suggests that the conserved sequence
motif has an important function, perhaps in mediating
the interactions of pilins with other pilins or with some
component of the assembly machinery (Sakellaris et al .,
1996).
In addition to its role as a structural component of CS1
pili, CooD is also required for pilus assembly because
CS1 pili are not made in the absence of CooD (Froehlich

684 H. Sakellaris and J. R. Scott
et al ., 1994). Unlike all the other Coo proteins, CooD can-
not be detected in cell-free extracts of wild-type strains of
ETEC by immunoblotting, indicating that it is expressed at
a very low level (Sakellaris et al ., 1996). Its location in the
pilus tip, its low concentration in the cell, and the fact that
CooD is required for pilus assembly suggest that CooD
may be a rate-limiting initiator of CooA polymerization at
the cell surface. A similar function has been proposed
for the minor Pap pilin, PapK (Jacob-Dubuisson et al .,
1993) and the minor G-fimbrillin, GafD (Saarela et al .,
1995). If CooD is required for nucleation of CooA pilin,
the level of CooD expression should determine the num-
ber of pili produced by a cell.
In some cases, pilus-mediated binding of bacteria to
host tissues is determined by the major pilin subunit, as
in K99 pili (Jacobs et al ., 1987), and in other cases by a
minor tip pilin, as in Pap pili (Lindberg et al ., 1987). It
has been suggested for CFA/I pili that the major subunit
is the adhesin (Buhler et al ., 1991). Experiments relying
on monoclonal antibody inhibition of pilus binding (Buhler
et al ., 1991) are open to the concern that the large anti-
body molecule may sterically interfere with a nearby adhe-
sin without actually recognizing and binding to it. However,
Buhler et al . also found that the major pilin subunit of CFA/
I pili interfered with haemagglutination. Unless this subunit
interacts in the haemagglutination reaction with the tip pro-
tein, this result suggests that the major subunit acts as an
adhesin. In addition, Buhler et al . found that the CFA/I
major subunit agglutinates erythrocytes in a Staphylococ-
cal coagglutination test. However, this reaction was much
weaker than the haemagglutination test, as it had to be
observed microscopically. Based on these results, it has
been proposed that the major CS1 pilin is also an adhesin
(Marron and Smyth, 1995). In very recent experiments, we
have found that a point mutation in CooD, which does not
interfere with pilus formation, abolishes haemagglutination.
This suggests that CooD is involved in adherence. There-
fore, it appears that it is not yet clear what serves as the
adhesin for this class of pili.
CooB
CooB is a 28 kDa protein that has an essential role in pilus
assembly (Scott et al ., 1992), but unlike CooD, is not asso-
ciated with the final pilus structure (Sakellaris et al ., 1996).
Instead, CooB is located mainly in the periplasm, where
it forms intermolecular complexes with either the major
pilin, CooA, or minor pilin, CooD (Sakellaris et al ., 1996;
Voegele et al ., 1997). Interaction with CooB protects the
pilins from proteolysis in the periplasm (Voegele et al .,
1997). In the absence of CooB, very little full-length CooA
is detectable in cell extracts, indicating that the entire pro-
tein is degraded. However, in the absence of CooB, a trun-
cated CooD product of < 25 kDa is produced, indicating
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that the degradation of CooD is limited (Voegele et al .,
1997). Whether the small form of CooD, which is not pre-
sent in pili, plays a role in pilus assembly remains to be
determined. As CooB interacts with and stabilizes pilins,
it plays a chaperone-like role for CS1 pilus biogenesis.
However, it does not have the highly conserved and invar-
iant residues that define a consensus sequence for peri-
plasmic pilin chaperones in the Pap-related pilus family
(Holmgren et al ., 1992). Furthermore, CooB has no signifi-
cant overall sequence homology to any previously defined
periplasmic or cytoplasmic chaperones. Thus, CooB defines
a novel class of periplasmic chaperone-like proteins whose
mode of action has not been investigated.
In addition to its role in stabilizing pilins, CooB binds to
and stabilizes CooC in the outer membrane (Voegele et
al ., 1997). In the absence of CooB, CooC is degraded to
an <70 kDa protein. Stabilization of an outer membrane
pilus assembly protein has not been reported in other
pilus families and may be unique to CS1 and its relatives.
In the Pap pilus family, degradation of unchaperoned
pilins is catalysed by the periplasmic protease DegP (Bakker
et al ., 1991; Striker et al ., 1994). In contrast, when CooB is
absent, CooA, CooC and CooD are still degraded in a
degP mutant strain (Voegele et al ., 1997; H. Sakellaris
and V. R. Penumalli, unpublished). This indicates that peri-
plasmic degradation of the CS1 pilin and assembly pro-
teins is determined by an as yet unidentified proteolytic
system.
A model for the assembly of CS1 pili
A combination of genetic, protein localization and functional
studies has allowed us to propose the following model for
the morphogenesis of CS1 pili (Fig. 2). All of the Coo pro-
teins traverse the cytoplasmic membrane via the Sec
secretion pathway. As the CooD and CooA pilins enter
the periplasm, CooB binds to each. This promotes correct
folding and/or prevents inappropriate polymerization of the
pilins, thus preventing their degradation. CooB also binds
to the CooC assembly/transport protein, maintaining it in
a stable and active conformation in the outer membrane.
We propose that a new pilus is initiated when a CooD–
CooB complex finds a free CooC–CooB complex in the
outer membrane. As CooD–CooB complexes bind to
CooC, CooB is released from either the pilin or CooC. In
this way, CooB recycles into the periplasm to bind newly
secreted pilins while the CooB–CooC interaction is main-
tained to keep CooC in an active conformation.
How does CooD initiate pilus assembly? The binding of
CooD to CooC may alter the conformation of CooC so that
it can then bind to CooA, which is found in the periplasm in
the form of CooA–CooB complexes. When CooA binds to
the outer membrane-located CooC, it displaces CooD from
CooC and CooA binds to CooD in the process. Growth
Q 1998 Blackwell Science Ltd, Molecular Microbiology, 30, 681–687

of the pilus may be driven by the high concentration of
CooA–CooB complexes in the periplasm displacing
CooA subunits from CooC, thereby allowing the addition
of new pilins to the base of the pilus. The pilus then
extends by repeated rounds of pilin–chaperone interac-
tions with CooC, leading to the displacement of pilins
from CooC to the exterior of the cell.
Despite the lack of any clear homologies between CS1
and the Pap-related pili, their assembly shares some com-
mon features. These include the interaction of pilins with a
periplasmic chaperone, the requirement for a large outer
membrane protein specific for pilus assembly and the
presence of a minor, tip-associated pilin essential for the
polymerization of major pilins at the cell surface. This
may represent a case of convergent evolution in which
structurally unrelated proteins perform similar functions
in pilus morphogenesis. Thus, the CS1 system comprises
a newly recognized set of tools used in the old trade of
pilus assembly.
Q 1998 Blackwell Science Ltd, Molecular Microbiology, 30, 681–687
13652958, 1998, 4, Downloaded from https://onlinelibrary.wiley.com/doi/10.1046/j.1365-2958.1998.01088.x by Cochrane Mexico, Wiley Online Library on [05/12/2023]. 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
CS1 pilus morphogenesis 685
Fig. 2. Model of CS1 pilus assembly. Initiation
of pilus assembly: All of the Coo proteins are
transported across the inner membrane via the
Sec pathway. As they enter the periplasm, the
chaperone, CooB, binds to the major and minor
pilins, CooD and CooA, to prevent misfolding
and degradation and/or to inhibit inappropriate
pilin–pilin interactions. CooB also binds to CooC
in the outer membrane to maintain its
conformation and prevent CooC degradation. A
CooB–CooD complex initiates pilus assembly by
binding to CooC. CooB is simultaneously
displaced and is recycled into the periplasm
where it may interact with other pilins. CooB–
CooA complexes displace CooD from CooC,
replacing it with CooA. Pilus extension occurs by
repeated interaction of CooB–CooA complexes
with CooC, which allows incorporation of CooA
at the base of the pilus and extension of the
pilus rod.
Acknowledgements
We would like to thank the Biomolecular Computing Resource
at Emory for facilitating sequence analysis. The work in our
laboratory was supported by Public Health Service Grant
AI24870 from the National Institutes of Health.
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