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| Proc Natl Acad Sci U S A. 2001 June 5; 98(12): 6911–6916. Published online 2001 May 29. doi: 10.1073/pnas.111551898. | PMCID: PMC34452 |
Copyright © 2001, The National Academy of Sciences Microbiology The chaperone/usher pathways of Pseudomonas
aeruginosa: Identification of fimbrial gene clusters
(cup) and their involvement in biofilm formation Isabelle Vallet, * John W. Olson, † Stephen Lory, † Andrée Lazdunski, * and Alain Filloux *‡*Laboratoire d'Ingéniérie des Systèmes
Macromoléculaires, Unité Propre de Recherche 9027, Institut
de Biologie Structurale et Microbiologie/Centre National de la
Recherche Scientifique, 31 Chemin Joseph Aiguier, 13402 Marseille Cedex
20, France; and †Department of Microbiology and
Molecular Genetics, Harvard Medical School, 200 Longwood Avenue,
Boston, MA 02115 Received November 21, 2000. |
Abstract Pseudomonas aeruginosa, an important opportunistic
human pathogen, persists in certain tissues in the form of specialized
bacterial communities, referred to as biofilm. The biofilm is formed
through series of interactions between cells and adherence to surfaces,
resulting in an organized structure. By screening a library of
Tn5 insertions in a nonpiliated P.
aeruginosa strain, we identified genes involved in early stages
of biofilm formation. One class of mutations identified in this study
mapped in a cluster of genes specifying the components of a
chaperone/usher pathway that is involved in assembly of fimbrial
subunits in other microorganisms. These genes, not previously described
in P. aeruginosa, were named cupA1–A5.
Additional chaperone/usher systems (CupB and CupC) have been
also identified in the genome of P. aeruginosa PAO1;
however, they do not appear to play a role in adhesion under the
conditions where the CupA system is expressed and functions in surface
adherence. The identification of these putative adhesins on the cell
surface of P. aeruginosa suggests that this organism
possess a wide range of factors that function in biofilm formation.
These structures appear to be differentially regulated and may function
at distinct stages of biofilm formation, or in specific environments
colonized by this organism. |
P seudomonas
aeruginosa, a common environmental Gram-negative bacterium, is
also an opportunistic human pathogen and is responsible for serious
damage to the respiratory tract of cystic fibrosis patients ( 1).
Nosocomial pneumonia in intubated and mechanically ventilated patients
is the second most common infection of hospitalized patients, and
P. aeruginosa is the key etiological agent of
hospital-acquired infections ( 2). The ability of P.
aeruginosa to attach to abiotic surfaces, to host tissues, or to
each other, and the subsequent differentiation of the microorganisms
into biofilm, can be considered a major virulence trait in a variety of
infections ( 3). Biofilm formation can take place on a variety of
surfaces, such as medical instruments, leading to many types of
nosocomial infections, and P. aeruginosa has been shown to
persist in biofilm in the lungs of cystic fibrosis patients ( 4).
Biofilms are characterized by a complex, highly structured, bacterial
organization ( 5). They are initiated by the attachment of a single
planktonic cell on a surface. Multiplication and the development of
microcolonies separated by water-filled channels follow this event. The
ability to form biofilm endows the bacteria with several important
characteristics, including a marked increase in resistance to
antibiotics ( 6). A genetic screen, developed by Kolter and coworkers ( 7, 8) provided
some of the tools to study the genetic determinants of biofilm
formation. By characterizing several P. aeruginosa mutants
defective in distinct stages of biofilm formation, a number of
components that participate in the initiation of biofilm formation and
progression to microcolonies and mature biofilm were identified ( 9).
Through the analysis of the phenotypes of different P.
aeruginosa mutants, it was proposed that type IV pili and
flagella, two organelles of motility, may play an important role during
the initial interaction of the bacterial cell with the surface by
counteracting repulsive forces. Furthermore, type IV pili appeared to
be required for the initial differentiation by promoting cell
aggregation and the formation of microcolonies. Once the bacterial
population reached a given threshold, cell–cell communication by means
of the quorum-sensing regulatory systems, more particularly by means of
the lasI-dependent N-(3-oxododecanoyl)homoserine
lactone, programmed the differentiation process and the maturation into
biofilm ( 10). The stabilization of the biofilm community is usually
accomplished by production of an exopolysaccharide ( 11), such as
alginate in the case of P. aeruginosa. Several physiological
factors, such as the global carbon metabolism regulator (Crc) of
P. aeruginosa ( 12), also play a role in biofilm formation.
The availability of carbon/energy sources was also reported as an
important signaling information determining the initiation of biofilm
formation in Pseudomonas fluorescens ( 7). In this study we adapted the method described by O'Toole and
Kolter ( 9) for identifying biofilm-defective mutants. We screened a
collection of Tn 5 mutants in a strain of P.
aeruginosa that was unable to produce type IV pili. Expression of
pili is important during various stages of biofilm formation, but
bacteria lacking these organelles can still interact with abiotic
surfaces ( 9). This screen, therefore, identifies genes specifying new
adhesion factors. Among the series of biofilm-defective mutants
obtained, one was affected in a gene encoding a protein similar to
periplasmic chaperones of the chaperone/usher pathway in a variety of
bacteria ( 13). This gene belongs to a cluster that we called
cupA. We proposed that the cupA genes encode the
components of a new class of P. aeruginosa adhesins, related
to the adhesins in other microorganisms ( 14). We showed that mutants
devoid of a functional CupA are defective in the formation of biofilm,
in a manner that is independent of the presence of type IV pili. Using
the available sequence of the PAO1 genome, we identified additional
cup gene clusters and tested their involvement in biofilm
formation. The CupB and CupC systems identified do not appear to play a
role in biofilm formation under the conditions tested. These
observations suggest that multiple factors are available to P.
aeruginosa to facilitate its binding to various surfaces and for
interbacterial adhesion as well. The wide variety of such attachment
mechanisms, which are apparently differentially regulated, may reflect
the complex needs of this organism during the colonization of widely
diverse environmental niches. |
Materials and Methods Bacterial Strains and Growth Conditions. The bacterial strains and plasmids used are listed in Table
1. Strains were grown at 37°C in L
broth (LB) or on LB agar plates. Plasmids were introduced into P.
aeruginosa by electroporation ( 15), and transformants were
selected on Pseudomonas isolation agar (PIA) containing
antibiotics. Antibiotics were used at the following concentrations
(μg/ml): ampicillin, 50 ( Escherichia coli);
carbenicillin, 500; tetracycline, 200; and gentamicin, 50 ( P.
aeruginosa).
Construction of a Tn5 Insertions Library in P.
aeruginosa. A random insertion library was created by using Tn 5G, a
modified transposon Tn 5 ( 16). The transposon was introduced
into P. aeruginosa PAKΔpilA, a strain containing a
chromosomal deletion of the pilin gene, by pRK2013-mediated conjugation
( 17). Biofilm Formation Assay and Quantification. The biofilm formation assay was performed according to O'Toole
and Kolter ( 9) with slight modifications. Screening was performed in
96-well polystyrene microtiter dishes containing 100 μl of M63
minimal medium supplemented with 0.2% glucose, 1 mM
MgSO 4, and 0.5% Casamino acids. The wells were
inoculated with individual clones, taken with a toothpick, from the
Tn 5 mutant collection isolated on agar plates, and the
dishes were incubated at 30°C for 10–12 h. The phenotype of putative
nonadherent clones was further checked in 24-well microtiter dishes. In
those wells, 1 ml of M63-derived minimal medium was inoculated with
10 8 bacterial cells from an overnight inoculum.
Bacterial cells bound to the wall of the wells were stained with
crystal violet 1% (Sigma), and for quantification they were suspended
in 400 μl of 95% ethanol followed by addition of 600 μl of water,
and the OD 600 was measured. Microscopic Analysis of Biofilm Formation. Cells were grown in 4 ml of M63 minimal medium supplemented with
glucose, MgSO4, and Casamino acids, in a 50-ml
Corning tube containing a glass cover slide. After 10–12 h of growth
at 30°C without shaking, the glass slide was removed and rinsed. The
remaining cells were visualized by phase-contrast microscopy using a
Zeiss PhoMi III microscope. Images were captured with a camera and
integrated with the Image Pro Plus
software (Media Cybernetics, Silver Spring, MD). Flagellar Motility Assay. Bacteria were inoculated with a toothpick on minimal agar plate (M63
with glucose, MgSO4, and Casamino acids)
containing 0.3% agar. The plates were then incubated at 30°C, and a
halo corresponding to the spreading of bacteria from the point of
inoculation was observed. Inverse PCR. PCR amplification of DNA regions bordering the Tn5 in
selected PAKΔpilA mutants was performed by using two divergent
oligonucleotides: OTn1, 5′-GCGCGGATCCTGGAAAACGGGAAAG-3′; and OTn2,
5′-CCATCTCATCAGAGGGTAGT-3′. Chromosomal DNA from each mutant was
prepared by using the Nucleospin C + T kit (Macherey-Nagel), digested
with AluI, TaqI, or XhoI, and
religated, and the circular DNA was used as matrix for PCR
amplification. The DNA fragments thus amplified were directly cloned
into the PCR2.1 vector with the TA cloning kit (Invitrogen) and
transformed into E. coli TOP10F′ (Invitrogen). DNA
sequencing was performed by MWG Biotech (Ebersberg, Germany). Genome Sequence Analysis. Plasmids Constructions and Molecular Techniques. The ladN66/cupA2 gene was PCR amplified with
the High Fidelity polymerase (Boehringer), from the chromosomal DNA of
P. aeruginosa PAK, using oligonucleotides ON1
(5′-CCAACGGAGCCACCAGCACCA-3′) and ON2 (5′-CAGGAAGAGCCGAGCAACAG-3′). The
DNA fragment was cloned into PCR2.1 and subcloned into the broad host
range vector pBBR1MCS4 as a HindIII/XbaI
fragment, yielding pBBRN66. Internal DNA fragments from the
cupA3, cupB3, and cupC3 genes were PCR
amplified by using the oligonucleotide pairs OMP1/OMP2
(5′-TCCAACCTACACCTATTCCCGCTAC-3′/5′-CCGTCGTAGAAATCGCTGGAGGAG-3′),
MUB1/MUB2
(5′-CCTGTCTGCTGGCACTGTTTC-3′/5′-AATAGCTGGGCACCGAGACATA-3′), and
MUT1/MUT2
(5′-AGGTGTCCGTCTATTCCAGGT-3′/5′-GGTACGGTTGCTACTGAACTTG-3′),
respectively. The fragments were cloned into PCR2.1, yielding pCRN67,
pCRB3, and pCRC3, respectively. Those plasmids, which do not replicate
in P. aeruginosa, were introduced by electroporation into
strain PAK or the nonpiliated PAK-NP strain, and mutants were selected
on PIA plates containing carbenicillin. The positions of the plasmid
insertion, within the chromosome of these mutant strains, were checked
by PCR with appropriate oligonucleotide pairs, including ON1, UBV
(5′-CCCTCCGTTTCCCCGCTTTTTA-3′) or UTV (5′-TCAGAAGAGCAGAGCAGAGCAG-3′),
for cupA3, cupB3, or cupC3,
respectively, and the universal or reverse M13 primers. |
Results and Discussion Screening Procedure and Strategy for Identification of New
Adhesins. Recent advances in genetic studies on biofilm formation by
microorganisms resulted in substantial progress in the understanding of
the molecular mechanisms involved in this process ( 8). In P.
aeruginosa, the involvement of two distinct surface appendages has
clearly been established ( 9). Those structures include the primary
organelles of motility: the flagellum and the type IV pili. In P.
aeruginosa, type IV pili appear to be involved in several
functions, including adhesion to epithelial cells ( 18, 19) and a
particular form of motility, called twitching motility ( 20). Their
biogenesis and function in twitching motility involve the assembly of
PilA monomers into a filament, a process that requires the products of
a number of accessory genes ( 21, 22). Type IV pili also appear to be important for adherence to abiotic
surfaces as well, because mutations in three genes associated with
pilus formation ( pilB, pilC, and pilY1) yielded
P. aeruginosa strains defective in attachment to polyvinyl
chloride (PVC) ( 9). These mutants formed monolayers on plastic, but
they lacked the ability to form microcolonies and to differentiate into
biofilm. To uncover adhesive phenotypes that may function under various
conditions, we modified the original assay of O'Toole and Kolter ( 9),
by growing the bacteria at lower temperature (30°C) in an enriched
minimal medium. A modest difference was observed between binding to
abiotic surfaces by wild-type P. aeruginosa PAK and the
isogenic nonpiliated mutant (PAK-NP). Indeed, the nonpiliated mutant
could attach to plastic with an efficiency of 88%, relative to the
wild type (Fig. 1), suggesting that under
these conditions, the contribution of type IV pili to overall adhesion
was reduced, allowing the identification of a novel adhesive system.
 | Figure 1Biofilm formation of wild-type PAK strain, PAK-NP
(pilA), and PAK-NF (fliC).
(A) Bars represent the measure of OD600 as a
result of four distinct experiments. The level of attachment of the
mutant strains is indicated as a percentage of attachment (more ...) |
We constructed and screened a collection of Tn 5 mutants
derived from the nonpiliated P. aeruginosa strain
PAKΔpilA. Each individual clone was grown in Casamino acids/glucose
medium, and bacteria were allowed to bind to the wells of a polystyrene
microtiter dish for a period of 10–12 h at 30°C. The mutants that
were not able to form a ring on the walls at the air–liquid interface,
as revealed by crystal violet staining, were considered nonadherent. In
the conditions used for this screen, a mutant that lacks flagella
because of a mutation in the fliC gene, which encodes the
structural subunit of the flagellum, PAK-NF, was unable to attach
efficiently (Fig. 1). This observation prompted us to analyze all of
the mutants selected by our screen onto plates containing 0.3% agar,
to determine whether the mutation affected flagellar motility (Fig.
2). Out of 4,140 clones tested, we
isolated 46 nonadherent mutants. From those, 29 were also deficient in
motility, as compared with the wild-type strain. The remaining 17
mutants had thus lost adherence independent of the function of type IV
pili and flagella. These mutants were named lad for
lost adherence.
 | Figure 2Flagellar motility. Motility of strains PAK, PAK-NP, and PAK-NF are
compared with motility of PAKΔpilA Tn5 mutant
derivatives. Strains R15, U60, and P48 appeared to be mutants affected
in flagellum biogenesis (see Table 2), whereas IC41, O51, (more ...) |
Characterization of the Nonmotile Mutants. The mutants were analyzed by inverse PCR, using two oligonucleotides,
OTn1 and OTn2, to amplify adjacent DNA fragment to the transposon
insertion site, followed by sequencing of the cloned product (see
Materials and Methods). To verify this approach, five of the
nonmotile mutants were analyzed first. Each insertion was located in
genes involved in flagellar biogenesis (Table
2) ( 23). Among those strains, mutation in
genes essential for flagellum structure ( flgF) ( 24),
flagellum assembly ( flhB) ( 25), or regulation of
flagellar gene expression ( fleS) ( 26) were found.
O'Toole and Kolter ( 9) also previously showed that a nonmotile mutant
possessing an incomplete flagellum ( flgK) was unable
to adhere to the wall of a microtiter dish. The adhesion defect was
severe in P. aeruginosa, since microscopic analysis revealed
a complete absence of bacteria adhering to plastic ( 9). In contrast,
the lack of functional flagella in E. coli does not totally
prevent surface attachment ( 27). Interestingly, our screen identified
genes that are necessary either for the assembly of flagella or for
flagellar rotation ( motA and motY) ( 28, 29). The
requirement for flagellar rotation is an indication that during
formation of biofilm by P. aeruginosa, the most
important function of the flagellum is not in adhesion, but in
facilitating bacterial motility.
 | Table 2Gene characterization from P. aeruginosa
mutants affected in flagellar motility |
Characterization of the lad Mutants. Among the 17 motile mutants obtained from the Tn 5
library, most of the transposons were found in genes that were
annotated as hypothetical unknown genes. One gene encoded a putative
component of regulatory systems, the product of another was similar to
a permease from an ABC transporter, whereas another putative
lad gene product shared 39% identity with the putative
protein Y4iJ from Rhizobium sp. NGR234 ( 30). Finally, one of
the lad genes ( ladN66) was identified through two
different Tn 5 insertions in the same coding sequence. This
gene encodes a protein belonging to a family of periplasmic chaperones
involved in pilus assembly via the so-called chaperone/usher pathway
( 31). Those chaperones are periplasmic proteins that adopt an Ig-like
fold for recognition and further assembly of pilin subunits ( 32).
Surface exposure required the function of a second accessory component,
called usher, that forms a channel into the outer membrane through
which the pilin subunits are sequentially assembled ( 33). This highly
conserved pathway in Gram-negative bacteria has been involved in the
assembly of more than 30 adhesive organelles ( 14). These organelles are
mostly fimbrial structures that may adopt different morphologies. Two
main classes could be discerned. The thick and rigid pili (7 nm in
diameter), such as the P or type 1 pili from E. coli, and
the thin and flexible pili (2–5 nm in diameter), such as the F17 pili
from E. coli. In addition, very thin or afimbrial adhesins,
also called atypical structures, are assembled via the
chaperone/usher pathway. The exact composition and the architecture
of those structures has not been well characterized, but they are most
probably composed of monomers or simple oligomers of the afimbrial
subunit assembled at the cell surface. Similar structures have been
proposed for Yersinia species, namely the Caf1 antigen of
Yersinia pestis ( 34). The periplasmic chaperones involved in the chaperone/usher
pathway could be distinguished into two structurally and functionally
distinct subfamilies. These two groups differ by the length of the loop
that connects the F1 and G1 β-strands of domain 1 ( 14). Chaperones of
the FGL family have a loop with a length of over 20 aa, whereas
chaperones from the FGS family have a shorter loop. The FGL chaperones
assemble atypical or nonfimbrial adhesins, whereas the FGS chaperones
assemble fimbriae. The amino acid sequence analysis revealed that the
ladN66 gene product belongs to the FGS family, which
suggests that it may be involved in pili formation rather than in
assembly of nonfimbrial adhesins. So far, we have not clearly
identified LadN66-dependent fimbrial structures by electron microscopic
analysis (data not shown). The ladN66 gene was amplified by PCR using the primers
ON1 and ON2 and was cloned under control of the lac promoter
into pBBR1MCS4, yielding pBBRN66. This plasmid was then introduced into
the corresponding mutant PAN66 by electroporation. This strain was
analyzed for its ability to attach to inert surfaces as described
previously. We observed that it attaches even more efficiently as
compared with the PAK-NP strain (data not shown), indicating that the
lad phenotype of the PAN66 mutant strain was complemented by
the introduction of this unique gene. The lack of attachment of the
mutant PAN66 was therefore due solely to the disruption of the
ladN66 gene by the transposon. Biofilm Formation Phenotype of PAN66 Mutant. More detailed analysis of the defects conferred by the mutations in the
ladN66 gene was obtained through microscopic analysis of
bacteria attached to glass cover slides (see Materials and
Methods). As illustrated in Fig. 3,
only a few cells of PAN66, which could be found in clusters, are
attached to the glass cover slide, whereas PAK-NP is capable of forming
a significant biofilm on this support. The direct visual inspection of
the biofilm formation phenotype of the PAN66 mutant revealed that,
compared with the parental strain, only very few bacteria could make a
stable attachment with the abiotic surface, and they are not able to
form a structured biofilm. This observation thus confirmed the results
of our initial screening procedure in microtiter dishes.
 | Figure 3Microscopic analysis of biofilm formation. Bacteria grown in M63
medium, supplemented with glucose, MgSO4, and Casamino
acids, were allowed to attach to glass cover slides. The development of
biofilm on a solid surface was visualized by phase-contrast (more ...) |
Characterization of the cupA Gene Cluster. The availability of the P. aeruginosa PAO1 genome
sequence ( http://www.pseudomonas.com) allowed us to identify
the gene cluster associated with ladN66. Flanking
ladN66 were genes that encode additional components of the
chaperone/usher pathway in other bacteria, including genes encoding
two pilin-like subunits, an usher, and a second chaperone (Fig.
4). Because of these similarities the
genes belonging to this cluster have been renamed cup for
“ chaperone– usher pathway.” This
gene cluster contains five genes ( cupA1– cupA5),
with cupA2 being the previously identified ladN66
gene.
 | Figure 4Organization of the cup gene clusters. The three
cup gene clusters have been represented to scale. Genes
encoding fimbrial subunits are represented as black arrows, genes
encoding periplasmic chaperones are represented as gray arrows, and
genes encoding (more ...) |
Comparison of the deduced protein sequences of the
cupA cluster showed sequence relatedness to other adhesive
system; however, the most closely related proteins were not always from
the same bacterium (Table 3). For
example, CupA1 shares sequence similarity with the F17A fimbrial
subunit of E. coli, whereas the sequence of CupA4 shares
sequence similarity with a protein from Yersinia pestis,
which is homologous to the HifA fimbrial subunit from Haemophilus
influenzae ( 35). CupA2 and CupA5 are both related to the members
of the periplasmic chaperone family, with the closest identity to
F17a-D of E. coli, and FhaD of Bordetella
pertussis, respectively. CupA3 appears to be the usher component
of the system, based on its high similarity to FocD from E.
coli. Since the analysis of the relatedness of the CupA proteins
to other fimbrial systems with defined structures, such as the thick or
thin filaments ( 14), revealed no consistent pattern, we should consider
the possibility that the CupA proteins participate in the assembly of a
type of adhesive organelle not previously recognized.
| Table 3Homologies between Cup components and components of known
chaperone/usherpathways |
We also examined the organization of genes that specify adhesins of the
chaperone/usher family in other bacteria. We observed that a gene
cluster in Y. pestis ( yp36–40) showed strictly
the same genetic organization as the cupA gene cluster ( 36).
Moreover, each of the yp genes encoded components having
precisely the same homologies as those found with the CupA proteins,
including the unique sequence of one of the putative fimbrial subunit
(Yp39). Interestingly, the yp36–40 locus is located
upstream of a high-pathogenicity island (HPI). cupA3 Is Involved in Biofilm Formation. To demonstrate that the whole chaperone/usher pathway is involved in
biofilm formation, and that the nonadherent phenotype is not
exclusively associated with a defect in the CupA2 chaperone, we
introduced a mutation in the gene encoding the usher component
( cupA3). An internal DNA fragment of the cupA3
gene was amplified by PCR and cloned into a nonreplicative plasmid in
P. aeruginosa, and, after electroporation and selection in
P. aeruginosa PAK-NP, a mutant strain, PAN67, was obtained.
This strain, carrying a disrupted cupA3 gene, showed an
adhesion-deficient phenotype, with only 10% of the bacteria adhering
relative to the parental PAK-NP (Fig. 5),
which is even lower than the adherence of PAN66 (14%). Interestingly,
when the same mutation was introduced into the wild-type PAK strain
(PAN67B), the level of attachment was also drastically reduced,
comparable to the level observed with PAN67, despite the presence of
type IV pili at the surface of those cells (data not shown). This
result confirmed that the CupA-dependent attachment is independent of
the presence of type IV pili. It also showed that CupA-dependent
adhesins are more important, or required at an earlier stage than type
IV pili, for attachment in these particular conditions. The involvement
in biofilm formation of pili assembled by the chaperone/usher pathway
has already been described for other organisms ( 37). Pratt and Kolter
( 27) reported that E. coli type I pili are essential for
initial attachment, and that the lack of those structures abolished the
attachment to the surface, in contrast to flagella-defective strains.
 | Figure 5Quantification of biofilm formation of the PAN66 and PAN67 strains.
Bars represent the measure of OD600 as a result of four
distinct experiments. The level of attachment of the mutant strains is
indicated as a percentage of attachment of the wild-type (more ...) |
The Fimbrial Gene Clusters cupB and cupC,
and Their Role in Biofilm Formation. By analyzing the PAO1 genome, we could identify two additional gene
clusters encoding a complete set of components belonging to the
chaperone/usher pathway. The cupB gene cluster contains
six genes (Fig. 4). CupB1 and CupB6 are homologous to fimbrial
subunits. CupB5 (1,018 aa) is homologous to particular type of adhesive
molecules, such as the filamentous hemagglutinin (Fha) from
Bordetella pertussis ( 38). CupB2 and CupB4 are
chaperone-like proteins with their highest homology to FimB/FhaD
precursor from B. pertussis. Finally, CupB3 belongs to the
usher family (Table 3). The cupC gene cluster contains three genes encoding CupC1,
CupC2, and CupC3 (Fig. 4). CupC1 is homologous to fimbrial subunits,
CupC2 to chaperone-like proteins, and CupC3 to usher proteins (Table
3). We subjected the genes of these two additional putative adhesion
systems to mutagenesis by insertional interruption of the genes
encoding the usher components, cupB3 and cupC3.
Internal DNA fragments were obtained by PCR, using MUB1/MUB2 primers
for cupB3 and MUT1/MUT2 primers for cupC3, and
cloned into PCR2.1, yielding pCRB3 and pCRC3, respectively. Those
plasmids were electroporated into the wild-type strain of P.
aeruginosa PAK, and recombinant clones were selected on PIA plates
containing carbenicillin. One mutant from each experiment was selected
and named PAB3B and PAC3B, respectively. The position of the insertion
was verified (see Materials and Methods) and the attachment
phenotype was analyzed as previously described. In contrast to PAN67B,
neither PAB3B nor PAC3B was altered in their ability to form a biofilm
in microtiter dishes. The same observation was made when the mutations
were introduced into the PAK-NP strain (PAB3 and PAC3). Thus, mutations
within genes located in these two clusters did not affect the
attachment capabilities of P. aeruginosa. Therefore, under
the conditions of our assays, CupB and CupC systems do not participate
in bacterial attachment to the polystyrene surface. Alternatively they
may not be expressed, even in the wild-type P. aeruginosa,
under the conditions that were used to grow the bacteria. It is worth
noting that E. coli attachment and biofilm formation involve
different surface appendages. Indeed, under growth conditions that were
different from those promoting type I pili expression, curli filaments
appear to predominate and play an essential role in early stages of
adherence ( 39). |
Conclusions Bacterial cell-surface organelles, such as flagella and
fimbrial structures, have a key role for attachment of microorganisms
to surfaces. The identification of CupA2 as a chaperone of the
chaperone/usher pathway brings a new insight into the P.
aeruginosa strategies for attachment to surfaces. Indeed, assembly
of fimbrial adhesins other than type IV pili has not previously been
considered for this organism. Their function might not be redundant but
synergic, specific for certain stages of attachment, or adapted to
particular growth and environmental conditions. Interestingly, the
cupB gene cluster encodes a protein with homology to
filamentous hemagglutinin (Fha) from B. pertussis. This
large protein, also known as fimbrial hemagglutinin, is clearly
different from the pilin subunits but is also found attached to the
surface of B. pertussis via the chaperone/usher pathway
( 38). Moreover, it was shown that Fha mediates attachment of B.
pertussis to the upper respiratory tract ( 40). This observation
also supports the idea that the three cup clusters
identified here assemble different types of adhesins. These adhesins
may provide P. aeruginosa with high adaptive capacities to
colonize totally different surfaces. One may imagine that complex
regulatory networks, that might involve two-component regulatory
systems for sensing environmental stimuli, are participating in the
differential expression of all these adhesive structures. In addition
to elucidating the structure and the precise function of the CupA,
CupB, and CupC-associated adhesins, full realization of the complexity
and the specificity of the adherence mechanisms used by P.
aeruginosa requires dissection of these networks controlling their
expression. Infections with P. aeruginosa are a major
burden for human health, and the understanding of the complete array of
adhesive mechanisms used by the bacterium to colonize tissues as well
as abiotic surface such as those of catheters or other medical devices
is fundamental to understand the infection process. |
Acknowledgments We thank G. O'Toole for helpful discussions. We are grateful to N.
Bomchil and A. Bernadac for help with microscopy. I.V. is supported by
a grant from the Ministry of Research and Technology. Research in the
laboratory of A.F. is supported by grants from the French Cystic
Fibrosis Foundation (AFLM), from the Programme de Recherche
Fondamentale en Microbiologie, Maladies Infectieuses et Parasitaires
(no. 00N60/1562), and from the Programmes Internationaux de
Coopération Scientifique (no. 848). Research in the laboratory of
S.L. is supported by Grant AI21451 from the National Institutes of
Health. |
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