Department of Microbiology and Immunology,
University of Kentucky Chandler Medical Center, Lexington, Kentucky
40536-0084
Received 12 September 1997/Returned for modification 14 January
1998/Accepted 27 January 1998
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Legionella pneumophila
replicates in protozoan hosts in an aquatic environment
(17), which enhances bacterial survival in the
environment (5, 8) and increases infectivity for mammalian cells (13). Once the bacteria have entered a human host
through aerosolized droplets, the bacterium enters and replicates
effectively within the phagosome of alveolar phagocytes and epithelial
cells (12, 20, 36, 37). L. pneumophila is
capable of intracellular survival and multiplication within similar
specialized vacuoles in macrophages (26) and protozoan cells
(1). L. pneumophila also utilizes similar
mechanisms to parasitize both evolutionarily distant hosts
(18). The ability of L. pneumophila to
replicate intracellularly in these hosts is thought to be integral to
the pathogenesis of the organism. Additionally, the ability of
L. pneumophila to attach, enter, and replicate within
epithelial cells of the lung has also been described elsewhere
(12, 14, 22, 36, 38). L. pneumophila is able
to replicate within both type I and type II mammalian pulmonary
epithelial cells (12, 36). The ability of L. pneumophila to replicate within pulmonary epithelial cells may be
responsible for epithelial cell necrosis found in infected lung tissue
(30, 40).
The molecular determinants for attachment and entry of L. pneumophila into epithelial cells have not been well described. Binding studies have shown that the molecular determinants required for
attachment to macrophages and lung fibroblasts may include one or more
bacterial protein adhesins (22). In the presence of
complement, the L. pneumophila major outer membrane
protein facilitates attachment to monocytes and macrophages
(9). Opsonized bacteria may then be recognized by host cell
complement receptors and internalized by a pseudopod coil
(27). An opsonin-independent mechanism of attachment has
also been described for macrophages and lung fibroblasts (22,
42). A potential protozoan receptor for L. pneumophila attachment and invasion has recently been described (24, 51). However, the bacterial adhesin(s) involved in
complement-independent attachment and adherence to protozoan surfaces
has not yet been described.
For various gram-negative pathogenic bacteria, proteins localized to
the surface of the bacterium have been found to facilitate attachment
and entry into cultured eukaryotic cells and to be required for
virulence. One example of such proteins is pilins or fimbrial proteins
which assemble into structures on the bacterial surface (28,
49). Specifically, type IV pilin genes are found in a number of
pathogenic bacteria including Neisseria species (34,
39) and Pseudomonas aeruginosa (29). The
type IV pili are characterized by a conserved hydrophobic
amino-terminal domain (25, 49). In addition, a short,
positively charged leader sequence is present prior to processing of
the prepilin protein. After processing, a conserved modified amino acid
(N-methylphenylalanine) is usually the first residue in the
mature pilin protein.
Pili have been previously demonstrated on the surface of
Legionella species by transmission electron microscopy
(41). The genetic components required for pilus
expression have not been previously described. Therefore, the
involvement of L. pneumophila pili in adherence and
intracellular replication has not been directly addressed. In this
paper, we describe the expression of various lengths of pili on the
surface of L. pneumophila, which may correspond to at
least two different sets of pilin genes. One of these pilin genes has
been isolated and characterized. The pilEL
gene of L. pneumophila is related to the type IV pili
of Neisseria and Pseudomonas. A mutation in the
type IV pilin gene results in the disappearance of the subset of long
pili expressed on the surface of L. pneumophila and a
defect for attachment to cultured epithelial cells, macrophages, and
protozoan cells.
Identification of L. pneumophila pilus expression.
L. pneumophila AA100 was examined by transmission
electron microscopy. The virulent L. pneumophila strain
AA100 has been previously described (3, 15) and is
a redesignation of strain 130b. L. pneumophila was
grown at 37°C with buffered yeast extract broth (16)
without shaking (to minimize possible shearing of pili) for 96 h.
Bacteria were fixed by addition (1:1) of 2% formaldehyde-saline. Samples were applied to copper Formvar grids (Electron Microscopy Sciences, Fort Washington, Pa.) for 2 min and negatively stained with
1% phosphotungstic acid (pH 6.5) for 2 to 4 min. Samples were examined
with a Hitachi 7000 transmission electron microscope (Hitachi, Inc.,
Tokyo, Japan). The bacteria expressed pili of various lengths
which were designated as short (0.1 to 0.6 µm) (Fig.
1A) or long (0.8 to 1.5 µm) (Fig. 1B).
The longer pili were found only infrequently (on approximately 10% of
bacteria) compared to the shorter pili (on approximately 40% of
bacteria). A large number of bacteria (50%) expressed no pili. Pili
found on the surface of L. pneumophila were
generally either long or short; simultaneous expression was not
apparent.
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FIG. 1.
Electron microscopic examination of expression of pili
by L. pneumophila AA100. L. pneumophila
wild-type strain AA100 expresses short (0.1 to 0.6 µm) (A) and long
(0.8 to 1.5 µm) (B) pili. Pili are indicated by small arrowheads.
Flagella are noticeably thicker than the pili observed and are
indicated by large arrowheads. Bars, 0.5 µm.
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Isolation of a pilin gene from L. pneumophila.
An
~800-bp fragment was amplified by PCR from L. pneumophila AA100 genomic DNA by using degenerate primers which
were originally designed to amplify regions of homology to members of
the family of two-component regulators such as PhoP/PhoQ and BvgA/BvgS
(23). One primer, TCR6 (5'CGCNNNCNNGCGCGATGNA),
was found to amplify an ~800-bp fragment. PCRs were carried out in
the presence of 4 mM MgCl2 and Taq polymerase
(Boehringer Mannheim, Indianapolis, Ind.) with standard concentrations
of other reagents according to the manufacturer's suggestions, with a
DNA Thermal Cycler 480 (Perkin-Elmer, Norwalk, Conn.). Oligonucleotide
primers were synthesized commercially (Gibco BRL, Gaithersburg,
Md.). Chromosomal DNA preparations, restriction enzyme digestion,
DNA ligation reactions, and in situ colony hybridization were performed
as described previously (44). Restriction and modifying
enzymes were obtained from Promega (Madison, Wis.) and New England
Biolabs (Beverly, Mass.). Plasmid DNA preparations were made according
to the manufacturer's specifications with a Qiagen (Chatsworth,
Calif.) Miniprep kit. Escherichia coli DH5
or HB101 was
used for vector manipulations and grown in Luria-Bertani medium
(35) with the appropriate antibiotic: chloramphenicol (40 µg/ml) or kanamycin (50 µg/ml). Electroporations were carried out
with a Bio-Rad Gene Pulser (Hercules, Calif.) as recommended by the
manufacturer.
The ~800-bp fragment was cloned into the vector pCR2.1 with the TA
cloning kit (Invitrogen, San Diego, Calif.). The resulting plasmid was designated pBJ110. Sequence analysis of the ~800-bp insert identified an open reading frame. The translated sequence was
similar to various type IV prepilin sequences including PilE from
Neisseria gonorrhoeae (34), PilE and PilA from
P. aeruginosa (29, 45), and fimbrial protein
Q from Moraxella bovis (32).
In order to isolate a larger fragment of DNA containing the open
reading frame, the ~800-bp L. pneumophila fragment in
pBJ110 was isolated from agarose gels and used to probe an
L. pneumophila chromosomal DNA cosmid library. Cosmids
containing the open reading frame were isolated from an L. pneumophila chromosomal DNA library constructed in the following
manner. L. pneumophila AA100 chromosomal DNA was
partially digested with Sau3A, and 35- to 50-kb sucrose gradient size-selected fragments were ligated into a BamHI
site in the cosmid pTLP6, a derivative of pTLP5 (7, 33). The
ligation mixture was packaged into 
phage with the use of a packaging mix (Stratagene, La Jolla, Calif.) and then transfected into
E. coli HB101. Of six cosmids that hybridized to the probe,
three were unique as assessed by restriction patterns generated during agarose gel electrophoresis (data not shown). Southern analysis of the
cosmids identified a common 2-kb ClaI fragment which
hybridized to the probe (data not shown). The fragment was isolated
from one of the cosmids (pCC3) and cloned into pBC-RI to generate
pBJ120. Plasmid pBC-RI is a derivative of pBC SK+
(Stratagene) from which the EcoRI restriction site has been
removed. Thus, plasmid pBJ120 contains a 2-kb ClaI fragment
(which includes pilEL) from pCC3 cloned into
pBC-RI.
Sequence analysis of the pilin gene.
DNA sequence obtained
from the 2-kb ClaI fragment of pBJ120 contained an open
reading frame of 447 bases, beginning with an amino-terminal methionine
(Fig. 2). Three methionines are present near the beginning of the open reading frame. However, only two potential ribosome binding sites are present: 7 bp from the first methionine and 7 bp from the third (Fig. 2). Translation of the longest possible open reading frame predicts a protein product of 149 amino acids in length with a molecular mass of 16.5 kDa.
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FIG. 2.
Nucleotide and deduced amino acid sequences of
L. pneumophila pilEL. Potential ribosome
binding sites are underlined; potential sites for the start of
translation are in boldface. The internal EcoRI site, which
is the site of the kanamycin gene insertion in BS100, is indicated by
the diamond. The position of cleavage of the leader peptide in type IV
pilin genes is indicated by the arrowhead.
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The open reading frame was compared to sequences deposited in GenBank
for identification of similar translated sequences. An area of the
amino-terminal region of the translated sequence was found to
have 38% identity and 71% similarity to the amino-terminal region of PilE from N. gonorrhoeae and 33% identity
and 54% similarity to PilA from P. aeruginosa
(strain PAK) (Fig. 3). Comparable levels of similarity were seen for other members of the type IV pilin family
of proteins as well (Fig. 3 and data not shown). The open reading frame
was then designated pilEL in L. pneumophila.
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FIG. 3.
Alignment of the deduced PilEL sequence with
those of type IV pilin proteins. The arrowhead indicates the site of
cleavage of the leader peptide between the conserved glycine and
phenylalanine in type IV pilin genes. PilA is the type IV pilin gene in
P. aeruginosa, PilE is in N. gonorrhoeae, and TfpQ is in M. bovis. Amino acids
homologous among all four proteins are in boldface.
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Type IV pili have been found in a number of pathogenic bacteria
including N. gonorrhoeae, Neisseria meningitidis,
M. bovis, P. aeruginosa,
Dichelobacter nodosus, Vibrio cholerae, and
enteropathogenic E. coli (49). Type IV pilins
have a number of common characteristics including homology at the amino
termini of the proteins. However, the pili can be separated into two
distinct groups based upon variations in this same region
(49). Group A prepilin proteins (found in N. gonorrhoeae, N. meningitidis, M. bovis, P. aeruginosa, and D. nodosus)
are cleaved between a glycine and a phenylalanine residue to remove the
prepilin leader sequence. The resulting mature pilin is N
methylated prior to assembly of mature pili on the bacterial surface
(25, 49). The group B pili (found in V. cholerae
and enteropathogenic E. coli) are proteolytically cleaved
between a glycine and a methionine or a leucine. The group B pili also
contain a longer leader sequence than the group A pili. In all cases,
an invariant glutamic acid is found five amino acid residues from the
amino terminus of the mature protein. In pilEL of L. pneumophila, the
phenylalanine is present at the appropriate position. However, the
conserved glycine residue found at the cleavage site of many type IV
pilins is not present in pilEL but has been
replaced by alanine.
The amino-terminal domain of type IV pili is hydrophobic and contains
an invariant glutamic acid residue five amino acids from the mature
amino terminus of the protein. The L. pneumophila PilEL amino-terminal sequence is also hydrophobic and
contains the invariant glutamic acid five amino acid residues from the putative cleavage site. The length of the pilin proteins with which
PilEL has similarity is also conserved. The type IV pilin proteins are approximately 150 to 170 amino acids in length, while the
longest possible PilEL open reading frame predicts
translation of a protein product 149 amino acids in length.
Based upon sequence homology, the L. pneumophila
pilEL gene is a member of the type IV prepilin genes.
The putative leader sequence precedes a phenylalanine, as is found in
the group A pili, but lacks the glycine present in all other members of
both group A and group B. The glycine has been replaced with an
alanine; both amino acids are neutral, polar, and similar in structure (alanine contains an extra methyl group). It is not known at this time
whether the replacement has any effect on the potential processing of
PilEL. However, replacement of the glycine at the leader
sequence cleavage site of P. aeruginosa pilA has been
studied in some detail (48). Among a number of replacements
of glycine, only replacement with alanine resulted in processing of
PilA into the mature pilin protein product. Therefore, on account of
the degree of homology of pilEL to various
members of the type IV prepilin gene family, including the presence of
the phenylalanine, the conservative replacement of the glycine with an
alanine at the putative cleavage site of the leader sequence, and the
presence of the invariant glutamic acid in the sequence, we propose
that the group A type IV prepilins be expanded to include
pilEL.
In addition to the pilEL locus, an
L. pneumophila locus which contains open reading frames
encoding homologs of P. aeruginosa PilB, PilC, and
PilD, which are required for type IV pilin processing and assembly and
type II protein secretion, has been described (31). Southern
analysis indicates that the two type IV pilin loci are unique, as
hybridization under low-stringency conditions does not reveal binding
of the pilBCD locus to cosmid or plasmid DNA containing
pilEL (data not shown). Because multiple
unique cosmids containing the pilEL locus
were analyzed, this data also indicates that the loci are not located
adjacent to each other. Whether the pilEL
and pilBCD loci act together to assemble a pilus structure is not known at this time.
Generation of an L. pneumophila pilEL
mutant.
A pilEL isogenic mutant
was generated in L. pneumophila AA100 by the insertion
of a Kanr cassette at the unique EcoRI site
(Fig. 2) in the pilEL open reading frame.
Plasmid pBJ114 is a derivative of pBJ120 in which a Kanr
marker was inserted into an internal EcoRI site within
pilEL. The Kanr gene was derived
from pUC-4K (Pharmacia Biotech, Piscataway, N.J.). The
plasmid pBOC20 contains a chloramphenicol resistance marker as
well as a sucrose sensitivity marker and has been previously described (6). The plasmid pBJ115 (derived from pBOC20) is a
shuttle vector designed to deliver the
pilEL::Kan mutation to the
L. pneumophila chromosome. Homologous recombination of
the mutation onto the chromosome generated strain BS100, as confirmed by Southern analysis (data not shown).
Pilus expression in a pilEL mutant.
To
confirm that pilEL was involved in
expression of pili, the wild-type strain AA100 and the
pilEL mutant BS100 were compared by
transmission electron microscopy. As described above, AA100 was found
to express pili of various lengths designated short or long. BS100
expressed only the shorter pili (Fig.
4C). Upon examination of BS100 harboring
a cosmid containing the pilEL locus (pCC3),
long pili were again displayed (Fig. 4D), at the same frequency as long
pili expressed by AA100. No long pili were expressed from BS100
containing the cosmid vector alone (data not shown). The
appearance of pili was documented for 50 to 100 bacteria for AA100,
BS100, and BS100(pCC3). Thus, the pilEL
locus is required for expression of the long pili on the surface of
L. pneumophila.
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FIG. 4.
Comparison of pili expressed in L. pneumophila AA100 and in the pilEL
mutant. L. pneumophila wild-type strain AA100 expresses
short (A) and long (B) pili. The pilEL
mutant strain BS100 expresses only short pili (C). Cosmid pCC3, which
contains the pilEL locus, is able to
reintroduce expression of long pili to BS100 (D). Pili are indicated by
small arrowheads; flagella are indicated by large arrowheads. Bars, 0.5 µm.
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L. pneumophila AA100 expressed both long and short pili
but not simultaneously. Expression of pili may be regulated by
conditions which allow expression of only one subset of pili at a time,
such as growth phase and/or phase variation. The
pilEL mutant, however, exhibited expression
of only the short pili. Expression of long pili by the mutant was
complemented by a cosmid carrying pilEL. Because the pilin mutant was generated by insertion of a resistance marker, the mutation may have caused polar effects downstream of the
pilEL gene. It is also possible that the
mutation has interrupted a gene which affects the length of pili by
either destabilizing the pilus structure or affecting secretion of
pilus structural components. However, based upon sequence homology with
other type IV pilin genes, pilEL is most
likely a type IV pilin structural gene.
Intracellular replication of the pilEL
mutant in mammalian and protozoan cells.
The phenotype of the
pilEL mutant during an intracellular
infection was determined in various cell culture systems and is summarized in Table 1. Intracellular
replication assays for L. pneumophila within mammalian
cells were performed. The macrophage-like cell line U937 was
maintained, and infections were performed, as described elsewhere
(18) with the following exceptions. Prior to infection, U937
cells were treated with phorbol 12-myristate 13-acetate for 48 h,
as described previously (3), to differentiate the cells into
nonreplicative, adherent, macrophage-like cells. For all infection
assays, L. pneumophila strains were grown for 48 h
on buffered charcoal yeast extract agar plates and resuspended in
tissue culture medium prior to infection. Intracellular replication assays were performed in triplicate with a multiplicity of infection (MOI) of 10 for 1 h and allowed to proceed for 0, 2, 24, or
48 h. Assays measuring replication kinetics of infection within
the macrophage-like cell line U937 revealed no defect for the
pilEL mutant BS100 compared to the wild-type
strain AA100.
The cytopathogenicity of L. pneumophila BS100 was also
determined. Cytopathogenicity assays were performed in triplicate with an MOI of 0.1. After the addition of bacteria to the U937 cell monolayer, the infection was allowed to proceed for 48 h. The supernatant from each well was removed, and Alamar Blue (AccuMed, Westlake, Ohio) was added according to the manufacturer's suggestions to determine the viability of the monolayer relative to uninfected control wells, as previously described (6). The level of
cytopathogenicity of BS100 was identical to that of AA100 for U937
cells (data not shown). BS100 was also able to replicate as efficiently
as AA100 in HeLa cells, an epithelial cell-derived cervical carcinoma
cell line. HeLa cells were maintained as described previously
(20).
Additionally, the growth kinetics of an intracellular infection of
BS100 within the protozoan Acanthamoeba polyphaga were determined as described elsewhere (18). Intracellular
replication assays were performed in triplicate with 105
A. polyphaga cells/ml and an MOI of 0.01. After addition of
bacteria to the monolayer, the infection was allowed to proceed for 0, 24, 48, and 72 h. Intracellular infections of the protozoan
A. polyphaga with BS100 were determined to be identical to
that with the wild-type strain AA100. Therefore, the
pilEL locus was not required for
intracellular survival or replication within mammalian macrophage
cells, epithelial cells, or the protozoan host A. polyphaga.
Adherence of the pilEL mutant to mammalian
and protozoan cells.
Because pili are known to be involved in
adherence of bacteria to mammalian cells (49), the
requirement of pilEL for attachment of
L. pneumophila to mammalian and protozoan cells was
investigated. Attachment to U937 macrophage-like cells and two
different epithelial cell lines (HeLa cells and WI-26 cells) was
tested. WI-26 VA4 cells (ATCC CCL-95.1) are derived from human
embryonic epithelial lung tissue and are a type I pulmonary epithelial
cell line. WI-26 cells were maintained in minimum essential medium with
Earle's salts, L-glutamine, and nonessential amino acids
(Gibco BRL) with 10% heat inactivated fetal bovine serum at 37°C
with 5% CO2. Attachment assays were performed with
L. pneumophila strains grown as described above and
also grown for 4 days without shaking to replicate growth conditions prior to examination by electron microscopy.
Attachment assays were performed in triplicate in 48-well tissue
culture plates with 5 × 105 tissue culture cells/ml
at an MOI of 20. Pretreatment of the monolayer with 1 µg of
cytochalasin D (Calbiochem, La Jolla, Calif.) per ml was performed to
inhibit bacterial uptake as previously described (18).
Bacteria were centrifuged with the monolayer at 1,000 × g for 5 min and allowed to attach for 20 min at 37°C and
5% CO2. The monolayer was washed five times with tissue
culture medium to remove nonadherent bacteria. To collect bacteria for enumeration, the monolayer was lysed by the addition of sterile water
and bacteria were diluted in sterile water and plated on buffered
charcoal yeast extract agar plates. The degree of attachment was
expressed as percent attachment and was calculated as follows: (CFU of
attached bacteria per milliliter 
CFU of added bacteria per
milliliter) × 100. The percent wild-type attachment was calculated as follows: (percent attachment of the mutant strain percent attachment of the wild-type strain) × 100.
The pilEL mutant exhibited a decrease in
attachment to both HeLa cells ([34.0 ± 20.1]% of wild-type
attachment) and WI-26 cells ([48.7 ± 30.1]% of wild-type
attachment) relative to that by AA100 (summarized in Table 1).
Attachment assays were performed with L. pneumophila
strains grown under two different growth conditions: those routinely
used for growth of cultures prior to infection assays and those used
for growth of L. pneumophila prior to electron microscopic examination for pili. Alteration of the growth conditions resulted in the same decrease in attachment for the
pilEL mutant. Adherence to the
macrophage-like cell line U937 was also decreased for the
pilEL mutant ([33.9 ± 16.0]% of
wild-type attachment) (Table 1).
The ability of the wild-type strain and mutant strain to adhere to
protozoan cells was determined. Attachment assays were performed in
assay buffer in triplicate at an MOI of 20 in 48-well tissue culture
trays (18, 19). Bacteria were centrifuged at 1,000 × g for 5 min and allowed to attach for 20 min at 37°C. The
monolayer was washed five times with assay buffer to remove nonadherent
bacteria. The monolayer was lysed by the addition of 0.05% Triton
X-100 (Sigma) as previously described (18). Bacterial
enumeration was determined as described above. Adherence of
L. pneumophila to A. polyphaga was assessed,
and a decrease in attachment of the pilEL
mutant was detected (Table 1). Pretreatment of the monolayer with 10 µM methylamine (Sigma) for 30 min to inhibit bacterial uptake by
A. polyphaga had no effect on the difference between the
wild type and the mutant for attachment. Therefore, since L. pneumophila is defective in attachment to mammalian and protozoan
cells, the pili may represent an adherence factor that binds a common
host cell component.
Prevalence of pilEL in
Legionella.
The occurrence of
pilEL among L. pneumophila serogroups and Legionella species
was examined by Southern hybridization analysis (Table
2). L. pneumophila
serogroup strains and Legionella species subjected to
Southern analysis listed in Table 2 were kindly provided by R. Benson,
B. Fields, and J. Pruckler from the Centers for Disease Control
and Prevention. Digestion of chromosomal DNA was performed with
ClaI, as the pilEL sequence in
L. pneumophila AA100 does not contain an internal
ClaI restriction site. The 2-kb ClaI fragment
from pBJ120 was used as a probe to identify the
pilEL locus. Labeling of DNA probes and
Southern hybridizations were performed as described previously
(5). High-stringency hybridization and washes were performed
at 60°C; low-stringency hybridization and washes were performed at
45°C.
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TABLE 2.
Legionella species and L. pneumophila serogroups and strains characterized by Southern
analysis for the presence of the
pilEL locus
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Southern analysis confirmed the presence of one DNA fragment
hybridizing to the pilEL locus under
high-stringency conditions in each of the 13 serogroups tested (Table
2). Hybridizing bands from a ClaI digest of chromosomal DNA
ranged in size from 2 to 6 kb (data not shown). Multiple strains of
L. pneumophila serogroup 1 were also tested and found
to be consistent with the serogroup data (Table 2 and data not shown).
Southern hybridization was performed on chromosomal DNA from various
Legionella species at low stringency to detect the presence
of pilEL (Fig.
5). Hybridization was observed for
L. wadsworthii, L. oakridgensis,
L. santicrucis, L. cherrii, and
L. parisiensis. Multiple bands were observed for each,
which may be due to multiple copies of the locus or the introduction of
an internal ClaI site within the
pilEL locus in these species. Southern
hybridization analysis of chromosomal DNA from Legionella
species performed under high-stringency conditions detected no
hybridization with pilEL; thus, the DNA
sequences of the pilEL loci in different
Legionella species are not identical.
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FIG. 5.
Presence of pilEL in
Legionella species. Chromosomal DNA from different
Legionella species was subjected to Southern analysis under
low-stringency conditions and probed with the
pilEL locus. L. pneumophila
AA100 chromosomal DNA hybridizes with a single 2-kb fragment
(arrowhead) under high-stringency conditions. Lanes 1 to 17, L. pneumophila, L. dumoffi,
L. longbeachae, L. gormanii,
L. micdadei, L. wadsworthii,
L. oakridgensis, L. feeleii,
L. sainthelensis, L. spiritensis,
L. jamestowniensis, L. santicrucis,
L. cherrii, L. parisiensis,
L. erythra, L. hackeliae, and
L. israelensis, respectively.
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In conclusion, the advantage that pilEL
confers on L. pneumophila may be related to attachment
of the bacteria to epithelial cells or macrophages during infection of
a human host. The pilin mutant showed an ~50% decrease in attachment
to human macrophages and epithelial cells. As multiple adhesion factors
are not uncommon in pathogenic bacteria and the role of
pilEL in attachment is only moderate, the
presence of other adhesion proteins required for attachment is likely.
Recently, multiple mutants of L. pneumophila which are
defective for attachment to both mammalian cells (18-20) and protozoa (24) have been described. As all the reported
mutants are defective for cytopathogenicity or replication within
macrophages, none are likely to have mutations in the
pilEL locus. Whether the long pili are the
ligand that allows bacterial attachment to the protozoan lectin
receptor (24, 51) has yet to be determined.
Another possible role for pilEL may include
enhanced survival in the environment. For example, expression of
pilEL increases adhesion to protozoan host
cells and may increase adherence to biofilms where L. pneumophila has been found in an aquatic environment (10). Additionally, expression under conditions
encountered during intracellular growth, either within
mammalian cells or within protozoa, has not been studied. However,
L. pneumophila is more invasive of mammalian cells
after intracellular replication within amoebae (13), and
intracellular specific expression of L. pneumophila
proteins has been observed during macrophage infection (2-6,
50). If increased expression of pilEL
occurs during intracellular growth within a mammalian or protozoan host
cell, an increase in attachment to or invasiveness of mammalian cells
may be partially attributed to pilus expression.
The type IV pilin structural protein gene (pilE) and a
gene required for pilus biogenesis (pilC) of
N. gonorrhoeae are required for natural DNA
transformation competence (11, 21, 43, 46, 52). Therefore,
the potential role for pilEL in competence
is being studied. The ability of L. pneumophila to
become competent for transformation of DNA and the dependence of the
competence phenotype upon pilEL have
recently been discovered (47). Thus, we propose that the
long pili described in this paper be termed CAP (competence- and
adherence-associated pili).
Nucleotide sequence accession number.
The nucleotide sequence
accession number is AF048690.
We thank M. R. Liles, V. K. Viswanathan, and
N. P. Cianciotto for allowing use of the
pilBCD clone prior to publication. We also thank Omar
Harb, Lian-Yong Gao, and Matthew Nilles for critical review of the
manuscript.
Y.A. is supported by Public Health Service grant 1R29AI38410. B.J.S. is
a recipient of Public Health Service National Research Service Award
TA-09509.
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