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Infection and Immunity, July 1999, p. 3662-3666, Vol. 67, No. 7
0019-9567/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.
Legionella pneumophila Contains a Type II General
Secretion Pathway Required for Growth in Amoebae as Well as
for Secretion of the Msp Protease
Laura M.
Hales, and
Howard A.
Shuman*
Department of Microbiology, College of
Physicians and Surgeons, Columbia University, New York, New York
10032
Received 11 December 1998/Returned for modification 18 January
1999/Accepted 19 April 1999
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ABSTRACT |
We report the identification of a set of Legionella
pneumophila genes that encode products with homology to proteins
of the type II general secretion pathway of gram-negative bacteria. A strain containing a deletion-substitution mutation of two of these genes was unable to secrete the Msp protease. This strain was unable to
multiply within the free-living amoeba Acanthamoeba castellanii yet was able to kill HL-60-derived macrophages.
Because Msp is not required for growth in amoebae, other proteins which are important for growth in amoebae are likely secreted by this pathway.
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TEXT |
Legionella pneumophila is
the gram-negative facultative intracellular pathogen responsible
for Legionnaires' disease. L. pneumophila is
able to infect and multiply within a variety of eukaryotic hosts,
including human mononuclear phagocytes, and a wide variety of protozoa
including the free-living amoeba Acanthamoeba castellanii. The bacteria are phagocytosed via a unique coiling mechanism and reside
in a specialized phagosome that does not acidify or fuse with
lysosomes. Following replication, the host cell lyses and the bacteria
are released and are able to initiate a new infection cycle (for
reviews, see references 1, 18, 46, and
47).
Identification of the lspFGHIJK genes.
As part of
an effort to identify regulatory proteins of L. pneumophila, we attempted to complement a mutant gene product from Escherichia coli (20, 21). Maintenance and growth
of E. coli and L. pneumophila and all DNA
manipulations were carried out as described previously
(39). A library of EcoRI-digested genomic DNA of
L. pneumophila Philadelphia-1 cloned into the vector
pMMB207 (31, 39) was used in a complementation screen.
DNA sequencing of the vector-L. pneumophila genomic DNA
junctions of a particular clone (plasmid pLM511) revealed homology to
the DNA sequence encoding xcpS (gspF).
The xcp genes encode proteins whose products function in the
main terminal branch (MTB) of the general secretion pathway (GSP) of
Pseudomonas aeruginosa (3). The GSP is a type II
protein secretion pathway that is highly conserved among gram-negative bacteria and was first described by Pugsley et al. 14,
15; for reviews, see references 34, 37,
38, and 42). Proteins secreted by
the GSP have an initial sec-dependent step for export across
the inner membrane. The proteins are then transported across the
outer membrane via an apparatus consisting of the protein products of
12 to 15 genes. A well-studied paradigm for this pathway is the
pullulanase secretion system of Klebsiella oxytoca
(37).
Plasmid pLM511 contains a single
EcoRI fragment
of
L. pneumophila DNA, which is 4,279 bp in
length. Sequence from both strands
of DNA was generated (DNA Synthesis
and Sequencing Facility of
the Comprehensive Cancer Center, College of
Physicians and Surgeons
of Columbia University). There are two partial
open reading frames
and five complete open reading frames contained on
the fragment
(Fig.
1). Because of the
homology to the GSP family of proteins,
we named the open reading
frames
lsp for
Legionella secretion
pathway. The
open reading frames containing homology to the GSP
family of proteins
were named
lspFGHIJK, corresponding to the
nomenclature of
the
pul operon homologs (
37,
38,
42).
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FIG. 1.
Schematic diagram of open reading frames (indicated by
arrows) encoded on pLM511 and construction of a deletion-substitution
that inactivates the lspGH genes. The orientation of the
arrowheads denotes the direction of transcription. The EcoRI
fragment contained in pLM511 is 4,279 bp in length and contains the
complete DNA sequence encoding the lspGHIJK genes and the
partial DNA sequence encoding the lspF and orf1
genes. For construction of a mutation in the lspGH genes, an
internal 587-bp NcoI fragment was deleted (indicated by  )
and a 2,118-bp gentamicin resistance cassette (hatched box) was
inserted in its place.
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Although some of the GSP family of proteins contain an additional two
to four genes downstream of
lspK (homologs of
pulLMNO),
the
L. pneumophila lsp operon
appears to end with
lspK. No open
reading frames encoding
homologs of other proteins in this family
were found on the contiguous
1 kb of DNA 3' to
lspK. A partial
open reading frame
(
orf1) is encoded by the downstream region,
but
orf1 contains no homology to sequences in the databases. At
this time, we cannot rule out the existence of an additional unlinked
region encoding proteins with homology to
pulLMNO located
elsewhere
on the
L. pneumophila chromosome. However, in
all other examples
of genes encoding proteins in the GSP family, the
genes are adjacent
and in essentially the same order on the
chromosome.
The
lspFGHIJK genes encode proteins with significant
homology to the gene products of the respective
xcp family
members (Table
1). Consistent with the
cognate GSP homologs, each of the LspGHIJK
proteins contains a putative
signal sequence, and the predicted
localization for all the
protein products is in the inner membrane
or periplasmic space. The
signal sequence consensus site contained
in the XcpTUVWX proteins for
cleavage by XcpA/PilD (GFXXXE [
11,
32]; also see
reference [
35]) is present in LspGHIJ (Table
1).
Additionally, the LspGHIK proteins are devoid of cysteines,
a trait
common among GSP family members (
2).
Construction of a strain containing a mutation in the
lspGH genes.
To help identify protein products
secreted by the L. pneumophila GSP, we
constructed a mutation in the lspGH genes. The 4,279-bp EcoRI fragment from pLM511 was subcloned into pBR322 to
generate pLM569. Plasmid pLM569 was digested with NcoI to
remove an internal 587-bp fragment (Fig. 1). The larger 3,692-bp
NcoI fragment containing the vector sequences was treated
with Klenow enzyme, and a ligation was performed between the
Klenow-treated fragment and a 2,118-bp HincII DNA fragment
encoding gentamicin resistance (a gift from David Figurski). This
resulted in plasmid pLM808 containing an gentamicin resistance cassette
inserted within the lspGH-coding region (Fig. 1).
Because the coding regions for the lspGHIJK genes overlap, such an insertion would likely be polar on the
lspIJK genes and would therefore represent a null
phenotype of the L. pneumophila GSP.
The 5,232-bp
EcoRV fragment containing
lspFGH::Gent
rIJKorf1
from plasmid pLM808 was subcloned into the
EcoRV site of the
vector
pLAW344 for allelic exchange (
50). The
resultant plasmid, pLM826,
was electroporated into the wild-type
strain
L. pneumophila JR32.
Allelic exchange of
the
lspGH::Gent
r mutation onto the
chromosome of JR32 was performed as described
previously
(
50) and generated strain LM1520. Southern blot analysis
confirmed the construction (data not
shown).
A complementing plasmid, pLM828, was constructed by cloning
the original 4,279-bp
EcoRI fragment containing the
lspFGHIJKorf1 genes from pLM511 into the vector
pMMB207
c (a
mobA Kan
s derivative of
pMMB207
b-Km-14 [
45]). Plasmid pLM828 was
electroporated
into strain LM1520, resulting in strain LM1559. Strain
LM1558
is strain LM1520 containing the vector pMMB207
c.
Identification of a protein secreted by the L. pneumophila GSP.
We were next interested in identifying a
protein secreted by this system. A prime candidate is the major
secretory protein (Msp) of L. pneumophila. Msp is a
38-kDa Zn2+ metalloprotease with caseinolytic and
hemolytic activities and is the most abundant protein found in culture
supernatants (17, 24). Msp contains homology to elastase, a
Zn2+ metalloprotease which is secreted by the
xcp-encoded GSP of P. aeruginosa (5,
25).
A simple test for the extracellular proteolytic activity of a
msp+ strain is a ring of casein hydrolysis
around a patch of wild-type
L. pneumophila
organisms grown on a agar plate containing casein
(
48). We
tested the ability of the strain containing the mutation
in
lspGH to hydrolyze casein. Strains LM1558 and LM1559 were
patched
onto buffered yeast starch extract (BYSE) medium containing
10
g of casein per liter as described previously (
48).
As a control,
we patched the wild-type strain JR32 onto the same
plate. We also
patched strains LS2102 (
L. pneumophila
mspA1::Tn
9) and its cognate
wild-type parent
LS2029 (
48) as Msp
and Msp
+
controls,
respectively.
A ring of hydrolysis was observed around the patch of JR32 growth but
not around the patch of LM1558 growth (Fig.
2). This
result indicates that the strain
LM1558 cannot hydrolyze casein.
Because the caseinolytic activity of
Msp accounts for virtually
all of the proteolytic activity of
L. pneumophila (
13,
48),
we conclude
that strain LM1558 is defective in the secretion of
Msp. Plasmid
pLM828 containing the wild-type
lspFGHIJK genes
(strain
LM1559) is able to complement the inability to hydrolyze casein
(Fig.
2). This result provides evidence that the Msp
phenotype of strain LM1558 is due to the loss of the
L. pneumophila GSP and not to an extraneous
mutation elsewhere in the genome.
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FIG. 2.
Casein hydrolysis of the strain containing a mutation in
the lspGH genes and the complemented strain. Various strains
were patched onto a BYSE agar plate containing casein and incubated for
3 days at 37°C as described previously (48). Patches: A,
Wild-type strain JR32; B, wild-type strain LS2029 (isogenic to LS2102);
C, msp mutant strain LS2102 (LS2029
mspA1::Tn9); D, LM1558 (JR32
lspGH::Gentr
pMMB207c::lspFGHIJKorf1); E, LM1559 (JR32
lspGH::Gentr pMMB207c).
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To confirm the results obtained in the casein hydrolysis
experiment, sodium dodecyl sulfate-polyacrylamide gel electrophoresis
(SDS-PAGE) was performed to analyze the presence of Msp in
L. pneumophila culture supernatants. Cell culture
supernatants of
the wild-type strain LS2029 and the Msp
strain LS2102 were analyzed for the presence of Msp, and the
results
compared to cell culture supernatants of strains JR32,
LM1558, and
LM1559. No Msp was observed in the culture supernatant
of strain
LS2102, as predicted (Fig.
3A). The
culture supernatant
from strain LM1558 contains low levels of Msp
activity compared
to that in the supernatant of the wild-type strain
JR32 (Fig.
3A). Plasmid pLM828 containing the wild-type
lspFGHIJK genes (strain
LM1559) is able to complement the
inability to secrete Msp into
the culture supernatant (Fig.
3A). Taken
together with the results
from the casein hydrolysis experiment, these
results confirm that
Msp is a substrate for the
L. pneumophila GSP. The fact that Msp
requires secretion by the GSP
confirms the functionality of the
system in
L. pneumophila. It has been shown that wild-type
E. coli
possesses a complete GSP operon, but this operon is not expressed
during growth under laboratory conditions (
19,
36).
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FIG. 3.
SDS-PAGE analysis of L. pneumophila
culture supernatants. Supernatants of L. pneumophila
overnight cultures were filtered through a 0.45-µm filter
(Millipore), and the proteins from the supernatant were precipitated in
10% trichloroacetic acid. SDS loading buffer was added to the protein
pellet, and an aliquot was electrophoresed on a 12% polyacrylamide
gel. The proteins were visualized by Commassie blue staining. Molecular
mass markers (in kilodaltons) are indicated on the left. (A) Lanes: 1, wild-type strain JR32; 2, wild-type strain LS2029 (isogenic to LS2102);
3, msp mutant strain LS2102 (LS2029
mspA1::Tn9); 4, LM1558 (JR32
lspGH::Gentr pMMB207c); 5, LM1559
(JR32 lspGH::Gentr
pMMB207c::lspFGHIJKorf1). (B) Culture
supernatants of Lsp (LM1558) and Lsp+
(LM1559) strains. The three proteins which are present in
Lsp+ but not in Lsp culture supernatants,
with approximate molecular masses of 74, 49, and 36 kDa, are indicated
on the right.
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During this analysis, we noticed that the protein profiles of the
culture supernatants of the Lsp
and Lsp
+
strains differed significantly (Fig.
3B). The proteins from an
SDS-PAGE
gel were electroblotted onto a polyvinyl difluoride
membrane
(Millipore), and three bands (p74, p49, and p36) were
excised
from the membrane. The samples were subjected to N-terminal
sequencing
for 8 cycles each (Protein Chemistry Core Facility, Columbia
University).
In this manner, the N-terminal amino acid sequences were
obtained
for p74 (AQPTACVN), p49 (YYTSQGSI), and p36 (KDVYEIKH). The
sequence
databases do not contain any proteins with homology to these
three
amino acid sequences. The proteins p74, p49, and p36 represent
examples of additional proteins that are likely secreted by the
L. pneumophila GSP (Fig.
3B).
Analysis of the intracellular growth phenotype of the strain
containing a mutation in the lsp genes.
We were then
interested in examining the ability of the strain containing a mutation
in the lspGH genes to replicate within eukaryotic hosts. We
first tested the ability of strain LM1520 for cytotoxicity of
HL-60-derived macrophages. The assay was performed as described
previously (27, 28). The cytotoxicity of strain LM1520 was
compared with the results obtained from the L. pneumophila wild-type strain, JR32, and the mutant strain 25D
(22). Strain LM1520 was able to kill macrophages in a manner
identical to that of wild-type strain JR32 (data not shown). This
finding indicates that the putative secretion system encoded by the
lsp operon, or a protein secreted by it, is not required for
killing of a macrophage-like cell line.
L. pneumophila also has the ability to multiply
intracellularly within the free-living amoeba
A. castellanii
(
12,
30,
40,
41). Therefore, strain LM1558 was tested for
its ability
to replicate within amoebae. Growth and maintenance of
A. castellanii was carried out as described previously
(
12,
30). The assay
for replication within amoebae was based
on previously described
methods (
12,
30).
L. pneumophila at a multiplicity of infection
(MOI) of 10 was added
to an adherent monolayer of 1.2 × 10
5 amoebae. After
incubation for 30 min at 37°C to allow for infection,
the wells were
washed three times with 0.5 ml of
Acanthamoeba medium buffer
to remove extracellular bacteria. A sample of the
infection supernatant
was removed once every 24 h for 4 days.
Colony forming units
(CFUs) of extracellular bacteria were quantitated
on ACES-buffered
charcoal yeast extract (ABCYE) plates. Wild-type
strain JR32
replicated 10
4-fold within 72 h while the mutant
strain 25D did not (Fig.
4A).
Strain
LM1558 containing a mutation in the
lspGH genes is clearly
defective for replication within amoebae. The 4,279-bp
EcoRI fragment
was able to complement the growth
defect in
A. castellanii (Fig.
4A). Therefore, either
the secretion apparatus itself or, more
likely, another protein
that is secreted by this system is required
for replication within
protozoa. In order to rule out Msp as this
protein, we tested the
ability of strains LS2029 (Msp
+) and LS2102
(Msp
) to replicate within
A. castellanii.
The results show that both
strains replicate approximately
10
4-fold in amoebae (Fig.
4B). This result indicates that
the growth
defect of strain LM1558 in amoebae is not due to the
inability
of this strain to secrete Msp and provides evidence for the
existence
of other secreted proteins that are important for growth in
amoebae.
An alternative explanation for our results is that the
deletion-substitution
mutation is polar on expression of
orf1, and it is the
orf1 gene
product that is
required for growth within amoebae. However, we
do not believe that
orf1 contributes to this phenotype because
the open reading
frames of the
lspGHIJK genes overlap and there
is
considerable distance between the end of
lspK and the
beginning
of
orf1 (314 bp). Additionally,
orf1
does not have homology to
genes in the GSP family, further suggesting
that it is not a member
of this operon. Therefore, we believe that it
is unlikely that
a mutation in
lspGH would affect the
function, if any, of
orf1.
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FIG. 4.
Intracellular growth of strains in A. castellanii. The net log growth of the various strains is plotted
as a function of time. Error bars represent standard deviation and may
not be visible. (A) Wild-type L. pneumophila strain
JR32 (open squares) and the mutant 25D (closed squares) are the
controls. Strain LM1558 (open circles) is strain LM1520 (JR32
lspGH::Gentr) containing the vector
pMMB207c. Strain LM1559 (closed circles) is strain LM1520 containing
the plasmid pMMB207c::lspFGHIJKorf1. (B)
Wild-type L. pneumophila strain LS2029 (open squares)
and the isogenic msp mutant strain LS2102 (filled
squares).
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In summary, our analysis shows that the
L. pneumophila
GSP or, a protein secreted by it, is required for growth within
amoebae.
Many of the proteins secreted by the GSP of gram-negative
bacteria
play a role in pathogenesis, as many of the organisms from
which
these components originate are plant or human pathogens. Our
evidence
indicates that Msp is secreted by the
L. pneumophila GSP. Indeed,
it was postulated that Msp might be a
virulence factor (
4,
6-10,
13,
29,
48). However, Msp is not
required for
L. pneumophila to either kill macrophages
(
29,
48) or multiply within
A. castellanii
(reference
29 and this work). Therefore, it is
likely
that one or more proteins other than Msp secreted by the
L. pneumophila GSP are required for replication within
A. castellanii. Several
organisms that have a GSP secrete
more than one protein, and
L. pneumophila secretes many
other exoenzymes, including acid and
alkaline phosphatases and lipases
(
16). Further work is needed
to identify other proteins that
are secreted by the GSP and to
determine the precise requirements
for replication within
A. castellanii.
The observation that
a set of genes (encoding the
L. pneumophila GSP)
is absolutely required for growth within
A. castellanii but
not in a macrophage-like cell line supports the protozoan host
as a
more restrictive model of
L. pneumophila intracellular
growth.
It has been shown previously that
L. pneumophila
possesses another type II secretion system involved in type IV pilus
biogenesis.
Liles et al. (
26) reported the identification of
the
L. pneumophila homologs of the
P. aeruginosa
pilBCD genes. We note that the
lsp operon reported
herein does not encode the homolog of the prepilin
peptidase, PulO,
that is required for the processing of the signal
sequences
present on several of the Pul proteins in
K. oxytoca (
33). The
xcp operon of
P. aeruginosa also lacks the O homolog
(
3; also
see references
23 and
43).
In the
xcp system,
the prepilin peptidase PilD (of the pilus
biogenesis system) substitutes
for the function of the missing O
protein in the
xcp system, in
effect performing the
signal peptide cleavage and modification
processes for both the
pil and
xcp gene products (
3,
32).
Unless
L. pneumophila encodes an as-yet-undiscovered
unlinked
O homolog, the
L. pneumophila pilD
gene product (
26) may function
for both pilus biogenesis
(
pil) and the GSP (
lsp) in the same
manner as
that observed for
P. aeruginosa. It remains to be
determined
if the
pilBCD system is required for the
pathogenesis of
L. pneumophila.
The
dot-icm
gene products are proposed to function as a novel
secretion system that
is required for inhibition of phagosome-lysosome
fusion and for
intracellular multiplication (
44,
49).
L. pneumophila is not unique in this aspect as multiple secretion
systems have
also been found in other prokaryotes, most notably
bacterial pathogens.
Therefore, it seems likely that
L. pneumophila is typical in its
acquisition of some common types of
secretion systems for use
in pathogenesis in eukaryotic
hosts.
Nucleotide sequence accession number.
The L. pneumophila lspFGHIJK sequence has been deposited in the GenBank
database under accession no. AF111940.
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ACKNOWLEDGMENTS |
We thank Tony Pugsley for insightful discussions; Pat Higgins for
E. coli NH757, the strain that was used in the
complementation experiment; David Figurski for the gentamicin
resistance cassette; and Carmen Rodriguez for laboratory maintenance.
L.M.H. was supported in part by NIH training grant AI-07161 and by
NRSA grant AI-09718. This work was supported by NIH grant AI-23549 to
H.A.S.
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ADDENDUM IN PROOF |
Liles et al. (M. R. Liles, P. H. Edelstein, and N. P. Cianciott,
Mol. Microbiol. 31:959-970, 1999) recently showed that a
pilD mutant strain is defective in the secretion of Msp, which further supports our hypothesis that PilD functions as the prepilin peptidase in the Legionella pneumophila GSP.
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FOOTNOTES |
*
Corresponding author. Mailing address: Department of
Microbiology, College of Physicians and Surgeons, Columbia University, 701 West 168th St., New York, NY 10032. Phone: (212) 305-6913. Fax:
(212) 305-1468. E-mail:
shuman{at}cuccfa.ccc.columbia.edu.
Editor:
P. J. Sansonetti
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Infection and Immunity, July 1999, p. 3662-3666, Vol. 67, No. 7
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Copyright © 1999, American Society for Microbiology. All rights reserved.
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