Multiple substrates of the Legionella pneumophila Dot/Icm system identified by interbacterial protein transfer

Abstract

Legionella pneumophila is an intracellular pathogen that multiplies in a specialized vacuole within host cells. Biogenesis of this vacuole requires the Dot/Icm type IV protein translocation system. By using a Cre/loxP-based protein translocation assay, we found that proteins translocated by the Dot/Icm complex across the host phagosomal membrane can also be transferred from one bacterial cell to another. The flexibility of this system allowed the identification of several families of proteins translocated by the Dot/Icm complex. When analyzed by immunofluorescence microscopy, a protein identified by this procedure, SidC, was shown to translocate across the phagosomal membranes to the cytoplasmic face of the L. pneumophila phagosome. The identification of large numbers of these substrates, and the fact that the absence of any one substrate rarely results in strong defects in intracellular growth, indicate that there is significant functional redundancy among the Dot/Icm translocation targets.

• bacterial pathogenesis
• protein translocation
• plasmid conjugation

Full text

Multiple substrates of the
Legionella pneumophila
Dot兾Icm system identified by interbacterial
protein transfer
Zhao-Qing Luo and Ralph R. Isberg*
Howard Hughes Medical Institute and Department of Molecular Biology and Microbiology, Tufts University School of Medicine, 150 Harrison Avenue,
Boston, MA 02111
Edited by John J. Mekalanos, Harvard Medical School, Boston, MA, and approved November 21, 2003 (received for review August 2, 2003)
Legionella pneumophila is an intracellular pathogen that multiplies
in a specialized vacuole within host cells. Biogenesis of this vacuole
requires the Dot兾Icm type IV protein translocation system. By using
a Cre兾loxP-based protein translocation assay, we found that pro-
teins translocated by the Dot兾Icm complex across the host phago-
somal membrane can also be transferred from one bacterial cell to
another. The flexibility of this system allowed the identification of
several families of proteins translocated by the Dot兾Icm complex.
When analyzed by immunofluorescence microscopy, a protein
identified by this procedure, SidC, was shown to translocate across
the phagosomal membranes to the cytoplasmic face of the L.
pneumophila phagosome. The identification of large numbers of
these substrates, and the fact that the absence of any one substrate
rarely results in strong defects in intracellular growth, indicate that
there is significant functional redundancy among the Dot兾Icm
translocation targets.
bacterial pathogenesis 兩 protein translocation 兩 plasmid conjugation
A
ctive modification of host cellular functions is essential for
a bacterial pathogen to establish a successful infection. Such
modification often is mediated by injecting effectors into the host
cytoplasm through specialized protein secretion systems (1).
Among these systems, conjugation-adaptive transporters, also
called type IV secretion systems (TFSS), have been identified in
a number of bacterial pathogens (2). Many of these TFSS are
dedicated DNA transfer apparatuses, whereas others allow
Gram-negative bacterial pathogens to translocate protein sub-
strates directly into the cytosol of eukaryotic cells. Only a few
protein substrates of TFSS that are translocated into host cells
have been identified (2).
Legionella pneumophila is an intracellular pathogen that
causes Legionnaire’s disease. After being phagocytosed by mac-
rophages, the bacteria multiply within a specialized vacuole that
is initially isolated from the endocytic pathway (3, 4), possibly by
intercepting early secretory vesicles (5). Biogenesis of this rep-
licative phagosome requires the TFSS transporter called Dot兾
Icm (6, 7). Substrates transported by this apparatus are believed
to directly promote the targeting pathway of the bacterial
vacuole (8). Two of these substrates, RalF and LidA, have been
identified (9, 10). RalF is a guanine nucleotide exchange factor
for multiple Arf proteins (9), whereas the biochemical activity of
LidA is unknown (10). There are clearly other unidentified
substrates of Dot兾Icm, as mutations that specifically eliminate
LidA cause negligible defects in intracellular growth and muta-
tions in ralF are proficient for intracellular replication (9, 10). A
comprehensive analysis of the identity and function of effectors
translocated by the Dot兾Icm apparatus is crucial in determining
how this bacterium establishes a replication vacuole. We report
here that proteins translocated from bacteria to host cells can
also be transferred between bacterial cells, allowing identifica-
tion of a large cohort of proteins transferred by the Dot兾Icm
apparatus.
Materials and Methods
Bacterial Strains and Growth Conditions. All L. pneumophila strains
used in this study are derivatives of the wild-type strain Lp02 (thyA,
hsdR, and rpsL) (11). Lp03 is an isogenic dotA
⫺
mutant (12). All
strains were grown on casamino acids yeast extract thymidine
(CYET) plates or in N-(2-acetamido)-2-aminoethanesulfonic acid
yeast extract (AYE) broth (11). Bone marrow-derived macrophages
were prepared as described (11). To assay for intracellular growth
within macrophages from A兾J mice (11) or within the amoebal host
Dictyostelium discoideum (13), L. pneumophila strains were grown
to postexponential phase, as judged by bacterial motility and cell
density (OD
600
⫽ 3.3–3.7).
Plasmid Constructions. A derivative of pBRR1MCS mob
⫺
(Cm
r
)
(14), which was suitable for reporting interbacterial protein
translocation (called pZL184; Fig. 1), was constructed in a
cloning process involving multiple steps. Sequences of oligonu-
cleotides used, sources of gene cassettes, and details of cloning
are found in Supporting Methods, which is published as support-
ing information on the PNAS web site. To express the Cre
fusions, we first constructed pZL180 (Amp
r
and thyA
⫹
), which
contains cre on the transfer deficient RSF1010 plasmid pJB908
(J. Vogel, Washington University School of Medicine, St. Louis),
and fusions were constructed as follows: Whole ORFs were
amplified by PCR and were fused to the 3⬘ end of cre on pZL180
for genes smaller than 2 kb, whereas for genes larger than 2 kb,
the 3⬘ regions encoding 500 or 700 amino acids of each ORF was
fused to cre.
Bacterial Two-Hybrid Screening. First, ralF was translationally fused
to the 3⬘ end of fragment T18 of the Bordetella pertussis cya gene,
encoding adenylate cyclase, on pUT18 (15). Sau3AI-generated
genomic DNA fragments from L. pneumophila strain Lp02 (11),
ranging from 800 base pairs to 5 kb, were inserted into pKT25
(15), resulting in a library of L. pneumophila proteins fused to the
C terminus of B. pertussis adenylate cyclase T25 fragment.
Escherichia coli strain BTH101 (15), expressing cya::ralF(C), was
used to identify RalF-interacting proteins by screening either on
LB medium containing 40
␮
g兾ml X-Gal or on the synthetic M63
medium (15) with lactose as the sole carbon source. Strains with
functional adenylate cyclase proteins were identified based on
the presence of detectable Lac
⫹
phenotypes on these media.
Proteins that interact with DotF were identified in a similar
manner by using either DotF or DotF (28–123) as the bait.
Bacterial Matings and Intercellular Protein Translocation. For RP4
Trb-mediated translocation of the Cre::MobA hybrid, ⬇2.5 ⫻
This paper was submitted directly (Track II) to the PNAS office.
Abbreviations: TFSS, type IV secretion systems; Sid, substrate of Icm兾Dot transporter.
Data deposition: The sequence reported in this paper has been deposited in the GenBank
database (accession nos. AY504668–AY504685).
*To whom correspondence should be addressed. E-mail: ralph.isberg@tufts.edu.
© 2004 by The National Academy of Sciences of the USA
www.pnas.org兾cgi兾doi兾10.1073兾pnas.0304916101 PNAS
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MICROBIOLOGY

10
8
cells from saturated cultures of S17–1 (Trb
⫹
) (16) or DH5
␣
(Trb
⫺
) expressing the fusion grown in LB broth were mixed with
a 15-fold excess of recipient strain XL1Blue carrying pZL184.
The mixtures were spotted onto 0.45-
␮
m nitrocellulose filters,
placed on LB plates, and incubated at 37°C for 3 h. The excisants
were selected on LB medium containing 5% sucrose and 30
␮
g兾ml kanamycin.
For L. pneumophila to L. pneumophila translocation, Lp02
(11) or Lp03dotA
⫺
(12) containing plasmids expressing the
appropriate Cre fusions were grown to postexponential phase in
AYE broth. For matings, 0.45-
␮
m nitrocellulose membranes
were first placed onto CYET medium containing 100 nM
isopropyl
␤
-D-thiogalactoside (IPTG) and the plates were incu-
bated at 37°C for 1 h before spotting with a mixture of ⬇3.5 ⫻
10
8
donor and a 15-fold excess of recipient Lp03(pZL184). The
mating plates were incubated at 37°C for 14 h and excisants were
selected on CYET ⫺5% sucrose, 30
␮
g兾ml kanamycin, which
kills both parents. Each mating was performed in triplicate and
repeated at least three times. Transfer frequencies were ex-
pressed as number of excisants (resistant to kanamycin and
sucrose but sensitive to gentamicin) per donor bacterium and the
number of donor bacteria was determined by counting colony-
forming units derived from appropriately diluted donor cells
plated onto CYET medium before mating.
For plasmid transfer, transconjugants were detected by plating
the protein translocation mating mixture onto LB containing 100
␮
g兾ml ampicillin and 30
␮
g兾ml chroramphenicol (for E. coli)or
onto charcoal yeast extract containing 5
␮
g兾ml chloramphenicol
(selecting Thy
⫹
and Cm
R
) for transfer in L. pneumophila). As a
positive control, the wild-type RSF1010 derivative pKB5 (11)
was used in both E. coli and L. pneumophila matings (6).
Construction of In-Frame Deletions and Complementation of the
Deletion Mutants.
In-frame deletions of L. pneumophila genes
were performed by a two-step allelic exchange strategy as
described (17). In each case, the deletion construct was designed
such that the intact gene was replaced by an ORF predicted to
express a 20-aa polypeptide consisting of the first 10 amino acids
and the last 10 amino acids of the gene. To perform comple-
mentation studies on deletion mutants, the gene of interest was
amplified by PCR and inserted into pJB908 or pBBRMCS2 (14).
Protein Purification and Antibody Preparation. The predicted ORF
of SidC was inserted into pQE30 and the resulting His
6
-SidC
fusion protein was purified from E. coli by using Ni-
nitrilotriacetic acid resin (Qiagen, Valencia, CA). Rabbit poly-
clonal serum was generated by the Pocono Rabbit Farm (Cana-
densis, PA; ref. 10). Antibodies were affinity-purified by using a
matrix containing purified (His)
6
-SidC covalently coupled to
Affigel-10 beads (Bio-Rad) (18).
Western Blot and Immunofluorescence Staining. Total L. pneumo-
phila proteins separated by SDS兾PAGE were transferred onto
Immobilon-P membranes (Millipore) and probed by Western
blotting, as described (16). Filters were probed with affinity-
purified anti-SidC antibody (diluted 1:5,000), or anti-Bacillus
subtilis isocitrate dehydrogenase polyclonal antibody (diluted
1:5,000; a kind gift from Dr L. Sonenshein, Tufts University
School of Medicine). Immunofluorescence staining was per-
formed with affinity-purified rabbit anti-(His)
6
-SidC antibodies
followed by a Texas red-conjugated goat anti-rabbit antibody
(Molecular Probes). Fixation and probing techniques were per-
formed as described (10). Postnuclear L. pneumophila phago-
somes were isolated as described (10).
Fig. 1. Interbacterial protein translocation by the Dot兾Icm system in the absence of DNA transfer. (A) Assay for interbacterial protein transfer. Translocation
of Cre hybrid protein from a donor bacterial strain is measured by removal of a floxed transcriptional terminator located between the trc promoter and the npt
II (kan
R
) gene on plasmid pZL184 harbored by a recipient bacterial strain. Bacteria harboring the intact reporter are unable to grow on media containing
kanamycin and sucrose. The translocation of Cre hybrid protein into the recipient strain leads to the excision, through recombination at the loxP sites, of the
DNA fragment that confers sucrose sensitivity (sacB) and reconstitution of a functional loxP-npt II translational fusion. S.D., Shine–Dalgarno sequence; npt II,
neomycin phosphotransferase; red diamond, transcriptional terminator. Arrows indicate the trc promoter and loxP sites. (B) Interbacterial transfer of a fusion
derived from the RSF1010 mobA gene. The mobA gene was fused to cre, and RP4-dependent protein translocation into a recipient E. coli strain was measured
by using E. coli S17–1 (15) as the donor selecting kanamycin resistance and screening for gentamicin sensitivity. Black bars, S17–1 Trb
⫹
donor; stippled bars, E.
coli DH5
␣
Trb
⫺
donor. (C). Plasmids harboring Cre fusions cannot be transferred to recipient cells. Plasmids expressing the designated proteins were harbored
in either E. coli S17–1 (Trb
⫹
)orL. pneumophila Lp02 (Dot兾Icm
⫹
; ref. 11), and the efficiency of plasmid transfer was measured by using either recipient E. coli
or L. pneumophila strains, respectively. As a positive control, the identical plasmids having an intact oriT and mob system were used to demonstrate transfer
proficiency of donor strains. Black bars, donor strain E. coli S17–1 (Trb
⫹
); gray bars, donor strain L. pneumophila Lp02 (Dot兾Icm
⫹
; ref. 11). (D) Transfer of
translocated Dot兾Icm substrates between bacterial cells. Protein transfer was performed as described in Materials and Methods, by using either Lp02 (Dot兾Icm
⫹
)
or Lp03(dotA
⫺
) expressing the designated protein fusions as the donor strains. Gray bars, donor strain L. pneumophila Lp02 (Dot兾Icm
⫹
); stippled bars, donor
strain L. pneumophila Lp03 (dotA
⫺
).
842
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www.pnas.org兾cgi兾doi兾10.1073兾pnas.0304916101 Luo and Isberg

Results
Interbacterial Protein Translocation by Dot兾Icm. To identify sub-
strates of the Dot兾Icm translocator, a screening strategy was
devised that allowed us to directly assay for protein transloca-
tion, using the Cre兾loxP system from bacteriophage P1 (for
review, see ref.19). Previous work (20) demonstrated that the
presence of Cre does not interfere with the translocation of the
TFSS substrates VirE2 and VirF in Agrobacterium tumefaciens.
A reporter suitable for monitoring translocation of Cre fusion
proteins between two prokaryotic cells was constructed in which
expression of the npt II gene depended on the excision of a floxed
cassette consisting of a gentamicin resistance gene, the sacB
gene, and a transcriptional terminator (Fig. 1A). By using this
system, E. coli and L. pneumophila could transfer a hybrid
protein in which cre was fused to the mobA gene of plasmid
RSF1010 (Fig. 1B). When the Cre::MobA hybrid protein was
expressed from a plasmid lacking its origin of transfer (oriT)in
the E. coli strain S17–1 (16), transfer of the fusion protein into
another E. coli strain could be detected based on excision of the
stopper sequence and expression of kanamycin resistance (Fig.
1B, and Fig. 6, which is published as supporting information on
the PNAS web site). No transfer occurred when the Trb
⫺
strain
DH5
␣
, which lacks conjugative transfer functions, was used as
the donor, or when a plasmid having cre alone was tested (Fig.
1B). Moreover, there was no transfer of the plasmid to the
recipient, indicating that the translocation of the fusion protein
occurred in the absence of DNA transfer (Fig. 1C; Cre::MobA
or Cre). L. pneumophila also was able to transfer the hybrid
protein in a Dot兾Icm-dependent manner (Fig. 1D; Cre::MobA),
indicating that the Dot兾Icm system is able to transfer proteins
interbacterially in the absence of DNA transfer (Fig. 1C).
Plasmid Mob proteins such as the MobA of RSF1010 are
believed to be transferred to recipient cells as protein–single-
stranded DNA complexes (21). However, our results demon-
strate that conjugation systems such as Trb and Dot兾Icm are able
to transfer MobA (and presumably similar proteins) in the
absence of DNA transfer. This result is similar to what had been
observed with the ColIb-P9 SogL protein (22).
Two proteins known to be transferred from bacterial cells to
mammalian cells could also be translocated interbacterially by
using this strategy. Full-length ralF and the 3⬘ half of lidA [called
lidA(C)] were each translationally fused to cre and the resulting
hybrids were examined for bacterium-bacterium translocation.
We found that wild type L. pneumophila, but not a Dot兾Icm-
deficient strain, could transfer Cre::RalF or Cre::LidA(C), based
on the ability of recipient strain to excise the floxed DNA
fragment and to express kanamycin resistance [Fig. 1D;
Cre::RalF and Cre::LidA(C)]. However, Cre itself could not be
translocated by wild-type L. pneumophila (Fig. 1D). The trans-
location proficiency of Cre::LidA(C) indicates that targeting
information resides in the C terminus of the protein, at least for
interbacterial transfer. We examined whether this finding was
also true for RalF by testing Dot兾Icm-mediated translocation of
Cre::RalF(C) and Cre::RalF(N) respectively. Only Cre:RalF(C)
was translocated at a detectable frequency (Fig. 1D). These
observations are similar to findings regarding A. tumefaciens Vir
proteins, in which secretion signals are localized to the C termini
of the substrates (20, 23).
Identification of Substrates of the Dot兾Icm Transporter. Because the
translocated proteins LidA and RalF could be transferred
interbacterially, we used the bacterial translocation assay to
identify proteins transferred by this transporter. To simplify the
screening of a randomly generated fusion library, we chose to
screen through a preselected pool of candidate substrates. We
hypothesized that some translocated substrates may interact with
specific components of the Dot兾Icm complex. Therefore, a
bacterial two-hybrid screen (15) was used to identify L. pneu-
mophila proteins that specifically interacted with the carboxyl
portion of RalF. From 41 positive clones sequenced, DotF,
predicted to be an inner membrane component of the Dot兾Icm
complex, was identified nine times independently. Interestingly,
in all cases, only the portion of DotF that spans amino acids
28–123 was obtained. The full-length DotF also gave a positive
two-hybrid readout, although at a somewhat lower level (Fig. 2).
A bacterial two-hybrid study was then performed by using Cya
fusions to the 3⬘ ends of L. pneumophila genes and baits of DotF
or its derivative, DotF (28–123). From ⬇150,000 candidates, we
isolated 148 clones that showed some level of interaction higher
than background, and these were sequenced and analyzed.
Although it was clear that some strains gave barely detectable
␤
-galactosidase activity and were of questionable significance,
this strategy greatly reduced the number of strains to be ana-
lyzed. Complete ORFs of these genes were retrieved from the
L. pneumophila genome database (http:兾兾genome3.cpmc.
columbia.edu兾⬃legion) and 68 genes were identified. Of these
genes, 20 candidates were eliminated based primarily on their
high hydrophobicity, which may have caused nonspecific inter-
actions with DotF. Forty-eight candidate genes were subse-
quently tested for interbacterial transfer (see Table 2, which is
published as supporting information on the PNAS web site, for
data on 17 such candidates from a single experiment). Table 1
shows eight clones that gave a positive signal by using the
Cre兾loxP reporter system. We designated these proteins sub-
strate of Icm兾Dot transporter (Sid) (Table 1). The efficiency of
interbacterial transfer varied, ranging from 10
⫺6
to 10
⫺5
ex-
cisants per input donor cell. Moreover, transfer of all of these
proteins depended on the Dot兾Icm apparatus, because mating
with dotA
⫺
strains expressing these fusions failed to cause the
excision of the floxed DNA fragment and the subsequent
expression of the kanamycin resistance marker (Table 1).
Characteristics of
sid
Genes. Sequence analyses revealed that with
the exception of sidB, the sid genes have no significant orthologs
present in the GenBank NR database (Table 1). SidB contains
Fig. 2. Interactions between DotF and the C termini of RalF and SidC,
respectively. E. coli strain BTH101 (14) containing the indicated plasmids was
grown overnight at 28°C in LB and the cultures were diluted 20-fold in the
same medium containing 100 nM IPTG. Cultures were grown for 14 h before
an appropriate volume was withdrawn for
␤
-galactosidase assay. Tested
strains are as follows: (column A) BTH101(pKT25dotF, pUT18C); (column B)
BTH101(pKT25, pUT18CRalF(C); (column C) BTH101(pKT25dotF,
pUT18CRalF(C); (column D) BTH101(pDotF (28–123), pKT25RalF(C); (column E)
BTH101 [pDotF (28–123), pSidC300(C)]k; and (column F) the leucine zipper
domain of the yeast GCN4 protein (14) was used as positive control in
BTH101(pKT25Zip, pUT18CZip).
␤
-galactosidase activity was expressed as
Miller units. Experiments were performed in triplicate for three independent
times. Data shown are from one representative experiment.
Luo and Isberg PNAS
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January 20, 2004
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vol. 101
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no. 3
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MICROBIOLOGY

a putative active site found in some lipases and shows a region
similar to a portion of the Rtx toxin from Vibrio cholerae (24).
In contrast, many of these proteins have one or more paralogs
present in the L. pneumophila genome (Table 1). The similarities
among the paralogs range from almost identical predicted
proteins (E ⫽ 0) to rather loose similarity (E ⫽ 5e
⫺2
to 2e
⫺8
;
Table 1). Interestingly, in some cases, subsets of paralogs are
organized into contiguous ORFs. For instance, sidC and its
homolog sdcA are separated by only 150 base pairs, whereas
sdeA, sdeB, and sdeC are closely clustered (Fig. 3). In addition,
a paralog of sidE (sdeD) is located directly upstream from what
appears to be the operon encoding sidC (Fig. 3B). Interestingly,
two putative genes, orf1 and orf3, located near the sidA and sdeC
regions are highly similar (E ⫽ 0; Fig. 3 A and B), although these
genes were not identified in the original two-hybrid assay.
SidC Is Translocated by Dot兾Icm Into the Cytosol of Host Cells. To
verify that proteins identified in our assay are translocated into
mammalian cells, affinity-purified antiserum against His
6
-SidC
was used to analyze expression and translocation of SidC by L.
pneumophila. As predicted, a protein of ⬇110 kDa was detected
in both a dot兾icm
⫹
strain and a dotA
⫺
mutant of L. pneumophila,
but not in a mutant missing sidC and its paralog sdcA [⌬(sdcA-
sidC)] (Fig. 3C). Moreover, expression of SidC was induced
⬇7-fold in cells grown to post exponential phase (Fig. 3C). The
translocated substrates RalF and LidA also show induced ex-
pression in post exponential phase (ref. 9 and G. M. Conover and
R.R.I., unpublished observations), and a large body of evidence
indicates that factors critical for intracellular survival of L.
pneumophila are similarly regulated (25, 26). When infected
macrophages were probed for SidC by indirect immunofluores-
cence microscopy, we found that1hafteruptake, ⬇86% of L.
pneumophila phagosomes stained positively for SidC (Fig. 4A),
with the protein localizing about the phagosomal membrane
(Fig. 4 C and D). In some cases, there was asymmetric diffusion
of the protein emanating from the phagosome surface, with most
infected cells showing protein only in the region near the
phagosome (Fig. 4D).
Because the above strategy only detects secretion of SidC and
does not demonstrate translocation across the phagosomal mem-
brane, a second approach was pursued (10). To demonstrate
SidC translocation across the phagosomal membrane by the
Dot兾Icm transporter, we prepared postnuclear supernatants
(PNS) from macrophages incubated with L. pneumophila and
probed intact phagosomes for SidC in the absence of perme-
abilization reagents. Approximately 85% of these phagosomes
stained positively for SidC (Fig. 4 B and E), whereas ⬍5%
stained positively with anti-L. pneumophila serum, demonstrat-
ing that the phagosomal membranes were intact. Less than 0.3%
of macrophages containing a L. pneumophila dotA
⫺
strain
stained positively for SidC (Fig. 4 A and C), which was consistent
with our data showing that interbacterial translocation of this
protein requires the Dot兾Icm apparatus. Similarly, in PNS, no
isolated phagosome containing a dotA
⫺
strain stained positively
for SidC (Fig. 4B). The antibody reactivity was specific, because
no macrophages harboring a mutant lacking sidC and its up-
Table 1. L. pneumophila proteins identified in this study that are translocated between
bacterial cells by the Dot兾Icm system
Gene
name
Accession
no.
Protein size,
aa
L. pneumophila
paralogs
E values of
paralogs
Orthologs in other
species (E value)
Translocation
frequency*
sidA AY504668 474 — NA — 7.2 ⫾ 1.2 ⫻ 10
⫺6
sidB AY504669 417 4 7e
⫺26
to 5e
⫺09
Rtx toxin兾lipase (e
⫺5
) 6.7 ⫾ 0.2 ⫻ 10
⫺6
sidC AY504673 918 2 0
†
— 3.3 ⫾ 0.7 ⫻ 10
⫺6
sidD AY504675 472 — NA — 2.3 ⫾ 0.2 ⫻ 10
⫺5
sidE AY504676 1,495 5 0–2e
⫺05
— 1.2 ⫾ 0.5 ⫻ 10
⫺6
sidF AY504681 912 NA — 6.2 ⫾ 0.2 ⫻ 10
⫺5
sidG AY504682 965 — NA — 8.2 ⫾ 0.3 ⫻ 10
⫺6
sidH AY504683 2,225 3 7e
⫺08
to 3e
⫺04
— 3.2 ⫾ 0.4 ⫻ 10
⫺5
SdeC
‡
AY504679 1,538 5 0 to 2e
⫺05
— 2.2 ⫾ 0.9 ⫻ 10
⫺6
Translocation assay was performed as described in Materials and Methods. NA, not applicable.
*Translocation frequency was expressed as numbers of excisants (resistant to both kanamycin and sucrose and
sensitive to gentamicin) per donor cell. No translocation occurred when the dotA
⫺
strain Lp03 was used as the
donor. The number of donor cells was determined by plating the appropriate dilutions of the donor culture onto
solid CYE medium. The rates of spontaneous mutants resistant to kanarnycin and sucrose of the reporter strain
appeared on the selective medium used to select excisants were approximately ⫺2.5 ⫻ 10
⫺9
per recipient.
†
An E value of 0 indicates that the paralogs are almost identical proteins.
‡
Paralogs are named for the gene identified in DotF two-hybrid assays. For instance, paralog A of sidE is called
sdeA.
Fig. 3. Clustering of sid genes, their paralogs into operon-like structures, and
growth phase regulation of sidC.(A) sidC and its homolog sdcA are separated
by only 150 bp, and the two genes are closely linked to a paralog of sidE(sdeD).
(B) Three paralogs of sidE are part of a contiguous region of the chromosome
that contains five significant ORFs. (C) Growth phase regulation of sidC.
Bacteria were grown in AYE broth to either an OD
600
⫽ 1.8 (exponential) or 3.7
(postexponential), harvested, and analyzed by SDS兾PAGE and immunoblot-
ting with affinity-purified anti-(His)
6
-SidC. Displayed are Lp02(dot兾icm intact;
ref. 11), Lp03 (dotA
⫺
; ref. 12), and Lp02(⌬sdcA, ⌬sidC), an Lp02 derivative
deleted for sidC and its upstream paralog. The isocitrate dehydrogenase
(ICDH) protein was used as a loading control by probing with antiserum raised
against B. subtilis ICDH.
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www.pnas.org兾cgi兾doi兾10.1073兾pnas.0304916101 Luo and Isberg

stream paralog stained positively for this protein [Fig. 4A;
⌬(sdcA-sidC)], nor did macrophages infected with a simple
deletion of sidC (data not shown). These results demonstrate
that during infection, SidC is translocated into mammalian cells
by the Dot兾Icm system, and that translocation occurs across the
phagosomal membrane. Because the transfer frequencies of
other proteins in our Cre兾loxP system were comparable to that
of SidC, we postulate that these proteins are also targeted to the
host cell by the Dot兾Icm transporter.
SdeC Is Required for Efficient Intracellular Growth by
L. pneumophila
.
To examine the importance of the Sid proteins in L. pneumophila
pathogenesis, we constructed in-frame deletions in some of these
genes and tested the resulting mutants for intracellular growth in
bone marrow-derived macrophages (10). Individual deletions of
sidA, sidD, sidF,orsidG, which have no detectable paralogs in
the available L. pneumophila database, resulted in strains with
intracellular growth properties that were difficult to distinguish
from the wild-type strain. Furthermore, a mutant lacking sidC
and its upstream paralog sdcA grew proficiently (data not
shown). Finally, a quadruple mutant lacking sidB and its three
paralogs has a defect in intracellular growth, but this result was
not significantly different from a mutant missing sdbA alone
(data not shown), suggesting that functional redundancy extends
beyond specific substrate families. We then constructed in-frame
deletion mutants lacking individual paralogs of sidE and exam-
ined intracellular growth of these mutants in bone marrow-
derived or in D. discoideum. Unexpectedly, only deletion of the
single paralog sdeC had a detectable growth defect (Fig. 5). In
D. discoideum, by using L. pneumophila strains harboring the
plasmid vector, the yield of viable counts after 48 h after
incubation was depressed 3-fold for the mutant lacking sdeC
(Fig. 5). Defective growth could be complemented in trans by a
single ORF containing sdeC (Fig. 5). A similar growth defect of
this mutant also was observed in macrophage (data not shown).
In contrast, mutants missing sidE, sdeA,orsdeB, respectively,
had no detectable growth defect in bone marrow-derived mac-
rophages (data not shown). As expected, a Cre::SdeC fusion was
found to be translocated between bacterial cells (Table 1).
Discussion
By developing a genetic assay for monitoring protein transloca-
tion using the cre兾loxP system, we conclusively demonstrated
that the Dot兾Icm TFSS transporter can perform interbacterial
transfer of proteins known to be targeted to mammalian cells, an
observation that we have exploited to identify a large number of
translocated proteins. The majority of the proteins we identified
have no significant orthologs in the database, suggesting that
biogenesis of the L. pneumophila replication compartment may
involve mechanisms that differ from other organisms, such as
Chlamydia trachomatis and Mycobacterium spp., which reside in
similar intracellular vacuoles. Alternatively, proteins with similar
functions in different microbial species may have evolved inde-
pendent of each other, and show little sequence similarity to each
Fig. 4. SidC is translocated by the L. pneumophila Dot兾Icm system to the host
cell and is localized about the phagosomal membrane. (A) Bone marrow-
derived macrophages from A兾J mice were infected with Lp02(dot兾icm intact),
Lp03(dotA
⫺
), or Lp02(⌬sdcA-⌬sidC) strain expressing GFP, respectively. One
hour after infection, cells were fixed as described (10), and SidC was probed
with anti-(His)
6
-SidC antibodies and Texas red-labeled secondary antibodies.
Stained macrophages were scored for translocation of SidC by counting
phagosomes that stained positively with anti-(His)
6
-SidC. Data shown are from
two independent experiments performed in triplicate in which at least 100
phagosomes were scored per coverslip. (B) SidC staining on PNS prepared from
L. pneumophila-infected U937 cells in the absence of permeabilization. Sam-
ple preparation, immunostaining, and data collection were performed as
described in ref. 10 or in A.(C) DotA-dependent translocation of SidC. (Left)
Bacteria expressing GFP associated with bone marrow-derived macrophage.
Strains used were Lp02(dot兾icm intact; Upper) and Lp03 (dotA
⫺
; Lower).
(Center) Immunoprobing of infected cells with anti-(His)
6
-SidC. (Right)
Merged images of GFP and anti-(His)
6
-SidC staining. (D) Limited diffusion of
SidC from L. pneumophila phagosome. Shown are images of Lp02(dot兾icm
intact) with murine bone borrow-derived macrophage. (E) SidC is translocated
across the phagosomal membrane. Shown are images of PNSs of Lp02(dot兾icm
intact)-infected macrophages. Bacteria and SidC are probed as above, with
bacteria marked by GFP and anti-SidC marked in red.
Fig. 5. sdeC is required for efficient intracellular growth. D. discoideum cells
were infected with a multiplicity of infection of 0.05, and growth of bacteria
was monitored as described (13). Total bacterial cells were washed from
individual microtiter wells at designated times, and the appropriate dilutions
were plated on charcoal yeast extract plates to obtain the colony-forming
units. Fold of growth was obtained by dividing colony-forming units at a given
time point by the input bacterial cell numbers. Strains tested are as follows:
black bars, Lp02(intact dot兾icm); gray bars, Lp02(⌬sdeC); striped bars,
Lp02(⌬sdeC) harboring pZL192 that carries the ORF of sdeC. Data shown are
from two independent experiments performed in triplicate.
Luo and Isberg PNAS
兩
January 20, 2004
兩
vol. 101
兩
no. 3
兩
845
MICROBIOLOGY

other. Many of the translocated proteins have paralogs within L.
pneumophila, often encoded in chromosomal regions devoted to
expression of translocated substrates of Dot兾Icm (Fig. 3 A and
B). These genes may have been organized to ensure proper
expression during exposure to environmental conditions that are
optimal for initiating intracellular growth.
The lack of intracellular growth defects observed in L. pneu-
mophila mutants lacking translocated substrates is reminiscent
of a L. pneumophila ralF mutant, which is indistinguishable from
the parental wild-type strain in regards to intracellular growth
(9). Nevertheless, a ralF mutant is unable to recruit Arf1 to the
surface of the replicative phagosome, a property that may play
some unknown role in the lifestyle of the organism (27). It is
possible that elimination of some of the genes here identified
similarly results in the formation of phagosomes with alterations
in either morphology or host protein content that have little
consequence with respect to growth in cultured cells. Alterna-
tively, the roles played by these proteins may be substituted by
other substrates of Dot兾Icm, or some of these proteins may be
important for growth in some untested host cell.
It is common that a bacterial pathogen codes for numerous
effectors but the presence of multiple paralogs of a specific
translocated effector in the same organism is only occasionally
found (28–30). The close similarity among proteins in a family
points to functional redundancy, which may provide an expla-
nation for the failure to identify these proteins in previous
genetic screens for bacterial mutants defective in intracellular
growth. In the case of the sidB family, however, functional
redundancy clearly extends beyond the identified paralogs, and
proteins of little similarity in sequence or function may be
redundant. It is plausible that each paralog may be adapted to
promote growth only in specific host cells. Because L. pneumo-
phila is a versatile pathogen that interacts with very diverse hosts
in the environment, the establishment of a successful intracel-
lular replication niche may require only a subset of translocated
substrates, with a single subset adapted for a particular host cell
type.
Sorting out the minimal complement of translocated proteins
necessary for intracellular replication will be a challenge for the
future, because the number of such proteins may be quite large,
based on the data displayed in Table 1. In addition to the
products of the genes identified in the original Cre兾loxP assay
and sdeC, we have tested four paralogs of Sid proteins for
interbacterial transfer, and all can promote transfer of Cre (data
not shown). Based on this finding, we speculate that it is likely
that all of the paralogs are capable of translocation. Further-
more, in another study we found an additional family of four
proteins that can be transferred (S. M. VanRheenen, Z.-Q.L.,
and R.R.I., unpublished results). Altogether, this brings the
minimum number of translocated proteins encoded by L. pneu-
mophila to 24. In fact, there are probably many more proteins
translocated by Dot兾Icm, as the DotF interaction screen was not
carried to saturation. The gene bank required in-frame fusions
to a Sau3AI restrictions site, which may not be present in all
genes encoding translocated substrates. Furthermore, the screen
described here focused on only the subset of DotF interactors,
and it appears that not all translocated substrates bind DotF at
detectable levels in this assay.
The other salient feature of the Dot兾Icm translocation system
that was uncovered here is the large size of the proteins that were
translocated. Many of the proteins identified in this study are
⬎90 kDa, with one predicted to be ⬎240 kDa. It appears likely
that TFSS have evolved to transport large molecules, such as
DNA–protein complexes, and these transfer systems may be the
preferred translocators for high molecule weight substrates.
In summary, the L. pneumophila TFSS is a transporter system
of striking flexibility. It is capable of promoting bacterial con-
jugation and translocation of proteins from bacteria into mam-
malian cells, as well as transporting these same proteins between
bacterial strains. Although the efficiency of interbacterial pro-
tein transfer appears to be many orders of magnitude lower than
the transfer from bacteria to macrophages (Table 1 and Figs. 1D
and 4 A and B), this property has allowed us to identify substrates
of the Dot兾Icm system. Understanding the functions of these
proteins should allow elucidation of the mechanisms underlying
the biogenesis of the L. pneumophila-replicative phagosome.
Finally, the methods we described here may be generalized to
identify mammalian effectors transferred by all TFSS.
We thank M. Tang for assistance in protein purification; S. Farrand, D.
Ladant, A. Vergunst, and J. Vogel for supplying plasmids; the Isberg
laboratory for helpful discussions; and Drs. Carol Kumamoto, Michael
Malamy, Susan VanRheeven, Marion Shonn, Isabelle Derre, and Mat-
thias Machner for review of the text. This work was supported by the
Howard Hughes Medical Institute. Z.-Q.L. is a Howard Hughes Medical
Institute Fellow of the Life Sciences Research Foundation, and R.R.I. is
a Howard Hughes Medical Institute Investigator.
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www.pnas.org兾cgi兾doi兾10.1073兾pnas.0304916101 Luo and Isberg