Download How is the intracellular fate of the Legionella pneumophila

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Vi e w p o i n t
How is the intracellular fate of the
Legionella pneumophila phagosome
determined?
Gil Segal and Howard A. Shuman
L
egionella pneumophila, the
causative agent of Legionnaires’ disease, is a facultative intracellular pathogen that is
able to infect, multiply within and
kill human macrophages, as well as
free-living amoebae. It evades the
microbicidal defenses of the phagocytes by maintaining the phagosome
pH near neutrality and preventing
phagosome–lysosome fusion. Once
inside the specialized phagosome,
the bacteria multiply exponentially
until the cell eventually lyses, releasing bacteria that can start new
rounds of infection1. Many of these
events are similar to those observed
in a variety of other intracellular
pathogens, including Toxoplasma
gondii, Leishmania donovani and
Mycobacterium tuberculosis.
The L. pneumophila icm/dot
genes and proteins
Recently, six papers describing new
genes and phenotypes involved in
the L. pneumophila–macrophage
interaction have been published2–7.
The icm/dot genes – which we
named icm (for ‘intracellular
multiplication’) and the Isberg lab
named dot (for ‘defect in organelle
trafficking’) – described in these
papers, together with previously
identified icm/dot genes8,9, probably account for most of the genes
that enable L. pneumophila to
grow within and kill human macrophages. In total, 23 icm/dot genes,
located in two unlinked regions on
the L. pneumophila genome, have
been identified (Fig. 1).
Sequence analysis of the Icm/
Dot proteins encoded by the genes
located in these two regions has
revealed some interesting information. Most of the proteins (14
of 23) are expected to be located
The following pair of articles,
the first by Gil Segal and
Howard Shuman, and the second
by James Kirby and Ralph Isberg
(Trends Microbiol. 6, 256–258),
explore the genetics and function
of the icm/dot genes of Legionella
pneumophila. This gene family is
implicated in several aspects of
virulence and appears to constitute
components of a conjugal transfer
system that has been adopted to
prevent phagosome–lysosome fusion
in the host cell and to mediate host
cytotoxicity by pore formation.
Whether these functions are
natural consequences or operate in
parallel remains to be discovered.
in the bacterial inner membrane
(Fig. 1), and four of them (IcmO,
IcmB, IcmF and DotB) contain an
ATP/GTP-binding site. However,
the most striking finding is that
four of the proteins (IcmP, IcmO,
IcmL and IcmE) contain significant sequence similarity to plasmidencoded genes involved in DNA
transfer. An additional protein
(DotB) was found to be homologous to a large family of nucleotide-binding proteins that includes
members of various conjugaltransfer systems10.
Phenotypes associated with
icm/dot mutants
Several phenotypes associated
with mutants in the icm/dot genes
G. Segal and H.A. Shuman* are in the
Dept of Microbiology, College of
Physicians & Surgeons, Columbia
University, 701 West 168th Street,
New York, NY 10032, USA.
*tel: ⫹1 212 305 6913,
fax: ⫹1 212 305 7323,
e-mail: [email protected]
Copyright © 1998 Elsevier Science Ltd. All rights reserved. 0966 842X/98/$19.00
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253
have been described. Strains in
which there are mutations in the
majority of these genes completely
lose their ability to grow within
and kill human macrophages, and
those with mutations in the minority of other genes have difficulty
in growing within or killing
macrophages2–6. Phagosomes containing mutants with defects in
several of the genes were tested for
colocalization with the late endosomal marker LAMP-1 (lysosomalassociated membrane protein 1),
which indicated that they are no
longer able to prevent phagosome–lysosome fusion5,6. Immediate cytotoxicity was found to be an
additional phenotype associated
with these genes7. The ability of
wild-type L. pneumophila to cause
immediate cytotoxicity for macrophages has been described in the
past11. This ability has now been
further characterized and shown
to be dependent on several of the
genes described above7. It is important to note that mutations in
all the genes tested resulted in strains
completely defective or attenuated
for all the following phenotypes:
intracellular multiplication, host
killing, LAMP-1 colocalization
and immediate cytotoxicity.
The Icm/Dot complex and
conjugal DNA transfer
The homology of several Icm/Dot
proteins to plasmid-encoded proteins involved in DNA transfer
prompted both our research group
and the Isberg group to test whether
wild-type L. pneumophila can
mediate plasmid DNA transfer.
Derivatives of the non-self-transmissible, heterologous IncQ plasmid
RSF1010 (Ref. 12) were tested,
and wild-type L. pneumophila was
PII: S0966-842X(98)01308-0
VOL. 6 NO. 7 JULY 1998
COMMENT
icmT S R Q
P
O
lphA M L K
E
G C D J
B
dotM
L
KJ I
G
F
O
H
E
N
icmV
dotD C
B
tphA
W
F
X
A
Lipoprotein
Cytoplasm
Inner membrane
Periplasm
Fig. 1. Linkage map of the two icm/dot regions. Region I contains the icmVWX–dotABCD genes, and region II contains the
icmT,S,R,Q,P,O–lphA–icmM,L,K,E,G,C,D,J,B–tphA–icmF or the dotM,L,K,J,I,H,G,F,E,N,O genes. Coding regions are indicated by
arrows, and the different tints indicate the predicted location of the protein in the bacterial cell. The icm letter codes are marked
above the gene arrows, and the dot letter codes are marked below the gene arrows.
found to conjugate these plasmids.
The conjugation process was dependent on several icm/dot genes3,5.
Conjugation is the first phenotype
to be identified, for which only a
subset of the icm/dot genes are required. This may be because conjugation is the only phenotype that
is not directly related to the ability
of L. pneumophila to grow within
and kill human macrophages.
Sequence similarity between
conjugation-related genes and genes
involved in pathogen–host cell interactions has been previously described in plants and animals13,14.
The conjugation system of the IncN
plasmid pKM101 exhibits a high
degree of sequence similarity and
similar gene organization to the virulence system of the plant pathogen
Agrobacterium tumefaciens, which
transfers DNA into plants, and to
the virulence system of the human
pathogen Bordetella pertussis,
which exports the pertussis toxin
into cells13. Two Icm/Dot proteins
(DotB and IcmE) show a low degree
of homology to A. tumefaciens Vir
proteins (VirB11 and VirB10, respectively)5. In addition, both the
Agrobacterium vir and Legionella
icm systems contain ~20 proteins
involved in the interaction of the
bacteria with the host cell; most of
these proteins are expected to be
located in the bacterial inner membrane and some of them contain a
nucleotide-binding site. Moreover,
both the A. tumefaciens vir system
and the L. pneumophila icm/dot
system transfer the RSF1010 plas-
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IN
mid between bacteria, probably as a
nucleoprotein complex (MobA–
ssDNA)3,5,15. The function of the
A. tumefaciens vir system in the
transfer of T-DNA from the bacteria into the plant cell nucleus has
long been known. The transfer
occurs as a nucleoprotein complex
containing the VirD2 protein covalently linked to the single-stranded
T-DNA, which is coated with the
VirE2 single-stranded binding protein16. The fact that both systems
share functional and organizational
similarities may indicate that secretion of a macromolecule (of an unknown nature) is the major function
of the L. pneumophila icm/dot system. This function might play a key
role in the ability of L. pneumophila
to prevent phagosome–lysosome
fusion and multiply within human
macrophages.
What is the natural substrate of
the icm/dot system?
It is unlikely that the natural substrate of the icm/dot system is
DNA, as prevention of phagosome–
lysosome fusion occurs within
30 min and there is probably not
enough time for DNA to be transferred and expressed within this
time period17. It is more likely that
L. pneumophila transfers an effector protein(s) into the host cell
(Fig. 2a). The major function of
this protein(s) is probably to inhibit
or modify the endocytic pathway
in a way that avoids fusion between
the L. pneumophila-containing
phagosome and the secondary
MICROBIOLOGY
254
lysosomes. After transfer, the effector protein(s) might be located
in the phagosome membrane, the
phagosome space or the macrophage cytoplasm (Fig. 2b).
We currently do not know
which, if any, of the Icm/Dot proteins are effector proteins, but
there are at least two candidates.
DotI/IcmL contains two amphipathic ␤-sheet regions6, structures
which are found in pore-forming
toxins. The pore-forming ability,
which was shown to be associated
with the Icm/Dot proteins7, might
be related to the transfer of this
protein into a host cell membrane.
Another candidate is the IcmG/
DotF protein, which contains a
coiled-coil domain. These domains
are known to be important in the
recognition of the vesicle and its
target in the SNARE system18. The
SNARE system plays an important
role in a variety of fusion events,
and IcmG/DotF might interact with
this system in such a way to prevent
phagosome–lysosome fusion. Alternatively, preventing phagosome–
lysosome fusion may be a result of
an earlier event that occurs during
phagocytosis, and this event may be
mediated by the Icm/Dot complex.
Membrane sorting of different receptors occurs during phagocytosis
of L. pneumophila19; the result of
this sorting might be a phagosome
that will not fuse with lysosomes.
Conclusions
Our current model of how the
Icm/Dot complex influences the
VOL. 6 NO. 7 JULY 1998
COMMENT
(a)
M
yt
a c o p lasm
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em
C
intracellular fate of L. pneumophila
is shown in Fig. 2. This model predicts at least two classes of Icm/
Dot proteins. One class of proteins
comprise or help to build the
transfer complex or transferosome.
The second class comprises ‘effector’ proteins, which are transferred
to the host cell and probably interact with components of the host
cell that are involved in phagosome formation and fate. Finding
out which Icm/Dot products correspond to the effector molecules
and how they interact with host
cell components is expected to shed
new light on our understanding of
the ways in which intracellular
pathogens survive and multiply
inside human cells.
Macrophage
e m bra
ra n e
r memb
Oute
lasm
Perip
Peptidoglycan
NTP
NTP
MobA
Effector
(b)
IN
SSBs
RSF1010–nucleoprotein
complex
Effector
Phagosome membrane
L. pneumophila
Fig. 2. A model for the Legionella pneumophila virulence transfer system. (a) Some of the
icm/dot gene products are expected to interact with one another to form a protein complex,
which will build a channel through the bacterial inner and outer membrane and will constitute
the Icm/Dot transfer complex or transferosome. Other icm/dot gene product(s) are expected
to be effector molecules. Some of the icm/dot gene products may be involved in the assembly of the complex, and the energy required for the assembly and/or transfer of the effector
molecule may be provided by nucleotide triphosphatase activities of several ATP/GTP-binding
proteins. Another substrate that is able to be transferred by the Icm/Dot transfer system is
the plasmid RSF1010–nucleoprotein complex, which has been shown to conjugate between
bacteria in an icm/dot-dependent manner. (b) There are three possibilities for the location of
the effector protein(s): in the phagosome membrane, in the macrophage cytoplasm and in
the phagosome space.
14 Censini, S. et al. (1996) Proc. Natl. Acad.
Sci. U. S. A. 93, 14648–14653
15 Beijersbergen, A. et al. (1992) Science 256,
1324–1327
16 Christie, P.J. (1997) J. Bacteriol. 179,
3085–3094
17 Wiater, L.A. et al. Infect. Immun.
(in press)
18 Hay, J.C. and Scheller, R.H. (1997) Curr.
Opin. Cell Biol. 9, 505–512
19 Clemens, D.L. and Horwitz, M.A. (1992)
J. Exp. Med. 175, 1317–1326
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Acknowledgements
Research in our laboratory is supported by a
grant from the NIH (AI23549). G.S. is supported
by a long-term fellowship from the EMBO.
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