Cellular microbiology and molecular ecology of Legionella–amoeba interaction

Abstract

Legionella pneumophila is an aquatic organism that interacts with amoebae and ciliated protozoa as the natural hosts, and this interaction plays a central role in bacterial ecology and infectivity. Upon transmission to humans, L. pneumophila infect and replicate within alveolar macrophages causing pneumonia. Intracellular proliferation of L. pneumophila within the two evolutionarily distant hosts is facilitated by bacterial exploitation of evolutionarily conserved host processes that are targeted by bacterial protein effectors injected into the host cell by the Dot/Icm type VIB translocation system. Although cysteine is semi-essential for humans and essential for amoeba, it is a metabolically favorable source of carbon and energy generation by L. pneumophila. To counteract host limitation of cysteine, L. pneumophila utilizes the AnkB Dot/Icm-translocated F-box effector to promote host proteasomal degradation of polyubiquitinated proteins within amoebae and human cells. Evidence indicates ankB and other Dot/Icm-translocated effector genes have been acquired through inter-kingdom horizontal gene transfer.

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www.landesbioscience.com Virulence 307
Virulence 4:4, 307–314; May 15, 2013; © 2013 Landes Bioscience
REVIEW
REVIEW
Legionnaire Disease
The first recognized outbreak of Legionnaire disease occurred
in 1976 during the 56th annual American Legion Convention
in Philadelphia.
1
There were 180 individuals who were diag-
nosed with severe pneumonia and of these individuals, 29 died.
Less than one year after the outbreak the causative agent of the
disease was isolated.
2
The bacterium, which was designated
Legionella pneumophila, is a gram-negative facultative intracel-
lular bacterium that proliferates within alveolar macrophages,
causing Legionnaire disease.
3
There are more than 50 species of
Legionellae, but L. pneumophila continues to be responsible for
more than 80% of cases of Legionnaire disease in most of the
world. The exception is Western Australia, where L. longbeachae
is the most predominant species in causing disease, but its patho-
genesis is distinct from L. pneumophila.
4-6
*Correspondence to: Yousef Abu Kwaik; Email: abukwaik@louisville.edu
Submitted: 02/14/13; Revised: 03/12/13; Accepted: 03/13/13
http://dx.doi.org/10.4161/viru.24290
Legionella pneumophila is an aquatic organism that interacts
with amoebae and ciliated protozoa as the natural hosts, and
this interaction plays a central role in bacterial ecology and
infectivity. Upon transmission to humans, L. pneumophila
infect and replicate within alveolar macrophages causing
pneumonia. Intracellular proliferation of L. pneumophila within
the two evolutionarily distant hosts is facilitated by bacterial
exploitation of evolutionarily conserved host processes
that are targeted by bacterial protein eectors injected into
the host cell by the Dot/Icm type VIB translocation system.
Although cysteine is semi-essential for humans and essential
for amoebae, it is a metabolically favorable source of carbon
and energy generation by L. pneumophila. To counteract host
limitation of cysteine, L. pneumophila utilizes the AnkB Dot/
Icm-translocated F-box eector to promote host proteasomal
degradation of polyubiquitinated proteins within amoeba
and human cells. Evidence indicates ankB and other Dot/Icm-
translocated eector genes have been acquired through inter-
kingdom horizontal gene transfer.
Cellular microbiology and molecular ecology
of Legionella–amoeba interaction
Ashley M. Richards,
†
Juanita E. Von Dwingelo,
†
Christopher T. Price and Yousef Abu Kwaik*
Department of Microbiology and Immunology; College of Medicine; University of Louisville; Louisville, KY USA
†
These authors contributed equally to this work.
Keywords: polyubiquitin, farnesylation, prenylation, effectors AnkB, Ankyrin B, proteasomes, cysteine, ppGpp, RelA, SpoT,
Ankyrin, Dot/Icm, pneumophila, Legionnaire
Legionnaire disease and Pontiac fever, which is a flu-like ill-
ness caused by Legionella, have only emerged within the past few
decades due to human alterations to the environment. These
alterations include the use of air conditioning systems, whirl-
pools, and water cooling towers that generate aerosols as a vehicle
to transmit Legionella from aquatic sources (Fig. 1).
3
To date there
has been no documented cases of L. pneumophila transmission
between individuals, and transmission from the environment to
the human host is considered to be the main mode of transmis-
sion of L. pneumophila. Once L. pneumophila infects the human
host, its intracellular lifecycle has a striking similarity to that
within the evolutionarily distant natural host, amoebae (Fig. 1).
7
Legionella–Amoebae Interaction
L. pneumophila is ubiquitous in freshwater environments as well
as many man-made water systems worldwide, often in close asso-
ciation with freshwater protozoa.
8
L. pneumophila replicate at
temperatures of 25–42 °C with an optimal growth temperature
of 35 °C. Consistent with what L. pneumophila would encounter
in the environment, motility and adherence to host cells are opti-
mal at temperatures below 37 °C.
9
When the temperature in the
aquatic environment is increased, the balance between bacteria
and amoebae can shift, which results in rapid multiplication of
Legionella.
10
The increase in the number of L. pneumophila in
the water as a result of proliferation within protozoa increases
the chance of transmission and disease manifestation.
3
Upon
decrease of temperature or exposure to environmental stress such
as chlorine, the amoebae differentiate into cyst,
10
and intracellular
L. pneumophila are capable of survival within the cyst
11
(Fig. 1).
Encysted amoeba is a highly resistant developmental stage that
contributes to the resistance of intracellular L. pneumophila to
different chemical and physical agents.
12
Therefore, the relation-
ship between L. pneumophila and amoebae plays an important
role in ecology and pathogenicity of the bacterium.
13
One mechanism by which the bacteria are released from
amoebae is within excreted vesicles similar to exocytosis of food
vacuoles.
14,15
L. pneumophila can still be cultured after 6 mo of
residence within excreted vesicles.
16,17
The number of bacteria
isolated from water sources of transmission to humans during
Legionnaire disease outbreaks is usually low or undetectable.
7,8
It is thought that the enhanced infectivity of L. pneumophila as

308 Virulence Volume 4 Issue 4
Numerous methods have been employed to attempt to eradi-
cate L. pneumophila from aquatic environments, with little
success. These attempts, which include chemical biocides, over-
heating water, and UV irradiation, have been successful for short
periods after which the bacteria can be again detected. It has been
suggested that in order to eradicate L. pneumophila from aquatic
environments continuous treatments effective against both the
bacteria and the protozoan host should be employed.
8,12 ,2 7, 33
Legionella-Like Amoebal Pathogens
There are Legionella-like species that cannot be grown on bac-
teriologic media but must be co-cultured with protozoa and are
referred to as Legionella-like amoebal pathogens (LLAP).
14
The
LLAPs are closely related to Legionella phylogenetically and
acquired their name because of their ability to infect and mul-
tiply within amoebae.
14
It is thought the LLAPs play a role in
community-acquired pneumonia and usually act as a co-patho-
gen but not as the sole pathogen.
34
Little is known about LLAPs
and future studies are needed to gain a better understanding of
the significance of these organisms in pulmonary infections.
Entry and Intracellular Trafcking of L. pneumophila
L. pneumophila infection of human alveolar macrophages is an
accidental infection and is thought to be a diversion from its
natural life cycle within amoebae. In the aquatic environment,
L. pneumophila resides in protozoa or in biofilms.
35,36
Amoebae
play a central role in the life cycle of L. pneumophila, and this
was first described in 1980.
37
Upon initial interaction between
L. pneumophila and amoebae, L. pneumophila is often engulfed
by coiling phagocytosis but other forms of internalization also
occur through a Gal/Gal-NAC specific receptor.
38,39
After inter-
nalization of L. pneumophila into the trophozoite of amoeba,
proliferation occurs within the Legionella-containing vacuole
(LCV) followed by bacterial release from the cell to seek a new
host (Fig. 1).
40-42
Once amoebae or mammalian cells engulf
L. pneumophila, the bacteria evade the default trafficking path-
ways into the lysosomal network (Fig. 1). The LCV recruits host
cell organelles, like mitochondria, ribosomes, and small vesicles
to its surface.
43
This accumulation begins during uptake into the
cell and is completed within a few minutes.
35
The ER-to-Golgi
vesicle traffic is intercepted by the LCV and the LCV membrane
becomes derived from the ER (Fig. 1).
13,43
The LCV becomes rap-
idly decorated with polyubiquitinated proteins within amoebae
and human cells.
44-46
Following maturation of the ER-remodeled
LCV and its decoration with polyubiquitinated proteins, rapid
replication of L. pneumophila commences (Fig. 1). During late
stages of intracellular proliferation, L. pneumophila escape from
the LCV to the cytosol where the bacteria finish the last 1–2
rounds of proliferation along with phenotypic modulations in
response to nutrient depletion in the host (Fig. 1).
8,40,47
The strategy used by L. pneumophila to avoid lysosomes and
to modulate cellular processes is dependent on the Dot/Icm type
IVB secretion system.
43,48,49
This secretion system injects ~300
effector proteins into the host cell, which accounts for ~10%
a result of growth within amoebae could compensate for the low
infectious dose in the water sources.
17-20
The ability of L. pneu-
mophila to parasitize human macrophages and to cause human
disease is thought to be a consequence of its prior adaptation to
intracellular growth within various protozoan hosts.
7,8
This is
most likely due to bacterial acquisition of eukaryotic genes dur-
ing its co-evolution with amoebae and adaptation to the intracel-
lular life within primitive eukaryotic hosts.
7,21-24
L. pneumophila and amoebae have been isolated from the same
source of infection during outbreaks of Legionnaire disease.
8
The
isolated amoebae have also been shown to support intracellular
replication of L. pneumophila.
25
It has been shown that L. pneu-
mophila that cannot be cultured in vitro using classical methods
can be resuscitated and proliferate if they are co-cultured with
amoebae.
8,26,27
Dictyostelium discoideum has been adapted as a
genetically amenable amoeba model system to decipher molec-
ular and cellular bases of L. pneumophila-amoeba interaction.
28
Therefore, amoebae are not only the environmental host for this
human pathogen, but constitute a genetically amenable model
system to study pathogenesis of L. pneumophila. These findings
demonstrate that studies on the L. pneumophila-amoeba interac-
tion will continue to contribute to our knowledge of the central
role of the amoeba host in the pathogenesis of these bacteria.
Amoebae Aid in the Persistence
of Legionella pneumophila in the Environment
Amoebae not only enhance the pathogenicity of L. pneumoph-
ila, but they also enable the bacteria to persist in the environ-
ment. Fourteen species of amoebae, with Hartmannellae and
Acanthamoeba being the most prominent, and two species of cili-
ated protozoa have been shown to support intracellular replica-
tion of L. pneumophila.
8
L. pneumophila infects the trophozoite
form of amoebae and the presence of the bacteria within amoebae
serves to protect the bacteria from harsh environments.
27
L. pneu-
mophila does not proliferate within encysted amoeba but when
conditions become unfavorable, protozoa can differentiate from
their trophozoite form into a cyst form that protects the organ-
isms and ensures their survival (Fig. 1). L. pneumophila released
from free-living amoebae also show an increased resistance
to harsh conditions compared with those grown in vitro.
29-32
When compared with bacteria grown in vitro, bacteria grown
in amoebae have changes in biochemistry, physiology, and viru-
lence potential.
12
These changes include an enhanced resistance
to chemicals and antibiotics, an altered fatty acid profile and pro-
tein profile, shorter size, motility, an increased ability to infect
amoebae and mammalian cells, an increase in environmental fit-
ness, and an increase in uptake.
8,19,20,26,27
In addition, the bacteria
found within vesicles excreted from amoebae are highly resistant
to biocides while the vesicle is resistant to freezing and sonica-
tion.
17
After prolonged starvation of L. pneumophila or treat-
ment with chlorine that renders L. pneumophila non-culturable
in aquatic environments, the bacteria can’t be cultured on rich
media but they can be resuscitated by infection of amoebae,
clearly indicating the remarkable protection of L. pneumophila
through its intracellular niche within amoebae.
26,27

www.landesbioscience.com Virulence 309
exponential phase of intracellular growth within macrophages
and Acanthamoeba,
47,59- 61
but the function of most of these pro-
teins has yet to be determined.
Growth phase-dependent regulation of bacterial virulence.
The intracellular lifecycle of L. pneumophila consists of a repli-
cative phase within the LCV, and a transmissive phase, exhib-
ited upon escape into the cytosol.
40,47,62,63
This biphasic lifestyle
is characterized by dramatic changes in the transcriptome, that
result in phenotypic modulations.
59-61
During the replicative
of the coding capacity of the genome of L. pneumophila.
7,50,51
Although a large number of effectors are injected into the host
cell, but with only few exceptions, deletion of individual effectors
does not result in reduced intracellular proliferation, suggesting
potential functional redundancy.
7,43,51,52
Strikingly, many L. pneu-
mophila effector proteins harbor eukaryotic protein domains,
which include ankyrin repeats, leucine-rich repeats, Sel-1,
U-box, F-box, and a C-terminal CaaX prenylation motif.
23,53-58
Expression of a large number of effectors is induced during the
Figure 1. The environmental life cycle of L. pneumophila within protozoa. (1) Flagellated L. pneumophila infect protozoa in the aquatic environment.
(2) The LCV evades the default endosomal–lysosomal degradation pathway and becomes rapidly remodeled by the ER through intercepting ER-to-
Golgi vesicle trac and becomes rapidly decorated with polyubiquitinated proteins in an AnkB-dependent manner. (3) Under unfavorable stress con-
ditions, such as nutrient deprivation, amoebae encyst, and bacterial proliferation will not occur due to nutrient limitation. Under growth-permissive
conditions for the amoeba, the LCV is decorated with polyubiquitinated proteins, which are targeted for proteasomal degradation leading to elevated
cellular levels of amino acids (AA) that power bacterial proliferation of the wild-type strain, while the ankB mutant is defective in this process and is
unable to grow despite formation of ER-remodeled replicative LCV. (4) During late stages of infection, the LCV becomes disrupted leading to bacterial
egress into the cytosol where the last 1–2 rounds of proliferations are completed. Upon nutrient depletion (see magnied box), RelA and SpoT are trig-
gered leading to increased level of ppGpp, which triggers phenotypic transition into a agellated virulent phenotype followed by lysis of the amoeba
and bacterial escape from the host cell. Excreted vesicles lled with bacteria are also released. The infectious particle is not known but may include ex-
creted Legionella-lled vesicles, intact Legionella-lled amoebae, or free Legionella that have been released from host cell. (5) Transmission to humans
occurs via aerosols generated from man-made devices and installations, such as cooling towers, whirlpools, and showerheads.

310 Virulence Volume 4 Issue 4
phase, the bacterium is undergoing exponential (E) growth; it
is non-motile and represses transmissive traits, such as lysosomal
evasion (Fig. 1).
64
The stringent-like response is triggered upon
transition of L. pneumophila into post-exponential (PE) growth,
when the bacteria become cytotoxic, motile, sodium-sensitive,
osmotic-resistant, and capable of lysosomal evasion.
63,64
These
phenotypic modulations are necessary to escape from the wasted
host and invade a new host to start a second cycle of intracellular
proliferation.
41,42,47,59,60,63-65
The transition between replicative and transmissive pheno-
types is highly orchestrated, and is governed by many factors that
are influenced by intracellular nutrient levels.
7,24,63,66
Upon amino
acid depletion, uncharged bacterial tRNAs activate RelA to syn-
thesize the bacterial alarmone 3',5'-bispyrophosphate (ppGpp),
a master regulator of numerous genes of L. pneumophila, which
triggers phenotypic modulations upon transition into the PE
phase.
63
SpoT, a bifunctional synthetase/hydrolase that responds
to a variety of stimuli, such as fatty acid starvation, also synthe-
sizes ppGpp leading to increased levels of the alarmone (Fig. 1).
67
RpoS and several global response two-component regulators,
such as LetA/S and PmrA/B, function as downstream cascades
of regulatory networks that govern phenotypic modulations at
the PE phase.
61,66,68-70
Small non-coding RNAs, such as RsmY
and RsmZ, are induced at the PE phase by the regulatory cascade
of networks triggered by elevated ppGpp levels.
66,68,71
The RNA
polymerase interacting protein, DskA, also responds to increased
levels of ppGpp and other stress signals to coordinate phenotypic
modulations of L. pneumophila at the PE phase and its transmis-
sion to a new host.
67
In addition to triggering flagellation and various virulence-
related traits, elevated ppGpp levels result in upregulation of the
type IV-secretion components and many of its exported effec-
tors.
59-61
One of the Dot-Icm-translocated effectors important
in the intracellular infection of amoebae and human cells is the
eukaryotic-like AnkB,
33,44,45
which is temporally and differen-
tially regulated at the PE phase.
33,35,61,72
Therefore, complex cas-
cades of regulatory networks govern phenotypic transition at the
PE phase and most or all of these networks are under the direct
or indirect control of ppGpp.
In addition to phenotypic modulations at the PE phase,
L. pneumophila undergoes a differentiation cycle that is dimor-
phic, cycling between a replicating form and a planktonic spore-
like cyst form, designated as mature intracellular form (MIF).
73-75
The MIF is near dormant metabolically, resistant to detergents
and antibiotics, and is more invasive.
73
The MIFs are detectable
in HeLa cells but do not form in macrophages, which is likely
due to early apoptotic lysis within 1–3 d of the infection, while
the MIFs are formed later in HeLa cells.
73
The MIFs of L. pneu-
mophila germinate following entry into a susceptible eukaryotic
host cell or in rich media in vitro. It is possible that the MIFs
contribute to the ecology of L. pneumophila during starvation in
the water system when nutrients are depleted and the amoebal
hosts are encysted and not susceptible to infection.
Exploitation of conserved eukaryotic processes by the
eukaryotic-like AnkB effector of L. pneumophila. L. pneumoph-
ila harbors a plethora of eukaryotic-like effectors that interfere
with host processes by mimicking eukaryotic functions.
21,23,53,54
Many translocated effectors of L. pneumophila are functionally
and structurally similar to eukaryotic proteins and interact with
and disrupt various eukaryotic processes such as signaling, pro-
tein synthesis, apoptosis, posttranslational modification, vesicu-
lar trafficking, ubiquitination, and proteasomal degradation.
43
Among the ~300 effectors of L. pneumophila, AnkB is the only
effector known to be indispensable for the intracellular infec-
tion of both human cells and amoebae, and the biological func-
tion of this effector has been deciphered. It is not surprising that
recent studies on the AnkB effector and its exploitation of mul-
tiple highly conserved eukaryotic processes may just be the tip of
the iceberg of our continued unraveling of L. pneumophila–host
interaction and its evolution from invading amoebae to invading
human cells and causing pneumonia.
The AnkB effector harbors multiple eukaryotic domains that
enable this protein to hijack a number of evolutionarily conserved
eukaryotic processes, and is essential for intracellular prolifera-
tion of L. pneumophila in amoebae and human cells and for viru-
lence in the mouse model.
33,44,45,76
The AnkB effector harbors two
Ankyrin domains (ANK), 33-residue repeats involved in protein-
protein interactions, and is the most common domain in eukary-
otic proteins.
56
AnkB also contains a C-terminal eukaryotic
CaaX motif (C, cysteine; a, aliphatic amino acid; X,I any amino
acid) that allows the protein to be lipidated through farnesylation
by the host farnesyltransferase (FTase), which anchors AnkB
into the LCV membrane (Fig. 2).
58,77-79
Farnesylation is a type
of prenylation that covalently links a 15-carbon lipid moiety to a
conserved cysteine residue within the C-terminus “CaaX” motif,
which confers hydrophobicity enabling the lipidated protein to
be anchored into the lipid bi-layer of eukaryotic membranes.
58
However, there is variation in the C-terminus CaaX motif of
AnkB, as some isolates, such as the Paris strain of L. pneumoph-
ila, have a truncated C-terminus without a CaaX motif.
45
The
biological relevance of this variation is still to be determined.
Ubiquitination of proteins is a highly conserved eukaryotic
post-translation modification that is mediated by three enzymes
(E1–E3); E1 is the activating enzyme which transfers a 76-amino
acid ubiquitin polypeptide to the conjugating enzyme (E2) while
the E3 ubiquitin ligase links ubiquitin to the target protein.
7,76
Polyubiquitin is formed by linking ubiquitin monomers through
one of the 7 lysine (K) residues of ubiquitin. Polyubiquitination
through K
48
linkages targets the modified protein for proteo-
lytic degradation by the proteasomes.
76
The AnkB effector is a
bona fide F-box protein that binds the E3 eukaryotic ubiquitin
ligase and functions as a platform for the assembly of K
48
-linked
polyubiquitinated proteins on the LCV (Fig. 2),
44,45
which occurs
within a few minutes of bacterial entry.
45,80
Proteasomal degrada-
tion of K
48
-linked polyubiquitinated proteins results in increased
cellular levels of amino acids (Fig. 2),
7,80
which are essential for
intracellular proliferation of L. pneumophila that is dependent on
amino acids as the major source of carbon and energy to feed the
tri-carboxylic acid (TCA) cycle.
81
The ankB mutant of L. pneumophila is severely defective in
intracellular proliferation in amoebae and human macrophages
due to the defect in assembly of K
48
-linked polyubiquitinated

www.landesbioscience.com Virulence 311
proteins decorating the LCV (Fig. 1).
7,45,80
Due to lack of pro-
teasomal degradation of K
48
-linked polyubiquitin during infec-
tion by the ankB mutant, cellular levels of amino acid do not
increase. This triggers a bacterial starvation response, mediated
by the induced expression of RelA and SpoT, and results in ele-
vated ppGpp levels.
7,80
Intracellular growth can be restored to the
ankB mutant within amoebae and human cells by supplement-
ing excess amino acids.
7,24,80
Thus, higher levels of cellular amino
acids are needed for intracellular replication of L. pneumophila.
Remarkably, supplementation of infected cells with certain sin-
gle amino acids, such as cysteine, reverses the growth defect of
the ankB mutant in amoebae and human cells. Interestingly, in
human cells cysteine is semi-essential and is the least abundant
amino acid, but in amoebae cysteine is essential.
24,80
Similar
to cysteine, supplementation of infected cells with pyruvate or
citrate to feed the TCA cycle, rescues the ankB mutant for intra-
cellular proliferation.
7,24,80
Interestingly, in vitro growth of L.
pneumophila in rich medium requires supplementation with 3.3
mM cysteine.
80
Therefore, AnkB is a remarkable example of an
effector involved in exploitation of multiple host processes that
are highly conserved in unicellular eukaryotes and mammals.
7
By promoting proteasomal degradation in amoebae and human
cells though the AnkB F-box effector, L. pneumophila generates
a gratuitous supply of cellular amino acids (Fig. 2), particularly
the limiting ones such as Cys, which is a metabolically favorable
source of carbon and energy for L. pneumophila to power intra-
cellular growth within amoebae and human cells.
24
Nutritional adaptation of L. pneumophila to amoebae.
Amino acids are the main sources of carbon and energy to feed
the TCA cycle of L. pneumophila, which has a defect in the gly-
colytic pathway, but carbohydrate metabolism plays a minor role
in contribution to central metabolism.
81
Cysteine and Serine are
particularly important to feed central metabolism of L. pneu-
mophila. Both amino acids are converted into pyruvate that feed
the TCA cycle, which is the primary pathway of carbon and
energy production in L. pneumophila.
24,80,81
L. pneumophila and
its primary host (Acanthamoebae or Dictyostelium discoideum) are
both auxotrophic for cysteine while L. pneumophila is also auxo-
trophic for arginine, isoleucine, leucine, methionine, valine, and
threonine.
7,24
Interestingly, toxicity of proteasomal inhibition in
human cells results from the lack of protein synthesis due to low
levels of limiting amino acids, particularly cysteine.
82
Therefore,
Figure 2. Nutritional and metabolic adaptation of L. pneumophila to the intracellular life within amoebae and human cells is facilitated by the AnkB
eector and its exploitation of multiple highly conserved eukaryotic processes. The AnkB eector is translocated into host cells by the Dot/Icm type
IV secretion system of L. pneumophila, and it is immediately farnesylated by the three host enzymes FTase, RCE1, and ICMT, that are recruited to the
LCV by the Dot/Icm system.
78
Farnesylation of AnkB results in its anchoring into the cytosolic face of the LCV membrane where it interacts with the
eukaryotic SCF1 ubiquitin ligase complex. The AnkB eector functions as a platform for the docking of K
48
-linked polyubiquitinated proteins to the
LCV. Proteasomal degradation of the K
48
-linked polyubiquitinated protein generates 2–24 amino acid (AA) peptides that are rapidly degraded by
oligo- and amino-peptidases. This generates a surplus of cellular amino acids within the cytosol of L. pneumophila-infected amoebae and human cells.
The amino acids are imported into the LCV through various host amino acid transporters present in the LCV membrane, including the neutral amino
acid transporter SLC1A5, which imports Cys, and subsequently into L. pneumophila through numerous ABC transporters and amino acid permeases
such as the threonine transporter PhtA.
94

312 Virulence Volume 4 Issue 4
through high nutritional dependence of L. pneumophila on host
limiting amino acids, such as Cys, and synchronization of amino
acid auxotrophy with its host, L. pneumophila synchronizes its
nutritional needs for growth with the availability of nutrients for
the host.
7,24
This remarkable nutritional adaptation could be what
allows the bacteria to be protected during amoebal encystation
upon nutrient depletion, leading to cessation of intracellular bac-
terial growth (Fig. 1). Encysted amoebae are thought to be pro-
tected against invasion by L. pneumophila.
75
Therefore, potential
growth and release of L. pneumophila from amoeba in an aquatic
environment in which amoebae have encysted in response to
nutrient depletion is unlikely to happen, since it would result in
free-living bacteria without a susceptible host, which may result
is eventual loss of bacterial viability. However, potential differen-
tiation into the MIF under starvation conditions may facilitate
continued presence of L. pneumophila in the aquatic environment
under these conditions.
73,74
Acquisition of eukaryotic genes through inter-kingdom
horizontal gene transfer. The long-term co-evolution of L. pneu-
mophila with various protists and metazoa has influenced the
genomic structure of this organism through inter-kingdom hori-
zontal gene transfer (HGT).
7,23,83,84
This long-term co-evolution
is likely what gave rise to the acquisition of eukaryotic host genes
encoding proteins with eukaryotic-like functions and structures.
Amoeba may act as a gene melting pot, allowing diverse micro-
organisms to evolve by gene acquisition and loss, and then either
adapt to the intra-amoebal lifestyle or evolve into new patho-
gens. Interestingly, mammalian F-box proteins do not have the
ANK domain, while F-box proteins from amoebae do.
7,24,5 6 ,76
Therefore, it is more likely that ankB had been acquired through
inter-kingdom HGT from a primitive eukaryotic host.
7,24,76
L. pneumophila is a naturally competent organism that takes up
DNA and can exchange DNA between bacteria through conju-
gation.
9,48,49
Long-term convergent evolution and modification of
the genes acquired through HGT, splicing of introns, acquisi-
tion of prokaryotic promoters and regulators, and translocation
motifs is likely what allowed eukaryotic-like proteins to become
translocated effectors with functional activities in the host cell.
83
It is to be expected that many of the eukaryotic-like proteins in
L. pneumophila are still undergoing convergent evolution through
modifications that might enable them to become translocated
and functionally active effectors.
7
Long-term co-evolution with its protozoan hosts has likely
contributed to the ability of L. pneumophila to cause dis-
ease in humans, perpetuated by changes in human lifestyle.
Understanding its association with amoebae will give us a bet-
ter understanding of how L. pneumophila causes human dis-
ease though exploitation of evolutionary conserved eukaryotic
processes.
7,24,80
Since L. pneumophila also exploits mammalian-
specific processes such as the inflammasomes and pro- and anti-
apoptosis,
85-92
it is likely that additional virulence properties have
been acquired by L. pneumophila to enhance its capacity to infect
humans. Since many other pathogens are detected within amoe-
bae, this primitive eukaryotic host may represent a reservoir for
many human pathogens.
93
Disclosure of Potential Conflicts of Interest
No potential conflicts of interest were disclosed.
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