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Annu. Rev. Microbiol. 2000. 54:567–613
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Copyright LEGIONELLA PNEUMOPHILA PATHOGENESIS: A
Fateful Journey from Amoebae to Macrophages
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M. S. Swanson and B. K. Hammer
Department of Microbiology and Immunology, University of Michigan Medical School,
Ann Arbor, Michigan 48109; e-mail: [email protected], [email protected]
Key Words stringent response, phagosome-lysosome fusion, virulence regulation,
intracellular pathogen, opportunistic
■ Abstract Legionella pneumophila first commanded attention in 1976, when investigators from the Centers for Disease Control and Prevention identified it as the culprit in a massive outbreak of pneumonia that struck individuals attending an American
Legion convention (84). It is now clear that this gram-negative bacterium flourishes
naturally in fresh water as a parasite of amoebae, but it can also replicate within alveolar
macrophages. L. pneumophila pathogenesis is discussed using the following model as
a framework. When ingested by phagocytes, stationary-phase L. pneumophila bacteria
establish phagosomes which are completely isolated from the endosomal pathway but
are surrounded by endoplasmic reticulum. Within this protected vacuole, L. pneumophila converts to a replicative form that is acid tolerant but no longer expresses
several virulence traits, including factors that block membrane fusion. As a consequence, the pathogen vacuoles merge with lysosomes, which provide a nutrient-rich
replication niche. Once the amino acid supply is depleted, progeny accumulate the
second messenger guanosine 30 ,50 -bispyrophosphate (ppGpp), which coordinates entry into the stationary phase with expression of traits that promote transmission to a
new phagocyte. A number of factors contribute to L. pneumophila virulence, including
type II and type IV secretion systems, a pore-forming toxin, type IV pili, flagella, and
numerous other factors currently under investigation. Because of its resemblance to
certain aspects of Mycobacterium, Toxoplasma, Leishmania, and Coxiella pathogenesis, a detailed description of the mechanism used by L. pneumophila to manipulate and
exploit phagocyte membrane traffic may suggest novel strategies for treating a variety
of infectious diseases. Knowledge of L. pneumophila ecology may also inform efforts
to combat the emergence of new opportunistic macrophage pathogens.
CONTENTS
LEGIONNAIRES’ DISEASE . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Epidemiology . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
An Airborne Respiratory Pathogen . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Histology . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Pontiac Fever . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
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Cell-Mediated Immunity . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
An Opportunistic Pathogen . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Amoebae, a Reservoir for Macrophage Pathogens . . . . . . . . . . . . . . . . . . . . . . . .
Contribution of Amoebae to Disease . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
REGULATION OF VIRULENCE . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Temperature . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Growth Phase . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Stringent Response Paradigm . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
THE INTRACELLULAR PATHWAY . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
The Nascent Phagosome . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Association with Endoplasmic Reticulum . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Maturation of the Replication Vacuole . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Transmission . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
LEGIONELLA PNEUMOPHILA VIRULENCE AS A PARADIGM FOR
OPPORTUNISTIC PATHOGENS OF MACROPHAGES . . . . . . . . . . . . . . . . . . .
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LEGIONNAIRES’ DISEASE
Epidemiology
Legionella pneumophila remains a significant cause of morbidity and mortality.
Indeed, the worst recorded outbreak of legionellosis occurred in February 1999,
at the Westfriese Flora Show in the Netherlands, where 231 people became ill and
21 died (236a). Legionellosis is usually acquired in the community, and it typically accounts for 2%–15% of all community-acquired pneumonias that require
hospitalization (39, 154, 164). Outbreaks of legionellosis remain newsworthy, but
usually <5% of the community-acquired Legionnaires’ Disease cases fit this description (154). The most common form of legionellosis is sporadic Legionnaires’
Disease, which often escapes diagnosis. In the United States, only ∼1000 cases
are reported annually (175), although 20,000 cases of Legionnaires’ disease are
estimated to occur (154).
Nosocomial legionellosis is often more severe, and its incidence more dramatic.
According to data from the passive surveillance system of the Centers for Disease
Control and Prevention, 23% of the legionellosis cases reported from 1980 to
1989 may have been nosocomial; of these, 37% were linked to outbreaks, often
in community hospitals. In this situation, the consequences of legionellosis are
grave; fatality rates can approach 50% (39, 154).
Although 42 species of legionellae have been described, >90% of the isolates
associated with Legionnaires’ disease are L. pneumophila (22, 154, 164). More
specifically, from 1980 to 1989, L. pneumophila serogroup 1 was identified in
71.5% of the cases in which a legionellosis strain was isolated (154). In addition, a large number of Legionella-like amoebal pathogens have been described
(191). Although the ecology and genetic composition of these Legionella-like
strains are similar to the legionellae, they are rarely associated with disease (191).
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Consequently, laboratory studies of Legionella pathogenesis have focused primarily on L. pneumophila, as does this review. A comprehensive description of the
genus Legionella is provided by Benson & Fields (22).
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An Airborne Respiratory Pathogen
People most often become infected with L. pneumophila after inhaling aerosols of
contaminated water droplets (165). For example, when flooding damaged
its main cooling system, a Memphis hospital was forced to activate emergency
backup equipment. An outbreak of nosocomial Legionnaires’ disease ensued, and
its duration correlated with the period when the secondary cooling system was
in operation. Furthermore, analysis of the airflow patterns and the location of
Legionnaires’ disease patients within the hospital was consistent with the hypothesis that a particular water-cooling tower was the source of aerosols contaminated
with L. pneumophila (63). However, as is typical of many outbreaks, the strain of
L. pneumophila isolated from the patients was also present in the hospital’s potable
water supply. Indeed, it is now well established that legionellae are ubiquitous in
both natural and engineered water supplies (79, 168, 230). As a result, it is difficult
to establish unequivocally that cooling towers are the source of infectious aerosols
in this and other outbreaks of legionellosis (165). In fact, a variety of equipment
that disperses water has been implicated as the source of infection, including clinical respiratory devices, whirlpools, showers, and even the mist machines found
in the produce sections of many grocery stores (16, 29, 130, 139, 165).
Histology
When inhaled into the lung, L. pneumophila can cause acute alveolitis and bronchiolitis. In patients with Legionnaires’ disease, the alveolar exudate typically
consists of equal numbers of polymorphonuclear cells and macrophages, with fibrin, red blood cells, proteinaceous material, and significant amounts of cellular
debris (99, 239). The many intact rod-shaped bacilli are predominantly intracellular, located within cytoplasmic vacuoles of cells that often cannot be identified
with certainty. Some bacteria are located in phagolysosomes, many are lying free
in the cytoplasm, and others reside within membrane-bounded structures resembling dilated endoplasmic reticulum (99). Histological studies of lung exudates
obtained from a guinea pig infection model revealed that the fate of intracellular
L. pneumophila depends on its phagocyte host. Within macrophages, >95% of
the L. pneumophila bacilli were intact; clusters of ≥20 bacilli were seen within
structures resembling endoplasmic reticulum. On the other hand, L. pneumophila
within neutrophils most often appeared partially degraded, with their membranes
disrupted (135). Consistent with these and numerous other histological reports,
many investigators have demonstrated intracellular growth of L. pneumophila in
cultured primary macrophages and a wide variety of cell lines, including some
derived from monocytes, fibroblasts, and epithelial cells (77).
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Pontiac Fever
L. pneumophila causes another illness, Pontiac fever, which differs from Legionnaires’ disease in several respects (100, 136, 218). It is a self-limiting, flulike
disease that has been recognized only when outbreaks occur. The attack rate
is high, but the mortality rate is zero. After an incubation period of ∼36 h, patients
suffer fever, chills, dry cough, myalgia, malaise, and headache, not pneumonia. It
is interesting that, although Pontiac fever patients seroconvert to L. pneumophila,
the microbe has never been isolated (164). Thus, Pontiac fever may be caused by
a bacterial toxin or may represent the sequelae of a robust immune response to the
pathogen. The pathogenesis of Pontiac fever will not be discussed further here.
Cell-Mediated Immunity
L. pneumophila is relatively resistant to innate and humoral immune responses. For
example, although it readily binds complement component C3, L. pneumophila
is resistant to complement-mediated killing, even when specific immunoglobulin is present (127). Moreover, L. pneumophila that have been treated with the
opsonins complement and specific antibodies interact efficiently with polymorphonuclear cells, but are not killed. In one experiment, after a serum-resistant
encapsulated Escherichia coli strain was incubated for 1 h with 10% fresh normal serum, L. pneumophila-specific antiserum, and polymorphonuclear cells, the
number of colony-forming units (CFUs) decreased by 2.5 logs. In contrast, a similar treatment reduced L. pneumophila CFUs by only 0.5 log (127). In a similar
manner, monocyte bactericidal activity is only modestly enhanced by complement
and specific antibody (127). Under some conditions, L. pneumophila may directly
impair the oxidative killing by phagocytic host cells; a toxin purified from culture
supernatant inhibited polymorphonuclear cell killing of E. coli and reduced the
oxidative capacity of the cells (86, 149, 197). After phagocytosis, it is important
that bacteria that had been subjected to opsonization with specific immunoglobulin
replicated intracellularly as efficiently as did L. pneumophila treated with normal
serum (127). Together, data from these leukocyte models of infection indicate
that complement, specific antibody, and polymorphonuclear cells are ineffective
at clearing legionellosis.
Instead, cell-mediated immunity controls lung infections of L. pneumophila
(reviewed in 85). This type of host response was implicated initially by comparing
the activity of peripheral blood mononuclear cells obtained from patients who had
recovered from legionellosis to those of age- and sex-matched controls (121).
The lymphoproliferation of patient cells stimulated with L. pneumophila antigen
was consistently more robust than that of control cells, although mononuclear
cells from both populations responded similarly to the nonspecific plant mitogen
concanavalin A, as judged by incorporation of 3H-thymidine. Moreover, when
compared to controls, supernatants obtained from cultured mononuclear cells of
former patients that had been exposed to L. pneumophila antigen readily activated
naı̈ve mononuclear cells to restrict L. pneumophila replication.
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LEGIONELLA PNEUMOPHILA PATHOGENESIS
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A critical role for interferon-γ (IFN-γ ) as the cytokine activator of macrophages
was later demonstrated in experimental human monocyte models. Cultures of human peripheral blood monocytes or alveolar macrophages support L. pneumophila
replication. After treatment with recombinant IFN-γ for 1 h, the activated phagocytes inhibit intracellular replication of L. pneumophila (25, 169). However, IFN-γ
activation does not enhance bacterial killing, even in the presence of specific antibody, nor does it prevent formation of L. pneumophila replication vacuoles (25).
Instead, activated macrophages restrict L. pneumophila growth by an irondependent mechanism. The capacity of IFN-γ -activated human peripheral blood
monocytes to inhibit replication of L. pneumophila is reversed when the cultures
are supplemented with iron transferrin (36). Thus, the size of the labile iron pool
in activated monocytes may not be sufficient to support L. pneumophila replication. Indeed, human peripheral blood monocytes activated by IFN-γ expressed
73% fewer transferrin-binding sites than nonactivated control cells, as determined
by Scatchard analysis of 125I-transferrin binding (37). In a similar manner, rat
alveolar exudate macrophages that were activated in vivo by exposure to inhaled
L. pneumophila expressed fewer transferrin receptors than did resident alveolar
macrophages (207). The cellular pathway that delivers iron to the pathogen vacuole has yet to be defined.
An Opportunistic Pathogen
Epidemiological studies of Legionnaires’ disease also indicate that a robust immune response is sufficient to clear L. pneumophila infections (84, 108; reviewed
in 217). For example, the hotel employees on duty during the 1976 Legionnaires’
convention generally were seropositive for L. pneumophila antibodies, but asymptomatic (84). Typically, those who become ill are of advanced age and have sustained damage to the host defenses that normally protect lungs from infection
(154, 239). Some of the most common risk factors for legionellosis are cigarette
smoking, emphysema or other chronic lung diseases, lung and hematologic malignancies, and clinical immunosuppression or cytotoxic chemotherapy (39, 154).
Thus, L. pneumophila is a classic opportunistic pathogen.
From the microbe’s perspective, infection of the human lung is invariably
calamitous. Although more than 1000 cases of Legionnaires’ disease have been
reported annually in the United States since 1987 (175), not a single instance of
person-to-person transmission has been observed. Thus, unlike many other human
respiratory pathogens such as Haemophilis influenzae, Bordetella pertussis, or Mycobacterium tuberculosis, L. pneumophila is not adapted to the human host. In the
absence of transmission to a new host, genetic variants that survive the strong selective pressure exerted by activated alveolar macrophages cannot be maintained;
mutations that promote survival and replication only in the human lung will not
persist in the species’ genome. Instead, the capacity of L. pneumophila to establish infection within the lung is the consequence of selective pressure applied by
a different class of professional phagocytes: amoebae.
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Amoebae, a Reservoir for Macrophage Pathogens
The evidence that amoebae have acted as an evolutionary incubator for the emergence of L. pneumophila as an opportunistic pathogen of alveolar macrophages
was obtained by epidemiological and cell biological and genetic studies. Pioneering work by Rowbotham (189) demonstrated the pathogenicity of L. pneumophila
for amoebae of the genera Acanthamoeba and Naegleria, common inhabitants of
soil and water. L. pneumophila enjoys a wide host range: >13 species of amoebae
and two species of ciliated protozoa can support its growth (reviewed in 77). It is
important to note that protozoa, which are susceptible to L. pneumophila infection,
frequently contaminate potable water supplies, especially heated reservoirs. For
example, in a survey of water samples obtained from the plumbing and cooling
towers of five hospitals in Paris, 71% were positive for amoebae, and 47% for legionellae (168). Furthermore, when these water samples were incubated, bacterial
multiplication occurred, provided the samples also contained amoebae. Indeed, a
number of epidemiological studies have linked water contaminated with both
L. pneumophila and protozoan phagocytes to outbreaks of legionellosis (16, 29, 79).
The capacity to replicate in protozoa also correlates with bacterial virulence. In
a survey of 17 legionellae isolates, Fields et al (78) found that each strain that
caused pathology in guinea pigs also replicated within the protozoan Tetrahymena
pyriformis. Therefore, as its natural reservoir, protozoan phagocytes amplify the
L. pneumophila population in fresh and potable water supplies.
The life cycle of L. pneumophila in amoebae strongly resembles that observed in
macrophages. As discussed below, in both protozoan and mammalian phagocytes,
coiling phagosomes engulf L. pneumophila. These vacuoles neither acidify nor
fuse with the lysosomal compartment. Instead, the membrane-bounded microbe
associates with endoplasmic reticulum and replicates in high numbers. Thus, in
both mammalian and protozoan phagocytes, L. pneumophila appears to interact
with host organelles in the same striking manner. The similar cell biology also
indicates that the virulence of L. pneumophila for alveolar macrophages is a consequence of its evolution as a parasite of amoebae.
Many of the L. pneumophila factors that promote its survival and replication
in macrophages are also required for growth in amoebae. For example, a peptidyl prolyl isomerase, the macrophage infectivity potentiator Mip, increases the
efficiency at which L. pneumophila infects human macrophages, Hartmannella,
and Tetrahymena (46, 47). Thirteen intracellular multiplication (icm) genes, identified by insertion mutations that conferred defective bacterial growth in monocytic U937 cells, are also required for replication within Acanthamoeba castellanii (201). Likewise, seven transposon mutants isolated as defective for flagellar production were avirulent in both Hartmanella spp. and in monocytic U937
cells (176). Finally, Gao et al (90) screened a large collection of transposon mutants for the capacity to kill U937 cells and A. polyphaga. The 89 protozoan
and macrophage infectivity ( pmi) mutants they isolated were defective for replication in both protozoan and mammalian phagocytes, although virtually all of
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these mutants replicated in minimal medium. Together with the epidemiological
and cell biological data, results of these genetic studies indicate that the L. pneumophila strategy that evolved to promote its growth in amoebae also works in
human macrophages.
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Contribution of Amoebae to Disease
L. pneumophila isolated from amoebae express a variety of phenotypes that could
increase both the incidence and complications of disease in humans. For example, the progeny that escape from lysed amoebae are highly motile (190), a trait
that presumably facilitates transmission and also correlates with the expression
of other virulence traits (38, 109, 176). Compared to bacteria cultured in broth,
cells obtained from A. castellanii enter human peripheral blood monocytes and the
monocytic RAW 264.7 and THP-1 cell lines more efficiently and by a pathway that
is complement independent (48, 49). In addition, bacteria grown in phagocytes are
more resistant both to chemical biocides important for maintaining a safe water
supply and to antibiotics used to treat pneumonia (17, 19). For example, 71%
of bacteria obtained from Acanthamoeba polyphaga survived a 24h exposure to
5-µg/ml rifampin, a dose that killed >99.9% of bacteria that had been cultured in
broth. Therefore, replication in amoebae not only may increase bacterial numbers,
but also virulence and resistance to antimicrobial agents.
The hypothesis that amoebae exacerbate lung infections has been tested directly
in experimental systems that integrate both the protozoan and mammalian hosts
of legionellae. Brieland, Engleberg, and their colleagues (30) developed a murine
model of legionellosis, a self-limiting infection that mimics closely key aspects of
the human infection. After inoculation into the trachea of A/J mice, L. pneumophila
replicate primarily within alveolar mononuclear phagocytes for ∼2 days, and the
number of CFU typically increases 10-fold. During the next 5 days, the infection
is gradually resolved by an IFN-γ and tumor necrosis factor-α (TNF-α)-dependent
immune response marked by infiltration of the alveoli by polymorphonuclear cells
and macrophages (30, 33). By 7 days, the architecture of the lung is essentially
normal.
Mice inoculated with a mixture of bacteria and amoebae develop more severe
disease than those infected with either L. pneumophila or Hartmannella vermiformis alone (31, 48). In particular, measures of bacterial CFU, lung pathology,
and animal mortality were significantly increased. Experimental evidence indicates that one way amoebae contribute to pathogenesis is as a host for bacterial
replication. After intratracheal inoculation of mice with L. pneumophila-infected
amoebae, the yield of bacteria in the lungs was nearly 100-fold higher than that obtained from mice similarly infected with either bacteria or amoebae alone (32, 48).
However, a similar dramatic increase in the bacterial yield was not observed in
parallel experiments performed with L. pneumophila mutants that were defective
for growth in cultured H. vermiformis, but competent to replicate in the human
monocytic U937 cell line (32). Thus, amoebae in the inoculum appear to function
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as a site for bacterial replication, effectively amplifying the infectious dose. When
amoebae are present in the inoculum, more TNF-α and IFN-γ is secreted in the
lung, yet fewer lymphocytes and mononuclear phagocytes are recruited (31, 32).
Accordingly, protozoa could worsen the lung damage caused by L. pneumophila
infection by more than one mechanism. By amplifying the number of L. pneumophila, amoebae could increase the dose of bacterial cytotoxin(s) produced in
the lung. Alternatively, amoebae may contribute to pathogenesis indirectly, by
triggering a hyperactive, ineffective immune response.
L. pneumophila pathogenesis clearly illustrates the capacity of amoebae to
serve as a selective pressure and a natural reservoir for opportunistic pathogens of
macrophages. Amoebae in the environment have also been found to harbor bacteria of several other pathogenic genera, including Mycobacterium, Sarcobium,
Vibrio, Pseudomonas, Burkholderia, Listeria, and Franciscella, as well as rickettsialike endosymbionts (reviewed in 34, 238). As shown for L. pneumophila,
the macrophage pathogen Mycobacterium avium can be cultured in A. castellanii
and A. polyphaga (50, 213), and the amoebae-grown bacteria are more virulent
in a mouse model of infection (50). Thus, amoebae species that flourish in natural and potable water supplies are likely to continue to be a source of emerging
opportunistic pathogens (34, 238).
REGULATION OF VIRULENCE
To survive, bacteria must continually sense and adapt to environmental change.
Indeed, many pathogens respond to extracellular signals by coordinately expressing multiple virulence proteins with varied functions (reviewed in 62, 157, 160).
For example, to colonize humans, Salmonella typhi must survive ingestion and
passage through the gastrointestinal tract, traverse specialized M cells of the intestinal epithelium, and survive within macrophages that populate the underlying
lymphoid tissue. Salmonellae also survive and replicate outside a mammalian
host, another indication of their versatility. By responding to different cues, including the supply of nutrients, O2, Mg2+, and iron, as well as pH and osmolarity
(82, 227a, 211), Salmonella spp. sequentially express specific virulence factors at
particular stages of the infection (reviewed in 104).
Compared with Salmonella spp., L. pneumophila leads a simple life. Extracellular growth has not been documented. Moreover, although this bacterium
can establish infections in the lungs of immunocompromised people, person-toperson transmission has not been documented (77). Instead, the life cycle of
L. pneumophila revolves around single-celled, freshwater protozoa. Therefore,
the selective pressures most likely to have determined the pattern of L. pneumophila gene expression are linked to its encounter with aquatic protozoa. By
this reasoning, particular virulence regulons may be dedicated to safe entry, robust
intracellular replication, survival in fresh water, and efficient transmission from
one phagocyte to another. Below, we first summarize the evidence that growth
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conditions determine the phenotype of L. pneumophila. Second, we describe a
model in which a stringent responselike mechanism induces virulence expression
when the amino acid supply is depleted.
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Temperature
Temperature is one condition that affects the motility, piliation, and virulence of
L. pneumophila cultured in bacteriological medium. Cells express more flagellin
RNA and protein and assemble more flagella when incubated at 30◦ C than at 37◦ C
(114, 171). Similarly, production of type IV pili and transcription of the pilBCD
pilin locus occurs when bacteria are cultured at 30◦ C, but is minimal at 37◦ C
(146). Adherence is also temperature dependent: twofold more L. pneumophila
associate with guinea pig alveolar macrophages when the bacteria are incubated
for 1 h prior to infection at 25◦ C, compared to incubation at 41◦ C (69). On the other
hand, L pneumophila cultured at 24◦ C were less virulent than bacteria cultured
at 37◦ C, as judged by measuring their 50% lethal dose (LD50) in a guinea pig
model of infection (155). The finding that motility and adherence is optimal at
temperatures less than 37◦ C is consistent with a dominant role for the aquatic
environment on the evolution of L. pneumophila as an intracellular parasite. In
nature, it is unlikely that L. pneumophila must adapt to temperatures as high as
37◦ C. Instead, temperature elevation may mimic an environmental stress to which
L. pneumophila responds.
Growth Phase
Conditions within the phagocyte vacuole clearly influence the L. pneumophila
phenotype (Figure 1). Compared to bacteria grown in microbiological medium,
L. pneumophila released from eukaryotic cells are short, thick, and highly motile;
they have a smooth, thick cell wall, a higher β-hydroxybutyrate content, different staining properties, and express a different array of proteins and genes
(3, 5, 49, 70, 190). Also, compared to bacteria isolated from broth, A. polyphaga—
grown cells have a different composition of membrane fatty acids, profile of
lipopolysaccharide and outer membrane proteins, and susceptibility to proteinase
K (18). After growth in phagocytes, L. pneumophila are also more resistant to
biocides and antibiotics (17, 19), more invasive for mammalian cells, and more
virulent in mouse models of infection (32, 48, 49). Finally, after replicating for
10–12 h within monocytic U937 cells, L. pneumophila begin to express stress
proteins (3). By analogy to Salmonella, it is likely that multiple environmental
signals determine the phenotype of intracellular L. pneumophila.
The growth phase has a dramatic effect on the phenotype of L. pneumophila cultured in phagocytes and in broth (Figure 1). By microscopic observation of infected
amoebae, Rowbotham (190) first noted that the intracellular life cycle of L. pneumophila consists of two distinguishable phases. After a period of replication,
L. pneumophila enter an “active infective phase,” marked by their synchronous
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Figure 1 Model for the Legionella pneumophila life cycle in amoebae. After a period of
intracellular replication, L. pneumophila converts to a virulent form and expresses a number
of traits that promote survival in the environment and transmission to a new host. If inhaled
into the lung, Legionella pneumophila can replicate within alveolar macrophages. In the
absence of a robust cell-mediated immune response, pneumonia ensues.
conversion to highly motile short rods that were observed to escape lysed host
cells and disperse in culture. Recently, this paradigm has been confirmed and bolstered by phenotypic and molecular studies of L. pneumophila cultured in broth
and in macrophages (38, 109). Unlike replicating cells, bacteria obtained from
postexponential phase cultures of L. pneumophila express a number of traits that
have been correlated with virulence, including sodium-sensitivity, cytotoxicity,
osmotic resistance, motility, and the capacity to evade phagosome-lysosome fusion. Similarly, during the replication period in macrophages, L. pneumophila are
sodium resistant and do not express the flaA gene or produce flagella; concomitant with the macrophage lysis that ends the infection cycle, intracellular bacteria
express flaA and become flagellated and sodium sensitive. Amino acid limitation appears to induce the virulent phenotype, because exponential phase cells
convert to the virulent phenotype when incubated in postexponential phase culture supernatant, except when the supernatant is supplemented with amino acids.
Accordingly, we have postulated that when nutrient levels and other conditions
are favorable, L. pneumophila replicates within its specialized vacuole. When
amino acids become scarce, intracellular bacteria coordinately express several
traits that facilitate escape from the depleted cell and transmission to a new host
(Figure 1; 38, 109).
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Stringent Response Paradigm
For E. coli, a limited amino acid supply triggers the stringent response, a developmental pathway that promotes long-term survival in adverse conditions (reviewed
in 40). A rapid arrest of the growth and synthesis of proteins and stable RNA
molecules is coordinated with the induction of the stationary-phase regulon by
guanosine 30 ,50 -bispyrophosphate (ppGpp). Uncharged transfer RNAs (tRNAs)
bound to the ribosome activate the ppGpp synthetase, RelA. The subsequent accumulation of ppGpp increases the amount of the stationary-phase σ factor RpoS
(97), and stationary-phase genes are expressed. By this mechanism, E. coli alters
its physiology to tolerate a nutrient-poor environment.
In L. pneumophila, a similar stringent-response pathway appears to coordinate expression of virulence with entry into the stationary phase (109; Figure 2).
Two key observations support this model. First, L. pneumophila accumulates
the second messenger ppGpp when cultured in conditions that induce virulence
expression; namely, in the postexponential phase and in response to amino acid
starvation. Second, in response to the gratuitous expression of the E. coli relA
gene, L. pneumophila accumulates ppGpp, and the bacteria then express a number
of virulence traits, independent of the cell density or nutrient supply. Therefore,
Figure 2 A stringent response model for Legionella pneumophila virulence regulation. When amino acids are limiting, uncharged tRNAs activate RelA, a guanosine 30 ,50 bispyrophosphate synthetase. Accumulation of ppGpp coordinates entry into the stationary
phase with expression of virulence traits that promote transmission to a new host. Some
effectors of virulence are predicted to be substrates for type II and type IV secretion systems.
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L. pneumophila belongs to a class of environmental microbes that have co-opted
the stringent-response signal transduction pathway to coordinate a survival strategy specific to its lifestyle. For Myxococcus xanthus, ppGpp accumulation initiates the formation of a multicellular fruiting body that subsequently differentiates
into hardy myxospores (111). Bacillus subtilis that is starved for amino acids
accumulates ppGpp, which induces expression of stress response proteins that
may promote sporulation (235). In Streptomyces coelicolor, ppGpp accumulation
plays a role in antibiotic production and the pigmentation characteristic of mature
spores (42). For L. pneumophila, when nutrients are limited within its host cell,
transmission to a new phagocyte is paramount.
By analogy to E. coli, the stationary-phase σ factor RpoS may coordinate
expression of the L. pneumophila virulence regulon. Indeed, RpoS is required
for maximal virulence of several pathogens, including Salmonella (74), Shigella
flexneri (208), toxigenic E. coli (231), and phytopathogenic Erwinia carotovora
(166). Hales & Shuman (107) recently cloned the L. pneumophila rpoS gene and
characterized an rpoS transposon insertion mutant strain. The mutant replicated
as well as wild-type L. pneumophila within monocytic HL60 and THP-1 cells,
but it was attenuated for virulence in A. castellanii cultures. According to the
stringent-response paradigm, RpoS functions primarily to coordinate entry into
stationary phase. Consequently, in L. pneumophila, RpoS may be dispensable
for replication but important for efficient transmission to a new phagocyte or for
survival in fresh water, traits that may be critical for efficient infection in amoebae
experimental models. Consistent with this hypothesis, one intracellular growth
mutant of L. pneumophila that is defective for expression of an entire panel of
virulence traits harbors an in-frame deletion allele of rpoS (11, 109). Therefore,
it is of interest to determine whether rpoS null mutants express postexponential
phase activities implicated in L. pneumophila transmission, including cytotoxicity,
osmotic resistance, motility, and evasion of phagosome-lysosome fusion. The
stringent-response paradigm provides a conceptual framework for the extensive
phenotypic variation that has been documented for L. pneumophila cultured under
different conditions (Figure 1).
THE INTRACELLULAR PATHWAY
The Nascent Phagosome
Coiling Phagosomes
Macrophages and amoebae engulf L. pneumophila within coils of plasma membrane (27, 124). However, this unusual mode of entry does not appear to be
necessary or sufficient for intracellular survival of L. pneumophila in professional
phagocytes. Heat-killed, fixed, and avirulent L. pneumophila are also ingested
within coiled phagosomes, but these particles are delivered to the endosomal
compartment (27, 123, 125). Conversely, L. pneumophila that have been opsinized
with specific antibody form conventional phagosomes, but evade lysosomes (124).
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LEGIONELLA PNEUMOPHILA PATHOGENESIS
579
In addition, although L. pneumophila replicate in H. vermiformis, coiling phagosomes have not been detected in these cells (2). Coiling phagocytosis also was not
observed for the virulent Knoxville 1 strain of L. pneumophila (183).
Coiling phagocytosis has been observed for a number of other microbes, including Leishmania donovani, Borrelia burgdorferi, various spirochetes, trypanosomatids, and yeasts (43, 186, 187). Based on their detailed ultrastructural studies of
coiled and conventional phagosomes, Rittig and colleagues (186) have proposed
that coiling phagosomes are a direct consequence of a perturbation to conventional
circumferential phagocytosis. According to this model, when the membranes of
pseudopods which surround a particle fail to fuse, whorls of closely apposed plasma
membrane form. Consistent with this hypothesis, some microbes that are engulfed
within coiled phagosomes are also known to inhibit its subsequent fusion with
lysosomes. For example, Leishmania promastigotes express on their surface an
abundant negatively charged lipophosphoglycan that blocks phagolysosome maturation (57, 226). Similarly, live and formalin-fixed L. pneumophila form coiled
phagosomes, and both particles delay phagosome maturation, presumably owing
to an inhibitory factor on the bacterial surface (AD Joshi, S Sturgill-Koszycki,
MS Swanson, unpublished observations). Whether the L. pneumophila surface
molecules that interfere with phagosome maturation also affect phagosome architecture can be investigated directly once this class of virulence factors is identified.
Isolation of the Nascent Phagosome
The composition of L. pneumophila coiling phagosomes differs markedly from
plasma membrane (Table 1). Newly formed L. pneumophila phagosomes contain
the plasma membrane proteins 50 -nucleotidase and complement receptor CR3 (51);
however, these vacuoles lack other protein residents of the plasma membrane,
including MHC class I and class II molecules and alkaline phosphatase (51, 52).
Moreover, as they age, L. pneumophila phagosomes lose some host proteins. The
majority of the L. pneumophila phagosomes lack 50 -nucleotidase activity 1 h after
formation and have reduced levels of CR3 (51). Accordingly, Clemens & Horwitz
(52) postulated that during phagocytosis of L. pneumophila, membrane proteins
are sorted rapidly in such a manner that the membranes that surround the bacterium
are markedly different from the plasma membrane.
Within minutes of formation, the coiling phagosome resolves to a vacuole
with a single membrane. However, the morphology of the nascent vacuole in
macrophages has an unusual feature: L. pneumophila phagosomes fixed 15 or 60
min after phagocytosis have numerous smooth vesicles attached to their cytoplasmic face (122). Based on kinetic studies of L. pneumophila phagosome maturation,
described in detail below, this population of small vesicles does not deliver cargo
from the endosomal compartment. Instead, vesicles could bud from the L. pneumophila vacuole, selectively removing host proteins critical for recognition or
activation of the endocytic fusion machinery, thereby preventing phagolysosome
maturation. Alternatively, the vacuoles attached to the L. pneumophila phagosome
may represent aborted membrane fusion or fission reactions.
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TABLE 1
Maturation of the Legionella pneumophila phagosome
Age
Features
<5 min
Coils
Contains
Lacks
27, 124
51
CR3a
50 nucleotidasea
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5 min–2 h
1–6 h
Attached vesicles
Mitochondria
Alkaline phosphatasea
MHC class Ia
MHC class IIa
51
51
51
122
50 nucleotidasea
Transferrin receptora
51
133f
MHC class Ia
MHC class IIa
51
51
Rab7b
LAMP-1b, c
Cathepsin Dc
Texas Red-ovalbumind
Alexa Fluor-streptavidind
CM-DiId
192
133f, 192
133f
133f
133f
133f
pH 6.1
Transferrin receptora
Acid phosphatasec
CD63b, c
Colloidsd
LAMP-1b, c
Texas Red-ovalbumind
∼4–12 h
Ribosomes
BiP e
ER antigene
LAMP-1b, c
Cathepsin Dc
Texas Red-ovalbumind
Fluorescein-dextrand
∼12–24 h
BiP e
a
Plasma membrane, early endosomes
b
c
f
Late endosomes
Lysosomes
d
e
Lysis
Endocytic probe
Endoplasmic reticulum
AD Joshi, S Sturgill-Koszycki, MS Swanson, unpublished observations
g
S Sturgill-Koszycki & MS Swanson, unpublished observations
126
52
27, 123
52
24, 27, 52, 123
52, 222
222
122
2, 221
221
220g
220g
220g
pH 5.5
LAMP-1b, c
Cathepsin Dc
Texas Red-ovalbumind
Fluorescein-dextrand
∼24–28 h
Reference(s)
2
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LEGIONELLA PNEUMOPHILA PATHOGENESIS
581
The vacuoles that harbor L. pneumophila differ from conventional phagosomes
in two other important respects: they do not acidify or fuse with lysosomes
(123, 126). Since the early studies of Horwitz, several laboratories, using a variety of methods, have established clearly that L. pneumophila phagosomes aged
5 min to 8 h do not acquire lysosomal markers (Table 1). Electron microscopic
studies indicated that lysosomes labeled by acid phosphatase cytochemistry or
electron-dense colloids do not fuse with L. pneumophila phagosomes (23, 52, 123).
Cryosection immunogold localization of CD63, LAMP-1 (lysosome-associated
membrane protein), LAMP-2, and cathepsin D demonstrated that bacterial phagosomes do not acquire these late endosomal and lysosomal proteins (52). Finally,
fluorescence microscopic assays confirmed that L. pneumophila phagosomes do
not contain LAMP-1 and demonstrated further that they do not acquire Texas
Red-ovalbumin preloaded into lysosomes by pinocytosis (222). Similarly, after
ingestion by A. castellanii, virulent L. pneumophila reside in vacuoles that do not
acquire lysosomal characteristics, including host acid phosphatase and ferritin that
had been delivered by endocytosis to the lysosomal compartment (27).
Moreover, young vacuoles containing virulent L. pneumophila appear to be
completely isolated from the endosomal compartment (Table 1). The majority of
phagosomes that are aged 5–90 min lack LAMP-1 and Rab7, a monomeric GTPbinding protein that acts as a positive regulator of fusion between the early and late
endosomal compartments (76, 192, 209, 222). Furthermore, vacuoles containing
virulent L. pneumophila aged 5–60 min do not interact with the early endosomal
compartment, as judged by the absence of transferrin receptors (52, AD Joshi,
S Sturgill-Koszycki, MS Swanson, unpublished observations) and their failure to
accumulate the endocytic tracers Texas Red-ovalbumin, the lipid dye CM-DiI,
or Alexa Fluor-streptavidin, markers that were readily detected in phagosomes
containing polystyrene beads or exponential phase L. pneumophila (AD Joshi, S
Sturgill-Koszycki, MS Swanson, unpublished observations). Therefore, to survive
in macrophages, L. pneumophila appears to employ a strategy similar to that
of Toxoplasma gondii, which triggers formation of a vacuole that is completely
separate from the endocytic network (162).
Virulent L. pneumophila blocks maturation of its own phagosome with no
apparent effect on phagolysosome formation elsewhere within the phagocyte.
Vacuoles harboring L. pneumophila remain at a neutral pH, whereas neighboring erythrocyte-containing phagosomes acidify below pH 5 (126). Also, after
infection with L. pneumophila, macrophages continue to deliver Saccharomyces
cerevisiae to phagolysosomes (53). Therefore, the L. pneumophila virulence factors that prevent its delivery to lysosomes must act locally, most likely by altering
the phagosomal membrane.
Host and Bacterial Factors that Affect Entry
The capacity of L. pneumophila to exploit both freshwater protozoa and a variety
of mammalian cells as a replication niche suggests that numerous factors are likely
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to mediate its adherence and entry. To date, >12 phagocytic and nonphagocytic
mammalian cell lines have been shown to support growth of legionellae (77), and
a number of bacterial and cellular factors that facilitate attachment and subsequent
entry of this pathogen have been identified in particular infection models.
Complement and Immunoglobulin Early studies by Horwitz & Silverstein (127)
focused on phagocytosis of L. pneumophila by peripheral blood mononuclear cells.
Opsonization of L. pneumophila with specific antibody and complement enhanced
by threefold its attachment to human monocytes, compared to adherence in the
presence of complement alone. The mechanism of binding does not appear to
influence significantly the intracellular fate of L. pneumophila: bacteria that were
phagocytosed in the presence of specific antibody and complement, or in the
presence of complement alone, replicated with an efficiency similar to untreated
bacteria (127).
When monocytes are incubated with L. pneumophila in the presence of serum,
phagocytosis occurs via CR1 and CR3, complement receptors that are present on
the surface of macrophages and several other mammalian cell lines (172). Complement component C3 present in immune and nonimmune sera fixes primarily
to the major outer membrane protein (MOMP), encoded by ompS (120), on the
L. pneumophila surface (21). In fact, C3 opsonization of purified MOMP reconstituted in liposomes induced phagocytosis by monocytes, suggesting that a
MOMP-C3 complex ligand is sufficient to mediate uptake of L. pneumophila via
the macrophage CR1 and CR3 receptors. MOMP may also have a complementindependent function: this abundant outer membrane protein also enhances bacterial binding to U937 cells in the absence of serum, and it increased the virulence of
L. pneumophila in chick embryo assays (144). Ultimately, the construction of an
L. pneumophila ompS mutant and assessment of its virulence phenotype in phagocyte and animal models of infections will provide a more detailed understanding
of the role of this dominant surface protein in L. pneumophila pathogenesis.
Because complement levels in the human lung are normally low, it is likely
that, at least early in infection, L. pneumophila attach to phagocytes by another
mechanism (185). In fact, in the absence of antibody or complement, this pathogen
still binds phorbol ester-treated U937 cells, monocytic cells that express Fc, CR1,
and CR3 receptors (188). Moreover, preincubation of these phagocytes with monoclonal antibodies (mAbs) directed against CR1 and CR3 did not inhibit binding
of L. pneumophila. Furthermore, L. pneumophila need not enter macrophages
by a complement-mediated route to establish an intracellular replication niche:
bacterial growth after complement-independent attachment has been observed in
guinea pig alveolar macrophages, phorbol ester-treated U937 cells, and MRC5
cells (98, 188). A bacterial protein associated with lipids or carbohydrates may
mediate binding to carbohydrates on the host plasma membrane, because bacterial attachment to U937 cells was inhibited after L. pneumophila was treated with
several proteolytic enzymes, or after both the bacterial and host cells were treated
with lipase and a carbohydrate oxidizing agent (98).
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LEGIONELLA PNEUMOPHILA PATHOGENESIS
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Complement-independent mechanisms must also promote phagocytosis of
L. pneumophila by aquatic amoebae. Its attachment to and invasion of the protozoa H. vermiformis and A. polyphaga is mediated by a protozoan 170-kDa lectin
that is inhibited by galactose/N-acetylgalactosamine (110, 227). It is interesting
that Entamoeba histolytica also encode a 170-kDa lectin that mediates its attachment to mammalian epithelial cells. Inhibition studies demonstrated the functional similarity of these lectins: L. pneumophila attachment to and invasion of
H. vermiformis was decreased in a dose-dependent manner by soluble galactose,
N-acetyl-D-galactosamine, or two mAbs specific to the 170-kDa protein of Entamoeba histolytica (182). The bacterial ligand(s) responsible for lectin binding
have yet to be identified.
Type IV pili Type IV pili, which mediate host cell attachment by pathogenic
Neisseria species (159), Pseudomonas aeruginosa (131), and other bacterial
species (219), may also function as L. pneumophila adhesins. An insertional mutation in the putative L. pneumophila pilin structural gene, pilEL, reduced the bacterial adherence to A. polyphaga and the mammalian monocytic U937 and epithelial
HeLa cell lines by ∼50%, although intracellular replication was not affected (215).
Because adherence of the pilEL mutant was attenuated in both mammalian cells
and amoebae, L. pneumophila pili may contribute to complement-independent
binding, perhaps as the ligand for the protozoan lectin.
Hsp60 The 60-kDa heat shock protein Hsp60 has also been implicated in attachment and entry of L. pneumophila to HeLa epithelial cells (96, 118, 119). Although
bacterial heat shock proteins typically serve as cytoplasmic chaperones (71),
L. pneumophila Hsp60 belongs to a large family of immunodominant protein
antigens, termed “common antigens”, many of which share cross-reactive epitopes and appear to be extracellular (225). Several other pathogens, including
Haemophilus ducreyi (88), Helicobacter pylori (66), Mycobacterium avium (180)
and S. typhimurium (73), appear to release proteins homologous to Hsp60 that
have been implicated in virulence. It remains to be determined whether the extracellular localization of Hsp60 proteins is a consequence of its release from cells in
certain growth conditions, bona fide secretion, or, as hypothesized for the Hsp60
homolog in H. pylori, bacterial cell lysis (173).
L. pneumophila Hsp60, encoded by htpB, is induced during growth in macrophages and in vitro in response to H2O2, heat, and osmotic shock (3, 96, 118, 119).
In L. pneumophila cultured in broth, immunogold labeling of Hsp60 indicated both
cytoplasmic and surface locations; heat-shock increased the amount of surfaceexposed Hsp60 epitopes modestly (95). In infected HeLa cells, extracellular Hsp60
protein can be detected lying free within replication vacuoles (96). Hsp60-specific
antibody inhibits entry by wild-type, but not avirulent L. pneumophila, and purified Hsp60 protein stimulates uptake of latex beads by HeLa cells (96). Specific host receptors for this class of Hsp60 proteins have not been identified.
Whether L. pneumophila requires Hsp60 to enter amoebae or macrophages or to
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establish its unique replication vacuole awaits phenotypic analysis of a defined htpB
mutant.
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Bacterial Factors that Establish the Isolated Vacuole
Dot/Icm Type IV Secretion System L. pneumophila is one of a growing list of
extracellular and intracellular bacterial pathogens that exploit type IV secretion
systems for virulence. The phytopathogen Agrobacterium tumefaciens (44) and
the animal pathogens Brucella suis (170), H. pylori (41), and B. pertussis (234)
all encode type IV secretion loci. Unlike type III secretion systems, which have
co-opted the flagellar assembly pathway, type IV systems are encoded by chromosomal loci homologous to operons dedicated to conjugal transfer of plasmid DNA
(reviewed in 80). How these systems contribute to bacterial pathogenesis and the
identity of their substrates is currently the focus of a great deal of research.
Membership in the family of type IV transport systems was defined originally
by the ability to transfer DNA to a recipient cell. For example, the plant pathogen
A. tumefaciens transfers RSF1010 plasmids by a process that requires a functional
type IV secretion apparatus (212). In like manner, L. pneumophila transfer of
RSF1010 plasmids to bacterial recipients depends on a functional type IV secretion
system (202, 229). The type IV secretion system of L. pneumophila is encoded
by 24 genes located within two separate regions of the bacterial chromosome
(Figure 3). Fourteen of these genes, termed dot (defective for organelle trafficking;
9, 23, 229) and icm (intracellular multiplication; 28, 177, 200, 202) share detectable
homology to the tra/trb genes of col1b-P9 plasmid, a member of the IncI class of
conjugal plasmids (Figure 3; 202, 229, 205). Whereas the majority of the dot/icm
genes are predicted to encode membrane-associated proteins, DotA is an integral
cytoplasmic membrane protein with eight membrane-spanning domains (193), and
IcmW is a small, soluble protein that resides in the cytoplasm (249). Together, the
Figure 3 Relationship between the Dot/Icm type IV secretion system loci and the transfer
region of IncI conjugal plasmid col1b-P9. The tra/trb plasmid genes are shown compared
with plasmid coordinates, and the homologous L. pneumophila genes are named underneath. On the bacterial chromosome, the dot/icm genes are located in two linkage groups,
designated Region I and II. (Adapted from a figure kindly provided by Dr. Joseph Vogel,
Washington University, St. Louis, MO.)
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LEGIONELLA PNEUMOPHILA PATHOGENESIS
585
Dot/Icm proteins are postulated to assemble and activate a secretion system in the
L. pneumophila membrane that exports plasmid and virulence factors.
Recently, Segal et al (201) identified a second L. pneumophila secretion system
related to type IV systems. Designated Lvh (for Legionella vir homologs), this
locus is dispensable for intracellular growth but can cooperate with the Dot/Icm
to transfer RSF1010 plasmids by conjugation. Insertions in several of the dot/icm
genes completely abolish conjugation, indicating that the lvh locus itself cannot confer conjugation. However, components of the lvh system may be able to
replace some Dot/Icm factors for conjugation, as judged by comparing the phenotype of particular single and double mutant strains. Deletion of the lvh locus in
the wild-type JR32 strain modestly reduces conjugation efficiency, ∼10-fold. In
like manner, dotB and icmE mutants donate plasmid at a somewhat reduced efficiency. On the other hand, double mutants carrying an lvh deletion and a dotB or
an icmE mutation are completely defective for conjugation. Thus, components of
the lvh system may substitute for dotB and icmE functions that are important for
conjugation, but not virulence (201).
The Dot/Icm type IV secretion system appears to act during phagocytosis to
establish the L. pneumophila replication vacuole. Every mutant of the dot/icm
family that has been examined is defective for evasion of the endocytic pathway
(9, 24, 125, 153, 192, 202, 222, 229, 236, 249). Each of these mutants is mistargeted to the endosomal pathway within the earliest period examined, in some
cases ≤5–30 min after infection (192, 236). For example, phagosomes containing
dotA mutants acquire the late endosomal and lysosomal marker LAMP-1 within
5 min of uptake (192). Thus, to evade delivery to the lysosomes, L. pneumophila
must alter its phagosome immediately.
Once a protected vacuole is established, L. pneumophila does not appear to
require the Dot/Icm machinery to maintain its replication niche. For example, by
using an inducible promoter to control dotA transcription, Roy and coworkers (192)
found that L. pneumophila which express DotA before contact with macrophages
but not after still replicate during the primary infection cycle. Furthermore, when
a dotA mutant resides within the same phagosome as a wild-type bacterium, it
can replicate (53). The hypothesis that Dot/Icm function is dispensable during the
replication period is consistent with the observation that virulence traits are not
expressed by exponential-phase L. pneumophila (38, 109).
The substrates of the Dot/Icm secretion complex that alter the fate of the
L. pneumophila phagosome have not been identified. Although related to conjugal DNA transfer complexes, type IV secretion systems also export proteins
that are effectors of virulence (237). For example, A. tumefaciens VirE2 protein
accompanies T-DNA (tumor DNA) during transfer (237), and pertussis toxin is
a protein substrate of the B. pertussis Ptl secretion system (234). In A. tumefaciens, RSF1010 plasmids appear to compete with T-DNA for secretion by the
type IV apparatus, and they attenuate bacterial virulence (26). Similarly, L. pneumophila strains that encode a functional RSF1010 mobilization system replicate
intracellularly and kill macrophages less efficiently than those that do not encode
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this system (203), suggesting a competition between conjugal plasmids and the
putative virulence factor substrates. By analogy to other type IV systems, the
L. pneumophila Dot/Icm conjugation complex is postulated to deliver virulence
proteins to phagocytes, to establish a protective-replication vacuole. As described
above, the Dot/Icm complex must act during phagocytosis to divert phagosome
maturation. Therefore, the putative effector molecule is not likely to be DNA.
The Dot/Icm complex is required by L. pneumophila to insert pores into the
host plasma membrane, a process that could perturb phagosome maturation (140).
Many dot and icm mutants have been shown to reside in vacuoles that acquire
endocytic markers; in addition, this class of mutants is noncytotoxic, and several
mutants have been shown specifically to lack pore-forming activity. Accordingly,
one model postulates that delivery of a small number of pores is sufficient to
retard phagosome maturation (140). Alternatively, at a high level of infection,
insertion of a large number of pores into the host plasma membrane causes rapid,
contact-dependent lysis of the phagocyte. How pore formation affects phagosome
maturation has been difficult to test directly, because neither the pore-forming
toxin nor its gene(s) have been isolated.
A related model postulates that the Dot/Icm-dependent pore serves as the conduit for the effector molecules that modify the nascent phagosomal membrane to
alter its course (249). Accordingly, mutants lacking such effectors are predicted to
retain cytotoxicity but fail to evade the endosomal compartment. By these criteria,
IcmW was an attractive candidate effector. However, cellular fractionation experiments indicate that this small, soluble protein resides in the bacterial cytoplasm.
Therefore, instead of acting as a substrate for type IV secretion, IcmW may regulate Dot/Icm activity, directly or indirectly (249). It is important to note that the
icmW mutant phenotype indicates that although pore-formation may be required
by L. pneumophila to establish an isolated phagosome, it is not sufficient.
Dot-Independent Activity By modifying L. pneumophila genetically, biochemically, and physiologically, and then studying the consequences on phagosome maturation, our laboratory recently determined that Dot-independent factors also retard
phagosome maturation (AD Joshi, S Sturgill-Koszycki, MS Swanson, unpublished
observations). Consistent with previous reports, when macrophages were fed either postexponential phase dotA or dotB mutant cells, or wild-type bacteria that
were first killed with formalin, each population of phagosomes rapidly acquired
LAMP-1. However, <20% of these vacuoles acquired lysosomal cathepsin D;
they also did not accumulate Texas Red-ovalbumin, CM-DiI, or Alexa Fluorstreptavidin, three fluorescent endocytic probes readily acquired by the majority
of phagosomes that contained nonpathogenic particles, such as E. coli, polystyrene
beads, or live or fixed exponential-phase L. pneumophila. Similarly, the capacity of L. pneumophila to evade lysosomal fusion in amoebae does not strictly
depend on bacterial viability or correlate with virulence: Whereas 93% of phagosomes containing Proteus mirabilis, a nonpathogenic control strain, fused with
lysosomes, as judged by colocalization with acid phosphatase, <40% of vacuoles
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harboring avirulent bacteria or formalin- or UV-treated virulent bacteria did so,
even ≤6 h after infection (27). Dot-dependent factors appear to isolate the L. pneumophila phagosome from a LAMP-1 containing compartment. However, because
formalin-killed bacteria and viable dot mutant bacteria reside for ≤4 h in vacuoles that do not acquire lysosomal characteristics, L. pneumophila also produce a
formalin-resistant, Dot-independent activity that retards its delivery to lysosomes.
Mip The virulence factor Mip is a 24-kDa surface protein that has garnered much
interest over the last decade (47, 72). In cultured macrophages, Mip appears to
promote efficient establishment of infection rather than intracellular replication
per se. During the initial infection period, ∼10-fold fewer viable mutant cells
associate with human monocytic U937 cells and alveolar macrophages compared
with wild-type bacteria; in contrast, their intracellular growth rate is identical to
that of wild-type bacteria (47). A similar pattern was observed for infections of
H. vermiformis and T. pyriformis (46). The requirement for Mip function appears
to be more stringent in amoebae and animal models of infection. After infection of
A. castellani, the yield of mip mutants is 50- to 100-fold lower than that of the wild
type (241), and the mip gene is expressed by replicating L. pneumophila (143). In
a guinea pig model of infection, mip null mutants are also less virulent than wild
type, as determined by lower morbidity and mortality (45). Thus, judging by a
variety of criteria, Mip contributes to L. pneumophila virulence.
The Mip protein exhibits peptidyl-prolyl-cis/trans isomerase (PPIase) activity,
as measured by cleavage of synthetic substrates, and this activity is inhibited by the
immunosuppressant macrolide FK506 (81). Because peptidyl prolyl isomerases
are characteristic of eukaryotes, Mip may target a host protein substrate, as documented for the Yersinia YopH virulence protein (103). However, Mip is not
exclusive to virulent L. pneumophila. Mip-like proteins have been identified in
both virulent and avirulent Legionella species (181), as well as in other intracellular pathogens, including Chlamydia trachomatis (150) and Trypanosoma cruzi
(163). Mip PPIase activity does not appear to be required for intracellular survival,
because a mip mutant can be complemented for intracellular replication by in trans
expression of mip genes bearing mutations that significantly reduce this enzyme
activity (241). As yet, no bacterial or host substrate for Mip has been identified,
and the mode of action of this virulence factor remains to be determined.
Phospholipases Bacterial phospholipases contribute to the virulence of a variety
of bacterial pathogens (reviewed in 210). For example, Listeria monocytogenes encodes two phospholipases that cooperate with listeriolysin to lyse the phagosomal
membrane and promote bacterial escape into the more hospitable cytosol (152).
L. pneumophila also possesses phospholipase activity, as measured by release of
p-nitrophenol from the synthetic substrate p-nitrophenylphosphorlycholine (13).
Baine hypothesized that alteration of the nascent phagosomal membrane by phospholipase could aid in establishment of a replicative niche by inhibiting fusion of
the phagosome with lysosomal compartments (14). Definition of the contribution
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by phospholipase activity to L. pneumophila virulence awaits analysis of defined
mutant strains.
Factors that impede L. pneumophila phagosome maturation are likely to interact
directly with the macrophage plasma membrane. For example, the multiacylated
trehalose 2-sulfates of M. tuberculosis bind the macrophage plasma membrane
and concentrate in lysosomes; these glycolipids inhibit phagolysosome formation
when either coupled to heterologous particles or added in soluble form to cultured
macrophages (102). Similarly, the promastigote form of Leishmania is covered
with a thick glycocalyx, composed primarily of the negatively-charged molecule
lipophosphoglycan, which inhibits phagosome maturation (226, 57). The surfaces
on these pathogens may retard phagolysosome formation by steric hindrance of
particular host receptors, altering transmembrane signaling molecules, or directly
blocking components of the fusion machinery.
Two other molecules are known to disrupt phagolysosome formation, although
neither have been linked to pathogenesis. The tetravalent lectin concanavalin A,
which can cross-link membrane glycoproteins, stimulates formation of endocytic
vacuoles that do not fuse with lysosomes (68). The weak base ammonium chloride,
which raises the pH of acidic organelles, including lysosomes, interferes with
phagolysosome formation (101). Accordingly, L. pneumophila could block fusion
with lysosomes by producing lectins or weak bases.
Association with Endoplasmic Reticulum
An unusual sequence of organelle associations follows establishment of the isolated
L. pneumophila vacuole (Figure 4, Table 1). Approximately 1 h after formation, the
cytoplasmic face of the L. pneumophila phagosome interacts with mitochondria;
by 4 h, the vacuole is surrounded by endoplasmic reticulum (ER; 122, 221). Within
amoebae, L. pneumophila also resides in vacuoles that associate transiently with
mitochondria before being enveloped by endoplasmic reticulum (2, 78, 90). In
macrophages, L. pneumophila replicates in the ribosome-studded vacuole for ∼20
h, then the macrophage lyses, and released bacteria begin a new round of infection.
Mitochondria also associate with vacuoles containing T. gondii, and both
T. gondii and B. abortus replicate in vacuoles decorated with rough ER (8, 58, 132).
Genetic and kinetic studies of L. pneumophila have correlated ER association and
intracellular replication (122, 221, 222). In none of these cases has a direct role for
mitochondria or ER in pathogen survival or growth been demonstrated. Indeed,
another pathogenic legionellae, L. micdadei, replicates within dilated vacuoles of
macrophages that do not associate with endoplasmic reticulum (92, 134, 232).
Autophagy
Theoretically, pathogens could exploit one or more of the activities of the ER to
obtain nutrients. In addition to its protein and phospholipid biosynthetic enzymes,
protein-conducting channels, and TAP peptide pores, the ER participates in autophagy, a ubiquitous mechanism critical for cellular homeostasis (67). When
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Figure 4 Model for the Legionella pneumophila pathway in macrophages. After being engulfed within coiled phagosomes, post-exponential-phase bacteria establish a vacuole that
does not acidify or interact with the endosomal pathway, but is surrounded by endoplasmic
reticulum. Within this protected vacuole, bacteria convert to an acid-tolerant, replicative form and no longer express virulence factors. Consequently, vacuoles merge with the
lysosomal compartment, an acidic, nutrient-rich replication niche. Once the local amino
acid supply is depleted, the progeny convert to the virulent form, expressing factors to
escape the spent host, survive and disperse in the environment, and establish a protected
replication niche in another phagocyte.
stressed, such as by nutrient deprivation or elevated temperature, cells increase
their rate of autophagy. Portions of the cytoplasm, including organelles, are sequestered within vacuoles derived from the ER, called autophagosomes. Next,
these vacuoles merge with the lysosomal compartment, wherein the contents are
degraded. By this process, a eukaryotic cell presumably reduces its metabolic load
and liberates molecules needed for vital cellular activities.
There is experimental evidence supporting an interaction between the autophagy pathway and vacuoles that harbor pathogens. The parasitophorous vacuoles of Leishmania mexicana appear to intersect with the autophagy pathway,
as judged by a slow accumulation of cytosolic markers in the vacuole which was
sensitive to 3-methyladenine (198). The ultrastructure of L. pneumophila replication vacuoles resembles that of autophagosomes, and elevated rates of autophagy
facilitate replication vacuole formation and enhance bacterial growth (221). By
stimulating autophagy, pathogens could increase the local supply of nutrients.
Alternatively, autophagy of pathogenic vacuoles may represent a form of quality control by the host phagocyte. The infectious forms of L. pneumophila and
Leishmania establish vacuoles that are isolated from the endocytic pathway. Later,
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these nonfusigenic vacuoles are engulfed by endoplasmic reticulum in a process
that resembles autophagy. Vacuoles bearing Leishmania eventually merge with
the lysosomal compartment. As described below, recent data from our laboratory
indicate that mature L. pneumophila replication vacuoles also acquire lysosomal
characteristics. Therefore, it is reasonable to postulate that the autophagy machinery can recognize certain nonfusigenic vacuoles and deliver these organelles to
lysosomes. Presumably, this class of vacuolar pathogens exploits the period when
delivery to lysosomes is blocked to convert to a replicative form which not only
tolerates, but thrives within the acidic and hydrolytic lysosomal compartment.
Maturation of the Replication Vacuole
Although many studies have analyzed the macrophage compartment where
L. pneumophila initially reside (Table 1), little is known about the vacuoles that harbor actively replicating bacteria. Although some macrophage pathogens, such as
Mycobacterium (10, 56), Toxoplasma (132), and Chlamydia (87) species, replicate
within compartments which remain separate from the lysosomes, Leishmania does
not. Similar to L. pneumophila, the growth phase of Leishmania determines its
competence to inhibit phagosome-lysosome fusion (57, 226). Motivated by its
similarity to Leishmania development, the resemblance of its replication vacuole
to autophagosomes, and the dearth of information regarding its mature replication vacuole, we investigated whether L. pneumophila eventually reside within an
endocytic compartment.
Acquisition of Lysosomal Characteristics
Indeed, during the time period when the yield of L. pneumophila colony forming units typically increases 10-fold, a significant proportion of the bacterial
vacuoles acquire lysosomal characteristics (Table 1; S Sturgill-Koszycki & MS
Swanson, unpublished observations). In particular, by 18 h post-infection, 70%
of the vacuoles contain LAMP-1, a late endosomal and lysosomal membrane
glycoprotein, and 50% contain the lysosomal enzyme cathepsin D, as judged by
fluorescence microscopic assays. Additionally, 50% of the replication vacuoles
accumulate the fluorescent endocytic probes Texas Red ovalbumin and fluoresceindextran. Finally, whereas nascent L. pneumophila phagosomes remain a neutral
pH, by 16 to 20 h after infection, replication vacuoles are acidic, averaging pH 5.5
(S Sturgill-Koszycki & MS Swanson, unpublished observations). Thus, as they
mature, L. pneumophila replication vacuoles appear to merge with the lysosomal
compartment.
Four lines of evidence indicate that fusion of the replication vacuole with the
lysosomal compartment promotes, rather than inhibits, L. pneumophila growth
(S Sturgill-Koszycki & MS Swanson, unpublished observations). First, acquisition
of lysosomal proteins correlates with bacterial replication: Virtually every vacuole
that contains greater than five bacteria also contains LAMP-1. Second, bacteria
within endocytic vacuoles are metabolically active, as judged by their capacity
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LEGIONELLA PNEUMOPHILA PATHOGENESIS
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to respond to IPTG by expressing from a Ptac promoter a gene encoding Green
Fluorescent Protein. Third, blocking acidification and maturation of the replication vacuole by treating infected macrophages with the proton ATPase inhibitor
bafilomycin A1 also arrests bacterial replication. Fourth, unlike exponential phase
broth cultures, replicating L. pneumophila obtained from macrophages 18 h after
infection are acid tolerant: Less than 0.01% of broth-grown bacteria survive a 4-h
treatment at pH 5.0, whereas ∼15% of macrophage-grown cells do so. Therefore,
in macrophages, L. pneumophila appear to reside and replicate within a lysosomal
compartment (S Sturgill-Koszycki & MS Swanson, unpublished observations).
Four previous observations are consistent with the interpretation that L. pneumophila replicates within phagolysosomes. First, the growth of L. pneumophila in
amoebae does not always correlate with a capacity to evade phagosome-lysosome
fusion: one environmental isolate evaded lysosomes poorly, yet replicated within
amoebae as well as the virulent control strain (27). Second, by 20 h after infection, virulent L. pneumophila reside in H. vermiformis vacuoles that no longer
have ribosomes and the ER protein BiP (2). Third, by 24 hours after infection, L. pneumophila vacuoles frequently contain granular and membranous material (2, 138, 245) consistent with an endocytic identity. Fourth, a pathogen
closely related to the legionellae, Coxiella burnetii (233), replicates within acidic
phagolysosomes (6, 113, 156, 199).
Similarity to Leishmania
For both L. pneumophila and L. donovani, the relationship between the pathogen
vacuole and the macrophage endocytic pathway changes dramatically during the
course of infection. Initially, an infectious, stationary phase cell expresses factors
which isolate the phagosome from the endocytic pathway; Leishmania promastigotes are coated with lipophosphoglycan (57), whereas L. pneumophila express
Dot/Icm-dependent and -independent factors of unknown identity (AD Joshi, S
Sturgill-Koszycki, MS Swanson, unpublished observations). Once the intracellular microbes convert to the replicative form, the inhibitory factors are no longer
expressed, and the vacuoles merge with the lysosomal compartment, wherein the
parasites replicate (226, 38; S Sturgill-Koszycki & MS Swanson, unpublished observations). Thus, L. pneumophila and L. donovani manipulate the macrophage
endocytic pathway first to survive, but later to exploit the lysosomes as a replication
niche.
Similarity to Coxiella Species
Coxiella burnetti, the etiological agent of Q Fever, is closely related phylogenetically to L. pneumophila (233). Although C. burnetti is an obligate intracellular pathogen, recent studies of L. pneumophila have revealed a number of
striking similarities between the two microbes. Both pathogens replicate within
human alveolar macrophages, where they undergo a marked developmental switch
(38, 112). In particular, C. burnetti converts between a replicative, large cell variant
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form and the stationary phase, small cell variant form. Moreover, C. burnetti encodes homologs of icmT, icmS, and icmK, three genes required by L. pneumophila
for intracellular growth (205). Finally, it is now apparent that both C. burnetti
and L. pneumophila replicate within the acidic, degradative environment
of macrophage phagolysosomes (6, 113, 156, 199; S Sturgill-Koszycki & MS
Swanson, unpublished observations). Given their genetic similarity and comparable life styles, L. pneumophila may function as a powerful experimental tool
to analyze C. burnetti pathogenesis, an effort hampered by an inability to culture
this infectious agent.
Model for Replication Vacuole Biogenesis
Taken together, current knowledge of L. pneumophila virulence regulation and
replication vacuole biogenesis support the following multistage model for the
L. pneumophila life cycle (Figure 4). When ingested by amoebae or macrophages,
L. pneumophila exports factors via the Dot/Icm secretion system to modify the
nascent phagosome and separate it completely from the endosomal pathway. Next,
endoplasmic reticulum engulfs the isolated phagosome, forming an autophagosomal vacuole. Within this protected niche, L. pneumophila converts to a replicative
form that is acid tolerant, does not require Dot/Icm function, and does not express
several virulence traits, including those factors which block fusion with the lysosomal compartment. Consequently, the autophagy machinery delivers the pathogen
to the lysosomal compartment, a harsh but nutrient-rich environment where bacterial replication occurs. Once the bacterial progeny have depleted the local amino
acid supply, the intracellular second messenger ppGpp accumulates, triggering expression of traits important for transmission of L. pneumophila to a new phagocyte.
In particular, a cytotoxin promotes escape from the spent host, osmotic resistance
increases survival in the extracellular environment, motility facilitates dispersal
and contact with a new host cell, and the capacity to evade phagosome-lysosome
fusion promotes survival within the next phagocyte, where the cycle repeats.
The apparent complexity of its intracellular pathway motivates speculation on
the evolution of L. pneumophila virulence. Presumably, ancestral legionellae were
ingested and efficiently digested within amoebae phagolysosomes. In response to
this considerable selective pressure, endosymbionts may have emerged by virtue of
their expression of a formalin-resistant surface component that retards phagolysosome maturation and promotes survival. Later, acquisition by horizontal transmission of the genetically linked dot/icm loci may have permitted the emergence of
pathogenic L. pneumophila. Now endowed with the capacity to avoid the endocytic
pathway completely for hours, L. pneumophila has a window of time sufficient to
alter its physiology. By activating regulons that confer acid tolerance and other
activities, the bacteria can exploit the highly dynamic and nutrient rich lysosomal
compartment as a replication niche. Thus, the complex intracellular life cycle of L.
pneumophila is the product of a relentless competition with the aquatic amoebae
with which it evolves.
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Bacterial Factors that Promote Intracellular Replication
Iron Acquisition Once established in the replication vacuole, iron acquisition
and assimilation appears to be critical for intracellular growth of L. pneumophila.
Interfering with the supply of intracellular iron, either by addition of chelators or by
γ -interferon activation of macrophages, inhibits intracellular bacterial replication
(36, 174). Consistent with these observations, an L. pneumophila iraAB mutant,
identified originally in a screen for strains defective for intracellular iron acquisition and assimilation, replicates poorly in U937 cells following a prolonged
lag phase (174). Moreover, a defined iraAB::tn(kan) mutant is also defective
for replication in a guinea pig model of lung infection (228). A nonclassical
siderophore, legiobactin, which has measurable iron-binding activity in a chemically defined medium has been described (145). Finally, L. pneumophila encodes
a homolog of Fur, an E. coli transcription factor which represses expression of
iron acquisition genes when ferrous iron is present (116, 12). A defined mutant in
the L. pneumophila Fur-regulated gene frgA was impaired for replication in U937
cells by 80-fold (117). Therefore, this intracellular pathogen likely responds to
iron limitation in part by altering its pattern of gene expression.
Protection from Stress During the exponential growth phase, L. pneumophila
expresses a cytoplasmic catalase:peroxidase, encoded by katB, which may be
important for its intracellular lifestyle (15). In E. coli, the homologous catalase:
peroxidase, KatG, which is induced by H2O2, mitigates the intracellular H2O2 generated by respiration during the exponential growth phase. Following phagocytosis
by macrophage-like THP1 cells, katB mutants display an apparent lag before replicating at a wild-type rate (15). Likewise, in primary macrophages derived from the
bone marrow of A/J mice, katB mutant cells exhibit a similar lag, but also a modest
replication defect, indicating a more stringent requirement for catalase-peroxidase
activity in this phagocyte model (B Byrne, MS Swanson, P Bandyopadhyay &
HM Steinman, unpublished data). Perhaps the lag which precedes replication
indicates that intracellular L. pneumophila induce a compensatory activity that
enables the bacteria to replicate in the absence of KatB. L. pneumophila katB
expression is not induced by H2O2 in vitro, suggesting that other conditions within
the phagosome may trigger katB expression.
L. pneumophila, like Salmonella (35) and Yersinia (244) species, express a
19-kDa GroEL-like General Stress Protein (GspA) after entry into macrophages
(3). GspA is dispensable for bacterial survival and growth in macrophage infection
models (4); whether it is required for transmission or for extracellular survival in
the environment is difficult to assess. This class of chaperone proteins is known
to be expressed by bacteria cultured in vitro in response to stress (3), suggesting
that the pathogen vacuole may be a stressful environment.
Type II Secretion System Type II secretion systems enable animal pathogens,
such as Vibrio cholerae and P. aeruginosa, to secrete toxins and proteases, and
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plant pathogens, like Erwinia chrysanthemi and Xanthomonas campestris, to secrete cellulases and pectinases (reviewed in 194). Proteins exported by type II
systems contain an amino terminal signal sequence that directs their delivery into
the periplasm by the Sec secretion machinery. Subsequent transport through the
outer membrane requires the general secretion pathway, a complex of 14 additional
proteins (194). L. pneumophila is the first intracellular pathogen found to carry a
chromosomal locus encoding a type II secretion system (146, 147).
A role for type II secretion in L. pneumophila pathogenesis was demonstrated by
analysis of a bacterial strain defective for expression of a type IV pilin biosynthesis
gene. The PilD prepilin peptidase, an enzyme required for IV pilus biogenesis,
processes proteins destined for secretion by the type II system. An L. pneumophila
pilin locus encodes homologs of Pseudomonas aeruginosa PilD, as well as PilB and
PilC (146). A nonpolar pilD:kanR insertion mutant was nonpiliated and defective
for secretion of at least three proteins, one of which is the zinc-metalloprotease
known as the major secreted protein (Msp). In broth, the pilD mutant grew as
well as wild type, but it was defective for growth within monocytic U937 cells,
Hartmannella vermiformis, and guinea pigs (147). Since msp and pilus mutants
replicate efficiently in macrophages (223, 215), additional type II secreted proteins
are predicted to contribute to L. pneumophila intracellular growth.
Lipopolysaccharide L. pneumophila produces an unusually hydrophobic lipopolysaccharide (LPS) which may facilitate its intracellular lifestyle (142). A
phase-variant mutant of L. pneumophila serogroup 1, designated 811, was isolated after repeat passage on the basis of its failure to bind the mAb2625, which
is specific for LPS of the virulent, wild-type strain (151). Unlike the wild type
and an mAb2625-positive revertant of mutant 811, mutant 811 was impaired for
growth within HL60 cells, although all strains entered HL60 cell with a similar
efficiency. Although virulent revertants of mutant 811 were obtained, suggesting
a single mutation confers the phenotype of interest, it remains possible that factors
other than LPS were altered as a consequence of repeated passage. Nevertheless,
it is curious that a phase variation, or switching between the mutant and the wildtype LPS phenotype, was promoted by growth in a guinea pig infection model.
Perhaps a developmental-phase variation of LPS contributes to L. pneumophila
pathogenesis.
Intracellular Growth Mutants Virulence factors have also been sought by a
combination of genetic and cell biological assays. For example, after screening
for mutants that are defective for growth in phagocytes, the fate of each mutant
in phagocytes can be determined by fluorescence or electron microscopic assays
(23, 222, 236). Alternatively, candidate genes are selected based on their position
within a chromosomal region known to be required for virulence; strains lacking
the candidate gene are constructed, and their fate in phagocytes is assessed. By
these approaches, many loci, termed dot (9, 23, 229), icm (28, 153, 200, 202), mak
(195), mil (91), and pmi (90), have been identified as important for intracellular
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growth. As yet, there are no virulence genes for which both the biochemical activity
and the mechanism of action in pathogenesis have been established unequivocally.
It is interesting that there is a paucity of avirulent mutants that evade endosomal
fusion but are defective for replication within the isolated vacuole (236). Perhaps
the factors required for replication within a modified phagosome are also required
for efficient growth in microbiological media. If so, phenotypic screening of a
collection of transposon mutants is unlikely to identify this class of virulence
genes.
Strategies to Identify Factors Expressed or Required During Infection Another strategy to identify L. pneumophila virulence factors is to isolate genes
whose expression is induced, or required, specifically during infection. This class
of factors has been sought by a number of molecular and genetic schemes, including two-dimensional gel electrophoresis (3), a promoter-trap genetic-screen and
genetic-enrichment protocol (179), differential-display polymerase chain reaction
(5), and signature-tagged transposon mutagenesis (70). By these techniques, many
factors that appear to be important for intracellular growth have been identified.
As predicted, a number of genes shown previously to be required for intracellular
growth were also identified by these strategies. For example, using signaturetagged mutagenesis, Edelstein and colleagues identified 16 transposon mutants
that were defective for growth in the lungs and spleens of guinea pigs; 12 of these
also failed to replicate in alveolar macrophages (70). Among the loci identified
by these mutations were proA, which encodes Msp, and five dot and icm genes.
Thus, additional molecular and genetic analysis of this class of factors is likely to
provide insight to the mechanisms of L. pneumophila pathogenesis.
Amoebae Versus Macrophages A number of L. pneumophila factors appear to
be critical for pathogenesis in one species of professional phagocytes, but not the
other. For example, when cultured with A. castellanii, rpoS mutants are avirulent;
yet, in the monocytic HL60 and THP-1 cell lines, the mutant replicated within
and killed host cells as efficiently as wild-type L. pneumophila (107). A similar
pattern was observed for the type II general secretion pathway: a null mutation in
the lspGH locus (Legionella secretion pathway) attenuated bacterial replication in
A. castellanii, but did not affect bacterial killing of monocytic HL60 cells (106).
Likewise, the growth defect of icmG, icmN, icmS, and tphF mutants was more
severe in cultures of A. castellanii than in mammalian HL60 cells (204). In addition, Brieland and colleagues identified, among a library of transposon mutants,
two strains that were avirulent in their H. vermiformis model, yet replicated as
efficiently as the wild type in monocytic U937 cells (32). Conversely, Fields et al
described legionellae isolates that replicated in T. pyriformis, but were attenuated
for pathogenesis of guinea pigs, as judged by bacterial survival after inoculation
into the peritoneal cavity (78). And Gao et al identified among a large collection of
transposon mutants a number of strains, termed mil mutants (macrophage-specific
infectivity loci), that were defective for replication in a monocyte cell line, but not
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amoebae (91). It is not surprising that L. pneumophila is sensitive to physiological
differences between infection models that remain undefined by experimentalists.
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Transmission
As secondary cases of Legionnaires’ disease have not been documented, it is clear
that L. pneumophila lacks virulence factors that promote transmission from one
person to another. Instead, the selective pressures of aquatic habits have determined
the L. pneumophila phenotype. Although monolayers of professional phagocytes
are proven tools to identify the host and bacterial determinants of intracellular
replication, analysis of transmission either within its natural reservoir or to human
phagocytes will require the development of experimental systems that more closely
mimic the complex ecology of L. pneumophila.
Based on the observation that amino acid starvation induces cytotoxicity, osmotic resistance, motility, and the capacity to evade phagosome-lysosome, we have
postulated that these traits facilitate transmission in the environment (38, 109).
Here we discuss these and other activities that are likely to promote bacterial
escape from phagocytes, extracellular survival, and dispersal in the environment.
Cytotoxins
To escape from a nutrient-poor vacuole, L. pneumophila may produce toxins
that lyse host membranes. Since 1977, when L. pneumophila was verified as the
causative agent of Legionnaires’ disease, numerous studies have described bacterial factors that are cytotoxic to a variety of types of mammalian cells. Two
L. pneumophila cytotoxins exhibit hemolytic activity on blood agar plates. A
39-kDa protein, termed legiolysin (encoded by lly), confers hemolytic activity
to recombinant E. coli K-12 (242). Yet, an L. pneumophila lly mutant remains
hemolytic, as judged by its colony phenotype on blood agar plates, and it replicates as efficiently as wild-type in both monocytic U937 cells and protozoan A.
castellanii (240), suggesting production of other toxins that are functionally redundant. The 38-kDa Msp, encoded by proA or mspA, is a zinc metalloprotease
(65, 178, 223). Several activities have been attributed to this enzyme, including
proteolysis on skim milk medium, hemolysis on canine erythrocyte medium, and
cytotoxicity for CHO cells (137, 178). Purified Msp also inhibits neutrophil and
monocyte killing of Listeria monocytogenes and neutrophil chemotaxis (184), and
it can induce the lung pathology typical of legionellosis (54). Although msp mutants had no apparent defect for growth within or killing of macrophages (161, 223),
their virulence was attenuated in a guinea pig model (161). Taken together, these
observations suggest that cytotoxic activities may contribute to L. pneumophila
transmission, perhaps mediating escape from the spent amoebae. In the lung, this
activity may be manifested as tissue damage.
L. pneumophila also exhibit a contact-dependent cytotoxicity. Husman &
Johnson determined that bacteria can kill guinea pig peritoneal macrophages and
J774.2 cells within 2–4 h of infection by a process that requires close contact,
but not entry or replication (129). More detailed studies by Kirby and colleagues
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reported that a contact-dependent cytotoxicity induces a necrotic cell death which
is marked by osmotic lysis (141). More specifically, L. pneumophila bacteria appear to insert, into the target cell membrane pores, with approximate diameters
of 1.5 nm, as calculated from the Einstein-Stokes molecular diffusion radius of
polyethylene glycols, which provide osmotic protection (141). Moreover, pore
formation requires Dot/Icm function and correlates with the capacity to evade
phagosome-lysosome fusion, as discussed above. As yet, neither the toxin nor its
respective gene has been isolated.
Apoptosis
Programmed cell death, or apoptosis, has also been postulated to play a role in
L. pneumophila pathogenesis. At 24 h after infection with L. pneumophila at a multiplicity of 10:1, monocytic HL-60 cells exhibited apoptotic nuclear morphology
and internucleosomal DNA cleavage (167). Neither the Msp zinc metalloprotease
nor the Mip peptidyl prolyl isomerase was required for the apoptotic response
(167). Under conditions in which bacterial replication and necrotic cell death
were minimal, L. pneumophila also induced apoptosis of HL60 cells, as judged by
annexin V binding to cells that were impermeable to propidium iodide (105). Abu
Kwaik and colleagues have demonstrated that this pathogen can kill the monocytic U937 cell line by a replication-independent and host protease-dependent
mechanism that is characterized by three hallmarks of apoptosis: fragmentation
of host DNA, cleavage of host poly (ADP-ribose) polymerase, and exposure of
phosphatidylserine, a phospholipid that is normally located on the cytoplasmic face
of the plasma membrane of healthy mammalian cells (89). Furthermore, when
cells were infected in the presence of Z-DEVD-FMK, a compound that inhibits
caspase-3 and several other cysteine proteases that mediate apoptosis (94), neither
host DNA nor poly (ADP-ribose) polymerase was degraded.
Induction of programmed cell death does correlate genetically with virulence.
Bacterial mutants that lack a functional Dot/Icm transport complex fail to evade
fusion with the endocytic pathway, replicate in macrophages and amoebae, or
express a pore-forming cytotoxin; they also fail to activate caspase 3 and to degrade DNA (89). A role for apoptosis in legionellosis was also postulated based
on genetic studies of mice, a species naturally resistant to L. pneumophila infections. The A/J strain of mice is permissive for L. pneumophila replication owing
to a recessive genomic mutation, lgn1, which has been mapped to a ∼350-kb region of chromosome 13 (60, 246, 248). Encoded in this region are six copies of
the murine homolog of the gene encoding neuronal apoptosis inhibitory protein
[NAIP (128, 247)]. This protein is expressed by macrophages and other tissues
and has been shown to inhibit apoptosis in neurons and other types of mammalian
cells (61, 148, 243). It is interesting that A/J mice tissues contain less NAIP RNA
and protein than do resistant, Lgn+ mouse strains (61). The level of NAIP also correlates with phagocytic activity: Lgn+ macrophages that have phagocytosed latex
beads or avirulent L. pneumophila contain more NAIP protein than macrophages
that have not been fed. Diez et al speculate that, to replicate in macrophages,
L. pneumophila must induce apoptosis. By this model, A/J mice cells readily
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undergo programmed cell death caused by abnormally low levels of NAIP; hence,
the A/J naip allele confers permissiveness for L. pneumophila infection (61). By
inducing apoptosis during growth in alveolar macrophages, L. pneumophila may
increase the likelihood of safe passage to a second phagocyte while being protected
from antimicrobial molecules that are abundant in the lungs (167).
Many questions remain about the role of apoptosis in L. pneumophila pathogenesis. Because apoptotic death of amoebae infected with L. pneumophila has
not been observed (105), the selective pressure that would maintain this putative
virulence trait is not obvious. Whether L. pneumophila must induce apoptosis
to replicate in macrophages remains to be established. Also, it has been difficult
to separate apoptosis from necrosis, because the high multiplicity of infections,
which are optimal for apoptosis induction by L. pneumophila, also induce substantial necrosis (89). Further complicating the interpretation of these experiments is
the likelihood that host cell permeabilization by the pore-forming toxin will also
expose phosphatidylserine and induce DNA damage (64). Mutants specifically
defective for inducing apoptotic cell death or for production of the pore-forming
cytotoxin would allow a direct test of the hypothesis that induction of apoptosis is
required for survival or replication in macrophages.
Motility
Motility likely facilitates bacterial transmission to a new host phagocyte. Consistent with a role in transmission, L. pneumophila become motile in the stationary phase in broth and within late-stage replication vacuoles of amoebae and
macrophages (38, 115, 190). Insertional mutants that are defective for expression
of flagella multiply at wild-type rates in U937 cultures (158, 176). However, flaA
mutants have a reduced capacity to invade cells, and after an intracellular replication cycle, the progeny are defective for lysing its host (59). Genetic studies indicate that flagellar synthesis and virulence are regulated coordinately (38, 109, 176).
Antioxidants
L. pneumophila encodes several antioxidants that may be important during transmission from one phagocyte to another. This class of enzymes can catalyze the
decomposition of reactive oxygen species that are either generated by bacterial
metabolism or are present in the environment. In animal models, superoxide dismutases contribute to the virulence of a number of bacterial pathogens including
S. typhimurium (74, 75), B. abortus (224), Shigella flexneri (83), and Nocardia asteroides (20). L. pneumophila produces a cytoplasmic iron superoxide dismutase
[FeSOD, encoded by sodB (196)], a periplasmic copper-zinc superoxide dismutase [CuZnSOD, encoded by sodC ) (214)], and a periplasmic catalase:peroxidase
[KatA encoded by katA (7)]. Similar to the RpoS-dependent expression of antioxidants in E. coli, KatA is expressed maximally during the postexponential phase
(15), consistent with a role in transmission. The CuZnSOD enzyme appears to
be important for persistence, not replication; sodC mutants survive poorly in the
stationary phase, yet the sodC mutant replicated at wild-type rates in phagocytic
cells (214). L. pneumophila sodB is an essential gene, because sodB-null mutants
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cannot be recovered in the absence of a plasmid-borne copy of wild-type sodB
(196); hence, its role in virulence is difficult to access. A specific role for these
enzymes in transmission has not been evaluated directly.
In the environment, L. pneumophila likely resides in complex communities with
a variety of bacterial and protozoan species that may persist either as planktonic
cells or within surface-adherent biofilms (34). Recent advances in understanding the molecular mechanisms of biofilm formation (55) will likely provide a
framework for studies to elucidate the molecular determinants of persistence and
transmission of L. pneumophila in the environment.
LEGIONELLA PNEUMOPHILA VIRULENCE AS A
PARADIGM FOR OPPORTUNISTIC PATHOGENS
OF MACROPHAGES
As illustrated by the body of literature discussed, L. pneumophila is an excellent experimental tool to investigate how vacuolar pathogens thrive within macrophages.
L. pneumophila replicates with a doubling time of ∼2 h both in cultured
macrophages and in bacteriological medium. Avirulent mutants can be isolated by
either chemical- or transposon-mediated mutagenesis followed by simple genetic
enrichment or screening procedures. The intracellular growth phenotype of L.
pneumophila strains can be assessed by either visual or quantitative assays, and
the fate of intracellular bacteria can be determined by a series of fluorescence microscopic assays. Cloned DNA can be transferred efficiently into L. pneumophila
by electroporation, conjugation, or natural competence (216). Relatively high
rates of homologous recombination facilitate precise genetic engineering; alternatively, stable replicating plasmids are available. Finally, A/J mice and guinea pigs
provide animal models of Legionnaires’ disease for in vivo analysis of putative
virulence factors.
Although macrophages efficiently internalize and degrade most microorganisms, L. pneumophila is one of a number of pathogens that exploit this host defense pathway. Post-exponential–phase L. pneumophila resemble M. tuberculosis
(10, 56), Chlamydia psittaci (87), T. gondii (132, 206), and L. donovani promastigotes (57); each pathogen can establish vacuoles within macrophages that do not
mature into phagolysosomes. During the intracellular-replication phase, L. pneumophila is similar to L. donovani and C. burnetti, parasites that thrive in acidic
phagolysosomes. Although many biochemical and cell biological features of the
vacuoles harboring these pathogens have been identified, the mechanisms that they
use to evade fusion remain obscure.
Thus, a biochemical description of the mechanism used by L. pneumophila to
evade phagosome-lysosome fusion and later to replicate within phagolysosomes
may suggest novel strategies for treating infections by a variety of intracellular
pathogens. In addition, the L. pneumophila factor(s) that interfere with lysosomal
fusion may be valuable reagent(s) for more detailed analyses of membrane traffic in
mammalian cells. Because macrophages are central effector cells in both humoral
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and cell-mediated immunity, detailed knowledge of macrophage membrane traffic
is likely to provide a myriad of opportunities for improving human health. Finally,
understanding the ecology of L. pneumophila in the natural and potable water
supply may inform the design of preventative measures to combat the emergence
of new opportunistic macrophage pathogens.
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ACKNOWLEDGMENTS
We thank members of our laboratory for their contributions to the ideas and data
presented here and Dr. Joseph Vogel for numerous stimulating discussions over
the years and for a schematic that was the basis for Figure 3. We are also grateful
for the rich intellectual environment provided by the members of the Department
of Microbiology and Immunology at the University of Michigan. Our research
is supported by grants R29AI40694-01BM and R01 AI44212-01 from the National Institute of Allergy and Infectious Diseases of the National Institutes of
Health.
Visit the Annual Reviews home page at www.AnnualReviews.org
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CONTENTS
THE LIFE AND TIMES OF A CLINICAL MICROBIOLOGIST, Albert
Balows
ROLE OF CYTOTOXIC T LYMPHOCYTES IN EPSTEIN-BARR
VIRUS-ASSOCIATED DISEASES, Rajiv Khanna, Scott R. Burrows
BIOFILM FORMATION AS MICROBIAL DEVELOPMENT, George
O'Toole, Heidi B. Kaplan, Roberto Kolter
MICROBIOLOGICAL SAFETY OF DRINKING WATER, U. Szewzyk,
R. Szewzyk, W. Manz, K.-H. Schleifer
THE ADAPTATIVE MECHANISMS OF TRYPANOSOMA BRUCEI
FOR STEROL HOMEOSTASIS IN ITS DIFFERENT LIFE-CYCLE
ENVIRONMENTS, I. Coppens, P. J. Courtoy
THE DEVELOPMENT OF GENETIC TOOLS FOR DISSECTING THE
BIOLOGY OF MALARIA PARASITES, Tania F. de Koning-Ward,
Chris J. Janse, Andrew P. Waters
NUCLEIC ACID TRANSPORT IN PLANT-MICROBE
INTERACTIONS: The Molecules That Walk Through the Walls, Tzvi
Tzfira, Yoon Rhee, Min-Huei Chen, Talya Kunik, Vitaly Citovsky
PHYTOPLASMA: Phytopathogenic Mollicutes, Ing-Ming Lee, Robert E.
Davis, Dawn E. Gundersen-Rindal
ROOT NODULATION AND INFECTION FACTORS PRODUCED BY
RHIZOBIAL BACTERIA, Herman P. Spaink
ALGINATE LYASE: Review of Major Sources and Enzyme
Characteristics, Structure-Function Analysis, Biological Roles, and
Applications, Thiang Yian Wong, Lori A. Preston, Neal L. Schiller
INTERIM REPORT ON GENOMICS OF ESCHERICHIA COLI, M.
Riley, M. H. Serres
ORAL MICROBIAL COMMUNITIES: Biofilms, Interactions, and
Genetic Systems, Paul E. Kolenbrander
ROLES OF THE GLUTATHIONE- AND THIOREDOXINDEPENDENT REDUCTION SYSTEMS IN THE ESCHERICHIA COLI
AND SACCHAROMYCES CEREVISIAE RESPONSES TO OXIDATIVE
STRESS, Orna Carmel-Harel, Gisela Storz
RECENT DEVELOPMENTS IN MOLECULAR GENETICS OF
CANDIDA ALBICANS, Marianne D. De Backer, Paul T. Magee, Jesus
Pla
FUNCTIONAL MODULATION OF ESCHERICHIA COLI RNA
POLYMERASE, Akira Ishihama
BACTERIAL VIRULENCE GENE REGULATION: An Evolutionary
Perspective, Peggy A. Cotter, Victor J. DiRita
LEGIONELLA PNEUMOPHILA PATHOGENESIS: A Fateful Journey
from Amoebae to Macrophages, M. S. Swanson, B. K. Hammer
THE DISEASE SPECTRUM OF HELICOBACTER PYLORI : The
Immunopathogenesis of Gastroduodenal Ulcer and Gastric Cancer, Peter
B. Ernst, Benjamin D. Gold
PATHOGENICITY ISLANDS AND THE EVOLUTION OF
MICROBES, Jörg Hacker, James B. Kaper
DNA SEGREGATION IN BACTERIA, Gideon Scott Gordon, Andrew
Wright
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81
129
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221
257
289
341
413
439
463
499
519
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615
641
681
POLYPHOSPHATE AND PHOSPHATE PUMP, I. Kulaev, T.
Kulakovskaya
ASSEMBLY AND FUNCTION OF TYPE III SECRETORY SYSTEMS,
Guy R. Cornelis, Frédérique Van Gijsegem
PROTEINS SHARED BY THE TRANSCRIPTION AND
TRANSLATION MACHINES, Catherine L. Squires, Dmitry Zaporojets
Annu. Rev. Microbiol. 2000.54:567-613. Downloaded from arjournals.annualreviews.org
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HOLINS: The Protein Clocks of Bacteriophage Infections, Ing-Nang
Wang, David L. Smith, Ry Young
OXYGEN RESPIRATION BY DESULFOVIBRIO SPECIES, Heribert
Cypionka
REGULATION OF CARBON CATABOLISM IN BACILLUS
SPECIES, J. Stülke, W. Hillen
IRON METABOLISM IN PATHOGENIC BACTERIA, Colin Ratledge,
Lynn G Dover
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