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Applied and Environmental Microbiology, September 1998, p. 3134-3139, Vol. 64, No. 9
Department of Microbiology and Immunology,
University of Kentucky Chandler Medical Center, Lexington, Kentucky
40536-0084
Received 28 April 1998/Accepted 5 June 1998
The intracellular pathogens Legionella micdadei and
Legionella pneumophila are the two most common
Legionella species that cause Legionnaires' disease.
Intracellular replication within pulmonary cells is the hallmark of
Legionnaires' disease. In the environment, legionellae are parasites
of protozoans, and intracellular bacterial replication within
protozoans plays a major role in the transmission of Legionnaires'
disease. In this study, we characterized the initial host signal
transduction mechanisms involved during attachment to and invasion of
the protozoan host Hartmannella vermiformis by L. micdadei. Bacterial attachment prior to invasion of H. vermiformis by L. micdadei is associated with
tyrosine dephosphorylation of multiple host cell proteins, including a
170-kDa protein. We have previously shown that this 170-kDa protein is
the galactose N-acetylgalactosamine
(Gal/GalNAc)-inhibitable lectin receptor that mediates attachment to
and invasion of H. vermiformis by L. pneumophila. Subsequent bacterial entry targets L. micdadei into a phagosome that is not surrounded by the rough
endoplasmic reticulum (RER). In contrast, uptake of L. pneumophila mediated by attachment to the Gal/GalNAc lectin is
followed by targeting of the bacterium into an RER-surrounded
phagosome. These results indicate that despite similarities in the
L. micdadei and L. pneumophila attachment-mediated signal transduction mechanisms in H. vermiformis, the two bacterial species are targeted into
morphologically distinct phagosomes in their natural protozoan host.
Along with Streptococcus
pneumoniae and Haemophilus influenzae,
Legionella species are some of the most common etiologic
agents of bacterial pneumonia (10). Legionella
pneumophila and Legionella micdadei are the two most
common Legionella species that are associated with the
majority of outbreaks of Legionnaires' disease (29). Upon
inhalation, both of these Legionella species are
phagocytosed by alveolar cells (30). After internalization,
both Legionella species replicate within pulmonary cells,
and this intracellular replication is believed to be the hallmark of
Legionnaires' disease (15, 22). While L. pneumophila is targeted into a rough endoplasmic reticulum
(RER)-surrounded phagosome that does not fuse with lysosomes, L. micdadei is targeted into a RER-free phagosome that is thought to
fuse to lysosomes in human monocytes (16). Interestingly, within the RER-surrounded phagosome, L. pneumophila
undergoes dramatic alterations in gene expression, which are thought to be required for bacterial adaptation to this intracellular niche (2-4, 6, 7). Gene expression by intracellular L. micdadei has never been examined.
Outbreaks of Legionnaires' disease occur through droplet transmission
of the bacteria from an environmental water source, predominantly air
conditioning cooling towers and showerheads (16). In the
environment, Legionella species are ubiquitous and are
parasites of at least 13 species of amoebae and ciliated protozoans
(16). Invasion by and intracellular replication of L. pneumophila in protozoans play major roles in the infectivity and
transmission of the Legionnaires' disease bacteria to humans (16). We have recently shown that L. pneumophila possesses genetic loci that are required for
survival and replication within both human macrophages and protozoans,
and these loci have been designated pmi (protozoan and
macrophage infectivity) loci (19, 20). However, there are
other distinct bacterial loci that are uniquely required for
intracellular replication within macrophages but not within protozoans,
and these loci have been designated mil (macrophage-specific
infectivity) loci (19, 21).
Uptake of L. pneumophila by monocytes occurs by
microfilament-dependent coiling and conventional phagocytosis
mechanisms, while uptake of L. micdadei occurs only by
conventional phagocytosis (30, 35). In contrast to uptake of
L. pneumophila by macrophages, entry of the bacterium into
the protozoan host Hartmannella vermiformis occurs by
different mechanisms and is not inhibited by the microfilament disrupting agent cytochalasin D (5, 20, 21, 24, 27). Recently, we demonstrated the presence of a 170-kDa galactose N-acetylgalactosamine (Gal/GalNAc)-inhibitable lectin in
H. vermiformis (24, 34). We have recently shown
that type IV pili, which are filamentous appendages expressed on the
bacterial surface, are involved in bacterial adherence to mammalian and
protozoan cells (32). Whether the type IV pili are the
ligands for the Gal/GalNAc lectin is not known.
Most studies have used L. pneumophila as a model pathogen
for studying Legionnaires' disease, and the mechanism of entry of L. micdadei into H. vermiformis and the
intracellular environment in which L. micdadei resides
within the host cell are not well-defined. Hence, we sought to address
these issues, which may be important for understanding the
intracellular life cycle and pathogenesis of L. micdadei in
its protozoan host, H. vermiformis.
Bacterial strain and culture.
Virulent strain AA100 of
L. pneumophila is a virulent clinical isolate that has been
described previously (3). The virulent strain L. micdadei Rivera was obtained from Barry Fields and Janet Purckler,
Centers for Disease Control and Prevention (Atlanta, Ga.). The strains
were grown on buffered charcoal yeast extract (BCYE) agar plates.
Protozoan culture.
H. vermiformis CDC-19 (= ATCC
50237) has been cloned and grown in axenic culture and has been used as
a model organism to study the pathogenesis of L. pneumophila
(1, 17). This strain was isolated from a water source during
an outbreak of nosocomial Legionnaires' disease in a hospital in South
Dakota (11, 17). The amoebae were maintained in American
Type Culture Collection axenic culture medium 1034 supplemented with
10% fetal calf serum at 37°C (17).
Infection protocol.
H. vermiformis was harvested and
infected with L. micdadei as previously described (24,
34). Briefly, amoebae were incubated overnight in culture flasks
in serum-free axenic medium. Then amoebae were harvested by
centrifugation and resuspended in fresh serum-free axenic medium.
Aliquots containing 2 × 107 amoebae/ml were infected
with 109 L. micdadei cells. After several
intervals of coincubation at 37°C, amoebal cell lysates were prepared
for immunoblot analysis as described below.
0099-2240/98/$04.00+0
Copyright © 1998, American Society for Microbiology. All rights reserved.
Signal Transduction in the Protozoan Host
Hartmannella vermiformis upon Attachment and Invasion by
Legionella micdadei
ABSTRACT
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References
INTRODUCTION
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References
MATERIALS AND METHODS
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References
Transmission electron microscopy. Monolayers were infected with L. micdadei or L. pneumophila by using a multiplicity of infection of 100 for 1 h, followed by extensive washing of extracellular bacteria with American Type Culture Collection axenic medium 1034 supplemented with 10% fetal calf serum. Ultrathin sections were prepared as described previously (21). Briefly, infected amoebae were fixed with 3.5% gluteraldehyde and then with 1% OsO4, dehydrated with ethanol, and embedded in Eponate 12 resin (Ted Pella, Redding, Calif.). Ultrathin sections were stained with uranyl acetate and then with lead citrate and were examined with a model H-7000/STEM electron microscope (Hitachi Inc., Tokyo, Japan) at 75 kV.
Preparation of cell lysates and Western blotting. After incubation of H. vermiformis with L. micdadei, infection was stopped by using cold stop buffer containing 1× phosphate-buffered saline (pH 7.2) and the phosphatase inhibitors NaF (5 mM) and Na3VO4 (1 mM) (Sigma Chemical Co., St. Louis, Mo.) (34). To examine the effects of methylamine on tyrosine phosphorylation, amoebae were pretreated with methylamine, and cells were also infected in the presence of this inhibitor, which blocks entry of the bacteria (see below). The cells were washed three times with stop buffer and pelleted by low-speed centrifugation at 735 × g for 2 min. The supernatant containing bacteria was discarded, and the amoebae were lysed with cold 1% Triton X-100 lysis buffer (20 mM Tris-HCl [pH 7.6], 150 mM NaCl, 10 mM NaF, 1 mM Na3VO4, 1 mM EDTA, 1 mM phenylmethylsulfonyl fluoride, 2 µg of aprotinin per ml, 2 µg of leupeptin per ml) for 30 min on ice (34). The detergent-soluble and -insoluble fractions were separated by centrifugation at 16,000 × g for 30 min at 4°C in a microcentrifuge. Proteins from detergent-soluble fractions were resolved on sodium dodecyl sulfate-10% polyacrylamide gel electrophoresis gels under reducing conditions. The blots were transferred onto Immobilon-P membranes (Millipore, Bedford, Mass.) in a transfer cell (Bio-Rad, Hercules, Calif.) for 1.5 h with 0.2 M Tris-0.025 M glycine buffer containing 20% (vol/vol) methanol. After the proteins were transferred, the membranes were incubated for 30 min in a blocking buffer containing 1.5% bovine serum albumin. The membranes were then probed with a 1:5,000 dilution of horseradish peroxidase-conjugated recombinant anti-phosphotyrosine antibody RC20H (Transduction Laboratories, Lexington, Ky.) for 30 min at room temperature. After extensive washing, the blots were developed with an enhanced chemiluminescence kit (DuPont NEN, Boston, Mass.) according to the manufacturer's instructions.
Inhibition of L. pneumophila uptake by sugars. H. vermiformis was infected with L. micdadei as described below (23). To analyze the effects of different sugars on invasion of H. vermiformis by L. micdadei, infection experiments were performed in triplicate in the presence of the following sugars: galactose (Gal), N-acetyl-D-galactosamine (GalNAc), glucose, mannose, and lactose (Sigma). Solutions of these sugars were prepared in assay medium and stored at 4°C (1).
The effects of sugars on invasion of H. vermiformis by L. micdadei were studied by examining the growth kinetics of L. micdadei in H. vermiformis in the presence of sugars. H. vermiformis cells were preincubated for 15 min with various sugars at a concentration of 100 mM prior to infection with L. micdadei. At several times after infection (1, 2, 3, 4, and 5 days) amoebae were lysed by adding a mild detergent (0.04% Triton X-100) (20). Lysis of the amoebae was monitored microscopically and was complete within 1 min. This treatment had no effect on the viability of bacteria (data not shown). Dilutions were plated onto BCYE plates for colony enumeration.Invasion of amoebae in the presence of methylamine. Uptake of Legionella strains by amoebae was studied by using gentamicin protection invasion assays (24, 31). Briefly, H. vermiformis was resuspended in axenic medium at a concentration of 107 cells/ml. The amoebae were incubated for 1 h in the presence of 100 mM methylamine (Sigma), an inhibitor of receptor-mediated endocytosis, prior to infection (13, 27). Following infection with 109 L. micdadei cells for 1 h, the extracellular bacteria were killed with 50 µg of gentamicin per ml, and the intracellular bacteria were released following lysis of the amoebae with 0.04% Triton X-100. Lysis of the amoebae was monitored microscopically and was complete within 1 min. Different dilutions of bacteria were plated onto BCYE plates for colony enumeration (34).
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RESULTS |
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Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References
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Tyrosine dephosphorylation of H. vermiformis proteins after infection by L. micdadei. In this study we examined the changes in host protein tyrosine phosphorylation status during attachment to and invasion of the protozoan host H. vermiformis by L. micdadei. Amoebae were coincubated with L. micdadei for different periods of time, and cell lysates were immunoblotted with anti-phosphotyrosine antibody (Fig. 1). Infection of H. vermiformis with L. micdadei resulted in prominent time-dependent tyrosine dephosphorylation of several host cell proteins, including 200- to 205-, 170-, 150-, 69-, and 55-kDa proteins (Fig. 1, arrowheads).
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L. micdadei-induced protein tyrosine dephosphorylation in H. vermiformis is reversible. Next, we examined whether attachment to and invasion of H. vermiformis by L. micdadei resulted in a permanent change in the tyrosine phosphorylation status of cellular proteins or whether the alteration in host cell processes was reversible. H. vermiformis was coincubated with L. micdadei for 30 min, and extracellular bacteria were washed away after this incubation period. H. vermiformis cells were subsequently incubated for an additional 15 min, and the patterns of tyrosine-phosphorylated proteins were determined in immunoblots probed with anti-phosphotyrosine antibody. The levels of all of the tyrosine-dephosphorylated proteins returned to the normal levels observed with uninfected cells (data not shown). Overall, these observations indicated that attachment and invasion by L. micdadei resulted in tyrosine dephosphorylation of multiple proteins in H. vermiformis. This bacterium-induced tyrosine dephosphorylation in H. vermiformis was reversible and dependent on the continuous presence of bacteria.
Invasion of H. vermiformis by L. micdadei
can be blocked by Gal and GalNAc.
Previous work in our laboratory
showed that a 170-kDa 
2 integrin-like Gal/GalNAc lectin is present
in H. vermiformis (Fig. 2, arrowhead) (34). This
lectin mediates attachment of L. pneumophila and undergoes
tyrosine dephosphorylation during this process (34). Invasion and intracellular replication of L. pneumophila in
H. vermiformis were effectively blocked by the Gal and
Gal/GalNAc monomers (34). To examine the functional
significance of the 170-kDa lectin of H. vermiformis in the
invasion of L. micdadei, blocking experiments were performed
in the presence of Gal and Gal/GalNAc. Amoebae were preincubated with
Gal or Gal/GalNAc for 15 min prior to infection with L. micdadei (Fig. 3). Mannose, lactose,
and glucose were used as negative controls to verify the specificity of
the sugars (data not shown). The presence of GalNAc had a dramatic
effect on invasion by L. micdadei. Gal had a less dramatic
effect but still sufficiently blocked the invasion of bacteria.
Mannose, glucose, and lactose had no effect on bacterial invasion, and
the number of intracellular bacteria at the end of the infection period
in the presence of these sugars was very similar to the number in the
untreated control (data not shown). The inhibition of bacterial
invasion by Gal and Gal/GalNAc was dose dependent (data not shown), and
these sugars were not toxic to H. vermiformis
(34). These results indicated that the 170-kDa Gal/GalNAc
lectin was involved in attachment to and invasion of H. vermiformis by L. micdadei.
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Attachment of L. micdadei to H. vermiformis is required to induce protein tyrosine dephosphorylation of host cell proteins. We examined whether bacterial attachment to the Gal/GalNAc lectin was required to cause changes in the pattern of protein tyrosine phosphorylation in H. vermiformis. In these experiments, H. vermiformis cells were incubated with L. micdadei in the presence of Gal, Gal/GalNAc, or mannose (Fig. 2, lanes 7 through 9). Incubation of H. vermiformis with these sugars does not alter the profile of tyrosine phosphorylated proteins in resting amoebae (34). H. vermiformis cell lysates were prepared after 20 min of infection and were analyzed by using immunoblots probed with the anti-phosphotyrosine antibody. The presence of Gal (Fig. 2, lane 7) or Gal/GalNAc (Fig. 2, lane 8) completely blocked bacterium-induced tyrosine dephosphorylation of the 170- and 69-kDa proteins (Fig. 2, lanes 7 and 8). These proteins have been identified as the Gal/GalNAc lectin (170 kDa) (34) and paxillin (69 kDa) (unpublished data). As expected, mannose failed to block tyrosine dephosphorylation of proteins in H. vermiformis (Fig. 2, lane 9). These results indicated that contact of L. micdadei with the Gal/GalNAc lectin on H. vermiformis was required to mediate early changes in the tyrosine phosphorylation of host cell proteins.
Invasion of H. vermiformis by L. micdadei is not required to induce protein tyrosine dephosphorylation. Since bacterial attachment was essential for induction of tyrosine dephosphorylation of amoebal proteins (Fig. 2), we examined whether entry of L. micdadei into H. vermiformis was also required to mediate these effects. Methylamine has been shown to block the entry of L. pneumophila into H. vermiformis (27). We examined whether methylamine blocks invasion by L. micdadei by using the gentamicin protection assay (see above). In this assay, following a period of infection, extracellular bacteria were killed with the antibiotic gentamicin, while intracellular bacteria were protected and subsequently enumerated (34). The data showed that like inhibition of invasion of H. vermiformis by L. pneumophila, invasion by L. micdadei was inhibited 99% in the presence of 100 mM methylamine (data not shown).
Next, we examined whether inhibition of invasion of H. vermiformis by L. micdadei blocked bacterium-induced tyrosine dephosphorylation. Incubation of resting H. vermiformis with methylamine did not affect the pattern of protein tyrosine phosphorylation (Fig. 4, lanes 1 and 2). Interestingly, inhibition of protozoan invasion by L. micdadei did not block bacterium-induced tyrosine dephosphorylation of H. vermiformis proteins. These results indicated that attachment but not entry of L. micdadei was sufficient to induce protein tyrosine dephosphorylation in H. vermiformis.
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L. micdadei is surrounded by a smooth phagosome in H. vermiformis. In H. vermiformis, L. pneumophila is targeted into an RER-surrounded phagosome (1). Transmission electron microscopy showed that after invasion of H. vermiformis by L. micdadei, the bacteria were targeted into RER-free phagosomes (Fig. 5). None of the approximately 100 phagosomes examined that contained L. micdadei was surrounded by RER; in contrast, 83% of the phagosomes containing L. pneumophila were surrounded by RER (1, 20). By 18 h postinfection, the amoebae were heavily infected with both Legionella species. These data showed that despite the fact that L. pneumophila and L. micdadei had similar mechanisms for utilizing the Gal/GalNAc lectin in bacterial attachment and invasion, these two species were targeted into phagosomes that were morphologically distinct, at least at the ultrastructural level.
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DISCUSSION |
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Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References
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This study focused on the initial signaling events that are activated upon attachment to and invasion of H. vermiformis by the Legionnaires' disease bacterium L. micdadei. We recently identified the Gal/GalNAc lectin of H. vermiformis as a potential receptor utilized by L. pneumophila for attachment to and invasion of protozoans (34). In this study we examined the role of the Gal/GalNAc lectin in the attachment to and invasion of H. vermiformis by L. micdadei. While entry of several pathogenic bacteria, including Salmonella typhimurium and Shigella flexneri, induces host tyrosine phosphorylation events (18), uptake of L. micdadei by H. vermiformis is associated with rapid and reversible tyrosine dephosphorylation of host cell proteins. These effects of L. micdadei on H. vermiformis are similar to the effects induced by Yersinia pseudotuberculosis during invasion of macrophages, which are mediated by a bacterial tyrosine phosphatase (28). Dephosphorylation of cellular proteins prevents uptake of Yersinia cells (18). In contrast, tyrosine dephosphorylation of H. vermiformis proteins appears to be associated with attachment and uptake of L. micdadei. These observations indicate that there is a novel mechanism for uptake of L. micdadei by H. vermiformis.
The Gal/GalNAc lectin of Entamoeba histolytica is
antigenically similar to the human 2 integrin and is involved in
cytoskeleton-mediated capping and shedding of antibody and
complement from the protozoan surface in order to evade the immune
system of the human host (8, 9). Utilization of the
Gal/GalNAc lectin on the protozoan host and its tyrosine
dephosphorylation upon bacterial attachment and invasion may provide a
selective advantage for L. micdadei in the regulation of its
uptake process. In contrast to the induction of tyrosine
phosphorylation of the receptor and other proteins upon engagement of
receptors with their ligands (12), attachment of L. micdadei to the Gal/GalNAc lectin was associated with tyrosine dephosphorylation of the lectin and other host proteins. We offer three
hypotheses to explain this observation. First, bacterium-induced tyrosine dephosphorylation of the lectin may disrupt communication of
the lectin with the underlying cytoskeleton in order to avoid shedding
of bacteria from the protozoan surface. This hypothesis is supported by
our recent observations that several lectin-associated proteins are
dissociated upon attachment of L. pneumophila to H. vermiformis (unpublished data). Some of these dissociated proteins may represent important cytoskeletal proteins involved in cell shape,
motility, and chemotaxis in amoebae. In addition, four protozoan
cytoskeletal proteins, including paxillin, vinculin, and
pp125FAK, undergo tyrosine dephosphorylation upon
attachment to L. pneumophila, indicating that disruption of
the cytoskeleton occurs (33). Second, dephosphorylation of
H. vermiformis proteins upon attachment to L. micdadei may downregulate host signals in order to prevent activation of phagocytic signals and microbicidal mechanisms that result in degradation of engulfed bacteria. Subversion of the bactericidal protozoan host cell processes by L. micdadei
may be supported by observations made with neutrophils (14).
Suppression of host cell functions, like superoxide anion production,
chemotaxis, and bactericidal activity, by L. micdadei was
observed when human neutrophils were allowed to attach to and ingest
bacteria (14). Third, dephosphorylation of H. vermiformis proteins, including the Gal/GalNAc lectin, by L. micdadei may increase the efficiency of bacterial uptake. Overall,
attachment to and invasion of H. vermiformis by L. micdadei mediated by the Gal/GalNAc lectin may be required to
accomplish more than one of the strategies proposed above to ensure
efficient uptake and subsequent targeting of the bacteria into a safe
phagosome in amoebae.
The Gal/GalNAc lectin of H. vermiformis is important for invasion by L. micdadei, as determined by blocking experiments performed with Gal and Gal/GalNAc. Recent studies in our laboratory have demonstrated the importance of the 170-kDa Gal lectin in the invasion of H. vermiformis by L. pneumophila (24, 34). The sugars Gal and Gal/GalNAc block invasion of H. vermiformis by L. pneumophila (24, 34). It is interesting to note that the two most frequent causative agents of Legionnaires' disease, L. micdadei and L. pneumophila, utilize a common host cell receptor to invade H. vermiformis.
Intracellular pathogens like L. pneumophila and Mycobacterium tuberculosis utilize the CR3 receptor to enter human macrophages, which prevents activation of oxidative bursts and other microbicidal mechanisms (36, 37). Uptake of many intracellular pathogens through the Fc receptor (instead of CR3) targets the internalized bacteria into lysosomes instead of a replicative niche (25, 26). Although L. pneumophila and L. micdadei utilize the Gal/GalNAc lectin for attachment and invasion of their protozoan host, H. vermiformis, the intracellular fates of the two bacterial species are different, at least at the ultrastructural level. Accordingly, a phagosome that contains L. pneumophila but not L. micdadei is surrounded by RER. These observations are similar to observations made with a phagosome containing both of these Legionella species in macrophages and epithelial cells (unpublished data). These results suggest that additional mechanisms (other than utilizing the Gal/GalNAc lectin) may be involved in the trafficking L. micdadei to the RER-free replicative phagosome. It is possible that L. micdadei resides in a relatively nascent phagosome, while L. pneumophila exists in a more mature phagosome that is surrounded by RER.
In summary, this study and our earlier report (34) revealed novel but similar signaling mechanisms that are triggered in the protozoan host H. vermiformis following attachment and invasion by the two Legionella species that have been most commonly implicated in outbreaks of Legionnaires' disease. Despite the similarities in what appears to be receptor-mediated signaling events, the two species are targeted into different compartments in the protozoan host. Additional studies should help explain the exact strategies utilized by Legionella spp. to enter and invade two evolutionarily distant hosts, human cells and environmental free-living amoebae.
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ACKNOWLEDGMENTS |
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Y.A. was supported by Public Health Service award R29AI38410. O.S.H. was supported by predoctoral National Research Service award 5T32CA09509.
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FOOTNOTES |
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* Corresponding author. Mailing address: Department of Microbiology and Immunology, University of Kentucky Chandler Medical Center, Lexington, KY 40536-0084. Phone: (606) 323-3873. Fax: (606) 257-8994. E-mail: yabukw{at}pop.uky.edu.
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REFERENCES |
|---|
|
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Abstract
Introduction
Materials & Methods
Results
Discussion
References
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| 1. | Abu Kwaik, Y. 1996. The phagosome containing Legionella pneumophila within the protozoan Hartmanella vermiformis is surrounded by the rough endoplasmic reticulum. Appl. Environ. Microbiol. 62:2022-2028[Abstract]. |
| 2. |
Abu Kwaik, Y.
1998.
Induced expression of the Legionella pneumophila gene encoding a 20-kilodalton protein during intracellular infection.
Infect. Immun.
66:203-212 |
| 3. |
Abu Kwaik, Y.,
B. I. Eisenstein, and N. C. Engleberg.
1993.
Phenotypic modulation by Legionella pneumophila upon infection of macrophages.
Infect. Immun.
61:1320-1329 |
| 4. | Abu Kwaik, Y., and N. C. Engleberg. 1994. Cloning and molecular characterization of a Legionella pneumophila gene induced by intracellular infection and by various in vitro stress stimuli. Mol. Microbiol. 13:243-251[Medline]. |
| 5. |
Abu Kwaik, Y.,
B. S. Fields, and N. C. Engleberg.
1994.
Protein expression by the protozoan Hartmannella vermiformis upon contact with its bacterial parasite, Legionella pneumophila.
Infect. Immun.
62:1860-1866 |
| 6. | Abu Kwaik, Y., L.-Y. Gao, O. S. Harb, and B. J. Stone. 1997. Transcriptional regulation of the macrophage-induced gene (gspA) of Legionella pneumophila and phenotypic characterization of a null mutant. Mol. Microbiol. 24:629-642[Medline]. |
| 7. | Abu Kwaik, Y., and L. L. Pederson. 1996. The use of differential display-PCR to isolate and characterize a Legionella pneumophila locus induced during the intracellular infection of macrophages. Mol. Microbiol. 21:543-556[Medline]. |
| 8. |
Adams, S. A.,
S. C. Robson,
V. Gathiram,
T. F. H. G. Jackson,
T. S. Pillay,
R. E. Kirsch, and M. W. Makgoba.
1993.
Immunological similarity between the 170 kDa amoebic adherence glycoprotein and human 2 integrins.
Lancet
341:17-19[Medline].
|
| 9. | Arhets, P., P. Gounon, P. Sansonetti, and N. Guillen. 1995. Myosin II is involved in capping and uroid formation in the human pathogen Entamoeba histolytica. Infect. Immun. 63:4358-4367[Abstract]. |
| 10. |
Bates, J. H.,
G. D. Campbell, and A. L. Baron.
1992.
Microbial etiology of acute pneumonia in hospitalized patients.
Chest
101:1005-1012 |
| 11. |
Breiman, R. F.,
B. S. Fields,
G. N. Sanden,
L. Volmer,
A. Meier, and J. S. Spika.
1990.
Association of shower use with Legionnaires' disease: possible role of amoebae.
JAMA
263:2924-2926 |
| 12. |
Clark, E. A., and J. S. Brugge.
1995.
Integrins and signal transduction pathways: the road taken.
Science
268:233-239 |
| 13. |
Davies, P. J. A.,
D. R. Davies,
A. Levitzki,
F. R. Maxfield,
P. Milhaud,
M. C. Willingham, and I. H. Pastan.
1980.
Transglutaminase is essential in receptor-mediated endocytosis of  2-macroglobulin and polypeptide hormones.
Nature
283:162-167[Medline].
|
| 14. |
Donowitz, G. R.,
I. Readon,
J. Dowling,
L. Rubin, and D. Focht.
1990.
Ingestion of Legionella micdadei inhibits human neutrophil function.
Infect. Immun.
58:3307-3311 |
| 15. |
Dowling, J. N.,
A. K. Saha, and R. H. Glew.
1992.
Virulence factors of the family Legionellaceae.
Microbiol. Rev.
56:32-60 |
| 16. | Fields, B. S. 1996. The molecular ecology of legionellae. Trends Microbiol. 4:286-290[Medline]. |
| 17. | Fields, B. S., T. A. Nerad, T. K. Sawyer, C. H. King, J. M. Barbaree, W. T. Martin, W. E. Morrill, and G. N. Sanden. 1990. Characterization of an axenic strain of Hartmannella vermiformis obtained from an investigation of nosocomial legionellosis. J. Protozool. 37:581-583[Medline]. |
| 18. |
Finlay, B. B., and P. Cossart.
1997.
Exploitation of mammalian host cell functions by bacterial pathogens.
Science
276:718-725 |
| 19. | Gao, L.-Y., M. Gutzman, J. K. Brieland, and Y. Abu Kwaik. Different fates of Legionella pneumophila pmi and mil mutants within human-derived macrophages and alveolar epithelial cells. Submitted for publication. |
| 20. | Gao, L.-Y., O. S. Harb, and Y. Abu Kwaik. 1997. Utilization of similar mechanisms by Legionella pneumophila to parasitize two evolutionarily distant hosts, mammalian and protozoan cells. Infect. Immun. 65:4738-4746[Abstract]. |
| 21. |
Gao, L.-Y.,
O. S. Harb, and Y. Abu Kwaik.
1998.
Identification of macrophage-specific infectivity loci (mil) of Legionella pneumophila that are not required for infectivity of protozoa.
Infect. Immun.
66:883-892 |
| 22. | Gress, F. M., R. L. Myerowitz, A. W. Pasculle, C. R. Rinaldo, Jr., and J. N. Dowling. 1980. The ultrastructural morphologic features of Pittsburgh pneumonia agent. Am. J. Pathol. 101:63-78[Abstract]. |
| 23. |
Harb, O. S., and Y. Abu Kwaik.
1998.
Identification of the aspartate--semiadehyde dehydrogenase gene of Legionella pneumophila and characterization of a null mutant.
Infect. Immun.
66:1898-1903 |
| 24. |
Harb, O. S.,
C. Venkataraman,
B. J. Haack,
L.-Y. Gao, and Y. Abu Kwaik.
1998.
Heterogeneity in the attachment and uptake mechanisms of the Legionnaires' disease bacterium, Legionella pneumophila, by protozoan hosts.
Appl. Environ. Microbiol.
64:126-132 |
| 25. |
Horwitz, M. A.
1983.
The Legionnaires' disease bacterium (Legionella pneumophila) inhibits phagosome-lysosome fusion in human monocytes.
J. Exp. Med.
158:2108-2126 |
| 26. |
Joiner, K. A.,
S. A. Fuhrman,
H. M. Miettinen,
L. H. Kasper, and I. Mellman.
1990.
Toxoplasma gondii: fusion competence of parasitophorous vacuoles in Fc receptor-transfected fibroblasts.
Science
249:641-646 |
| 27. |
King, C. H.,
B. S. Fields,
E. B. Shotts, Jr., and E. H. White.
1991.
Effects of cytochalasin D and methylamine on intracellular growth of Legionella pneumophila in amoebae and human monocyte-like cells.
Infect. Immun.
59:758-763 |
| 28. | Mecsas, J., and E. J. Strauss. 1996. Molecular mechanisms of bacterial virulence: type III secretion and pathogenicity islands. Emerg. Infect. Dis. 2:271-287. |
| 29. | Pasculle, A. W., J. C. Feeley, R. J. Gibson, L. G. Cordes, R. L. Myerowitz, C. M. Patton, G. W. Gorman, C. L. Carmack, J. W. Ezzell, and J. N. Dowling. 1980. Pittsburgh pneumonia agent: direct isolation from human lung tissue. J. Infect. Dis. 141:727-732[Medline]. |
| 30. | Rechnitzer, C., and J. Blom. 1989. Engulfment of the Philadelphia strain of Legionella pneumophila within pseudopod coils in human phagocytes. Comparison with the other Legionella strains and species. Acta Pathol Microbiol. Immunol. Scand. Sect. B 97:105-114. |
| 31. |
Sadosky, A. B.,
L. A. Wiater, and H. A. Shuman.
1993.
Identification of Legionella pneumophila genes required for growth within and killing of human macrophages.
Infect. Immun.
61:5361-5373 |
| 32. |
Stone, B. J., and Y. Abu Kwaik.
1998.
Expression of multiple pili by Legionella pneumophila: identification and characterization of a type IV pili gene and its role in adherence to mammalian and protozoan cells.
Infect. Immun.
66:1768-1775 |
| 33. |
Venkataraman, C.,
L.-Y. Gao,
S. Bondada, and Y. Abu Kwaik.
1998.
Identification of putative cytoskeletal protein homologues in the protozoan Hartmannella vermiformis as substrates for induced tyrosine phosphatase activity upon attachment to the Legionnaires' disease bacterium, Legionella pneumophila.
J. Exp. Med.
188:1-10 |
| 34. |
Venkataraman, C.,
B. J. Haack,
S. Bondada, and Y. Abu Kwaik.
1997.
Identification of a Gal/GalNAc lectin in the protozoan Hartmannella vermiformis as a potential receptor for attachment and invasion by the Legionnaires' disease bacterium, Legionella pneumophila.
J. Exp. Med.
186:537-547 |
| 35. |
Weinbaum, D. L.,
R. R. Benner,
J. N. Dowling,
A. Alpern,
A. W. Pasculle, and G. R. Donowitz.
1984.
Interaction of Legionella micdadei with human monocytes.
Infect. Immun.
46:68-73 |
| 36. |
Wilson, C. B.,
V. Tsai, and J. S. Remington.
1980.
Failure to trigger the oxidative burst of normal macrophages. Possible mechanisms for survival of intracellular pathogens.
J. Exp. Med.
151:328-346 |
| 37. |
Wright, S. D., and S. C. Silverstein.
1983.
Receptors for C3b and C3bi promote phagocytosis but not the release of toxic oxygen from human phagocytes.
J. Exp. Med.
158:2016-2023 |
Applied and Environmental Microbiology, September 1998, p. 3134-3139, Vol. 64, No. 9
0099-2240/98/$04.00+0
Copyright © 1998, American Society for Microbiology. All rights reserved.
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