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Previous Article | Next Article 
Infection and Immunity, March 2000, p. 1681-1686, Vol. 68, No. 3
Division of Microbiology and Infectious
Diseases, Western Australian Centre for Pathology and Medical Research,
Nedlands, Western Australia 60091 and
Department of Microbiology,2
Biomedical Confocal Microscopy Research Centre, Department
of Pharmacology,3 and Electron
Microscopy Unit, Department of Pathology,4
University of Western Australia, Nedlands, Western Australia 6907, Australia
Received 21 September 1999/Returned for modification 19 October
1999/Accepted 8 November 1999
Burkholderia pseudomallei causes melioidosis, a
potentially fatal disease whose clinical outcomes include rapid-onset
septicemia and relapsing and delayed-onset infections. Like other
facultative intracellular bacterial pathogens, B. pseudomallei is capable of survival in human phagocytic cells,
but unlike mycobacteria, Listeria monocytogenes, and
Salmonella serovar Typhimurium, the species has not been
reported to survive as an endosymbiont in free-living amebae. We
investigated the consequences of exposing Acanthamoeba
astronyxis, A. castellani, and A. polyphaga to B. pseudomallei NCTC 10276 in a series
of coculture experiments. Bacterial endocytosis was observed in
all three Acanthamoeba species. A more extensive range of
cellular interactions including bacterial adhesion, incorporation into
amebic vacuoles, and separation was observed with A. astronyxis in timed coculture experiments. Amebic trophozoites
containing motile intravacuolar bacilli were found throughout 72 h
of coculture. Confocal microscopy was used to confirm the intracellular
location of endamebic B. pseudomallei cells. Transmission
electron microscopy of coculture preparations revealed clusters of
intact bacilli in membrane-lined vesicles inside the trophozoite
cytoplasm; 5 × 102 CFU of bacteria per ml were
recovered from lysed amebic trophozoites after 60 min of coculture.
Demonstration of an interaction between B. pseudomallei and
free-living acanthamebae in vitro raises the possibility that a similar
interaction in vivo might affect environmental survival of
B. pseudomallei and subsequent human exposure. Endamebic passage of B. pseudomallei warrants further investigation
as a potential in vitro model of intracellular B. pseudomallei infection.
Burkholderia pseudomallei
causes melioidosis, a potentially fatal infection with septicemic,
subacute, and chronic forms that is endemic in northern Australia and
Southeast Asia. The acute septicemic form has a mortality rate of over
30% (31) and may present after a disease-free interval of
several decades (27). Survival of B. pseudomallei
within macrophages has been demonstrated in vitro and is proposed as an
explanation for delayed-onset disease (17, 24). Survival of
B. pseudomallei for extended periods in the absence of
nutrients and under acid conditions may allow persistence in a hostile
intracellular habitat (18, 32) but does not explain how
B. pseudomallei enters or escapes from professional phagocytic cells. A potential explanation is that B. pseudomallei has developed the capacity to invade and survive
within free-living amebae in the moist soil and surface water
environment where the bacterial species is normally found (5, 15,
29) and amebae are also likely to be present (25).
Several facultative bacterial intracellular pathogens, notably
Legionella pneumophila, Mycobacterium avium, and
Listeria monocytogenes, have been shown to survive as
endosymbionts in free-living amebae such as Acanthamoeba, Hartmanella,
and Naegleria (8, 20, 26). It has been suggested that growth
in an amebic intracellular environment might assist these bacteria to
adapt to survival in mammalian phagocytic cells (3, 7, 11,
26). Moreover, incorporation of bacteria in amebic cysts has also
been shown to confer resistance to adverse environmental conditions
such as exposure to biocidal agents (1). In addition, amebic
endosymbiosis is known to augment the virulence of L. pneumophila and M. avium (6-8). The
endocytosis of L. pneumophila by both
Acanthamoeba and macrophages has common features including
an unusual, coiling form of phagocytosis (3, 14). Although
evidence for the direct involvement of amebic endosymbionts in the
pathogenesis of human infection has yet to be found, molecular methods
and animal models indicate a possible role for free-living amebae as
determinants of bacterial virulence (4, 6-8, 11).
The best evidence in support of a putative endosymbiosis involving
B. pseudomallei and free-living protozoa is circumstantial: Ralstonia (previously Burkholderia)
pickettii, a close phylogenetic relative of B. pseudomallei, has been found in Acanthamoeba species from a hospital environment (23). More recently,
intra-amebic survival of Burkholderia cepacia has been
demonstrated in Acanthamoeba strains in vitro
(21). However, there has been no report of any interaction
between B. pseudomallei and free-living amebae to date, nor
has there been any report of simultaneous recovery of B. pseudomallei and Acanthamoeba species from the same
environmental location. During investigations into a recent cluster of
acute melioidosis cases in Western Australia, the strain of B. pseudomallei responsible for the outbreak was isolated from
potable water in the affected community (15). Several
Acanthamoeba species were also recovered from water
specimens collected during the initial outbreak and in further
environmental investigations over the following year, prompting the
present study into possible interactions between
Acanthamoeba species and B. pseudomallei.
Organisms.
The three amebic species used were
Acanthamoeba castellani CCAP 1534/1, A. polyphaga
CCAP 1501/3A, and A. astronyxis CCAP 1534/1. These were
obtained as axenic strains and maintained in axenic culture media (PYG
broth; Excel Laboratory Products, Bentley, Western Australia,
Australia) in tissue culture flasks incubated at 30°C. Culture media
were changed weekly. The supernatant fluid was kept in a sterile
plastic container at 20°C and used within 1 week of harvesting.
Amebic suspensions were examined by bright-field and phase-contrast
microscopy immediately prior to use. Viable trophozoite counts were
obtained with a Fuchs-Rosenthal hemocytometer and trypan blue vital stain.
0019-9567/00/$04.00+0
Copyright © 2000, American Society for Microbiology. All rights reserved.
Interaction between Burkholderia
pseudomallei and Acanthamoeba Species Results in
Coiling Phagocytosis, Endamebic Bacterial Survival, and
Escape
ABSTRACT
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
INTRODUCTION
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
MATERIALS AND METHODS
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
70°C and
subcultured via 5% horse blood agar to Trypticase soy broth (Excel
Laboratory Products), in which it was grown at 37°C for 18 h. In
previous experiments, it had been found that the logarithmic phase of
bacterial growth commenced 3 to 4 h after inoculation of the
growth medium. The pellicle was lifted aside with a sterile loop and a
1.0-ml aliquot of bacterial suspension transferred to 9.0 ml of fresh Trypticase soy broth. The mixture was incubated at 37°C for 1 h
to obtain rapidly motile bacteria in mid-log phase and then for 2 and
24 h. Bacterial motility was checked by examination of bacteria at
×400 magnification under phase-contrast illumination. All
aerosol-generating procedures were carried out in a biological safety cabinet.
Light microscopy. After it was ensured that amebic cells were present in all three identifiable forms (rounded trophozoites, trophozoites with acanthopodia, and thick-walled cysts) and no bacterial contamination had occurred, a 10-µl drop of bacterial suspension was combined with a 10-µl drop of amebic suspension. The interaction was followed under phase contrast at ×400 magnification for 120 min. Separate aliquots were kept in sterile containers at 20°C for 24 h and then reexamined under phase-contrast conditions. Each bacterial-amebic challenge was repeated on at least three occasions with each of the Acanthamoeba spp. The interaction between B. pseudomallei and A. astronyxis was repeated on three further consecutive occasions, when aliquots were taken at time (t) = 0, 20 min, 40 min, 60 min, and 24 h. On each occasion, 20 intact trophozoites were examined for the presence of surface-adherent bacilli, rapidly rotating bacilli, vacuoles containing single bacilli, multibacillary vacuoles (>2 bacilli per vacuole), polar tufts of bacilli (at least five), and extracellular bacterial tangles.
The interaction was followed using a confocal laser scanning microscope with simultaneous differential interference contrast microscopy (Bio-Rad, Hemel-Hempstead, United Kingdom). The vital stains SYTO 9 (1 µl of 3.34 mM in dimethyl sulfoxide; Molecular Probes, Eugene, Oreg.) and propidium iodide (1 µl of 20 mM in dimethyl sulfoxide; Molecular Probes) were added to 1 ml of microbial suspension and incubated for 5 min to assess bacterial viability in unfixed, wet-mounted preparations. Viable cells fluoresced bright green, and nonviable cells fluoresced red. Bacterial suspensions were centrifuged for 5 min at 1,000 × g and resuspended in sterile 0.89% NaCl solution; 24-h-old coincubated preparations were stained by direct addition of 1 µl of 3.34 mM SYTO 9 to 10 µl of cell suspension without a centrifugation or washing step. The internal structure of amebic cells was examined with the microscope's optical sectioning facility to confirm the intracellular location of bacteria. Time-lapse confocal microscopy was used to confirm movement of bacteria within amebic vacuoles.Transmission electron microscopy. After confirmation of phagocytosis by phase-contrast microscopy, suspensions of B. pseudomallei cocultured with A. astronxyis were centrifuged at 300 × g for 10 min. The supernatant was removed by pipette and replaced with fresh 2.5% glutaraldehyde. Cell suspensions were fixed in 2.5% glutaraldehyde in 0.05 M cacodylate buffer (pH 7.4) for 24 h, washed several times in 0.2 M cacodylate buffer (pH 7.4), resuspended in 10% albumin solution, placed in a microcentrifuge tube, and centrifuged; the resultant sediment of cells was fixed in 2.5% glutaraldehyde for a further 24 h. The tip of the tube was cut with a single-edged blade, and the block containing cells was postfixed in 1% osmium tetroxide, dehydrated in graded solutions of ethanol, infiltrated with Araldite, and embedded in polyethylene capsules. After retrimming of each block face, 50-nm ultrathin sections were cut, mounted on 200-mesh thin-bar copper grids, double stained in uranyl acetate and lead citrate, and examined in a Philips 410LS transmission electron microscope at an accelerating voltage of 80 kV.
Recovery of intracellular bacteria. Intracellular bacteria were recovered by a kanamycin-trophozoite lysis procedure as follows. A 5.0-ml coculture volume was set up with a suspension of 105 of trophozoites per ml in 0.9 N NaCl and a 1-h preparation of rapidly motile B. pseudomallei at 107 CFU/ml. Aliquots of 1.0 ml were removed at t = 0 and t = 1 h. These were centrifuged at 300 × g for 5 min, and the pellet was resuspended in 0.9 N NaCl with 100 µg of kanamycin sulfate (Sigma, St. Louis, Mo.) per ml. After 2 h of incubation at 20°C, the aliquot was centrifuged again at 300 × g for 5 min and resuspended in sterile, demineralized water. The sealed container was incubated in a waterbath at 37°C for 30 min and then vortex mixed for 30 s and sonicated for 15 m. The suspension was then centrifuged at 1,000 × g for 5 min and resuspended in 0.9 N NaCl. This suspension was spread in 50-µl aliquots on plate count agar in triplicate, using a spiral plating device. The remaining 850 µl was spread on a further plate count agar plate to detect very low bacterial counts. The same plating procedure was used with aliquots of the uninoculated amebic stock suspension, sterile demineralized water, and 0.9 N NaCl as negative controls. Plates were incubated in air at 37°C for 48 h before counting using a standard template (Don Whitley). Viable counts were obtained using the spiral plater interpretive tables. Viable counts were also performed at t = 0 and t = 60 min after inoculation of 0.9% NaCl with a 1-h B. pseudomallei preparation to determine whether bacterial growth occurred in the coculture medium during this period.
Statistical methods. Descriptive statistics and the unpaired t test were calculated with the assistance of statistical software (Prism, version 2.01; GraphPad Software, San Diego, Calif.).
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RESULTS |
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Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
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Early bacterium-ameba interactions.
When B. pseudomallei was added to a suspension of A. astronyxis, adhesion, endocytosis, and vacuole formation were all
observed, in that order. The sequence of cellular events could be
observed in individual amebic trophozoites and occurred in a 1-h time
frame when a 1-h preparation of highly motile B. pseudomallei was added to acanthapodium-bearing A. astronyxis trophozoites. This sequence of events was quantitated
in the timed coculture procedure where bacterial adhesion to the
trophozoite surface and rapid bacillary rotation were followed by the
appearance of vacuoles containing single bacilli, then multibacillary
vacuoles, and finally formation of external bacillary tufts (Table
1). In the initial adhesion event, one
end of a highly motile bacillus attached to the cytoplasmic membrane of
the amebic trophozoite (Fig. 1a). In most
instances, this was followed immediately by high-frequency helicopter
blade rotation of the entire bacillus around a fixed point on the
trophozoite surface. This lasted no more than a few seconds. Shortly
after bacillary rotation ended, adjacent pseudopodia curled round to enclose the bacillus. Formation of a complete cytoplasmic bridge around the bacillus resulted in its inclusion within a
membrane-lined vacuole. Pseudopod extension and curling were fast
enough to observe in real time. These events occurred repeatedly in
individual trophozoites, resulting in a collection of
bacterium-containing vacuoles (Fig. 1b). No endocytosis by fully
rounded trophozoites or cysts was observed. Using a 1-h motile
bacterial preparation, intracellular bacilli were easily demonstrated
by uptake of the fluorescent stain SYTO 9 (Fig. 1). Multiple vacuoles
packed with bacilli were observed inside rounded intact trophozoites.
No green fluorescent bacilli were observed moving freely in the
cytoplasm of intact trophozoites. Some intravacuolar bacteria were seen
to retain their motility for 1 to 2 h of continuous observation
after initial vacuole formation.
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Bacterium-ameba interactions after 24 h of coculture. Twenty-four hours after the start of coculture preparations with bacteria from 2-h preparations, motile bacilli were observed inside endamebic vacuoles that contained three or fewer bacilli. However, in cocultures using bacteria from 1-h preparations, amebae became enclosed in clumps or tangles of bacterial filaments not previously seen in any B. pseudomallei monoculture (Table 1; Fig. 1c and d). Many actively motile intracellular bacilli were present and were observed up to 72 h after coculture began (the maximum period observed). Vortex mixing of coculture suspensions released amebic cysts from bacillary tangles. Bacilli were observed in only a small proportion of amebic cysts (Fig 1d). Some rounded trophozoites were present. These contained vacuoles packed with highly motile bacilli, restricted by the vacuolar membrane (Fig. 1c). Vacuoles packed with bacilli were often observed adjacent to the external plasma membrane. In some cases these protruded or were in the process of releasing bacilli to form an external tuft similar in appearance to the polar tuft formed during endocytosis. In a few trophozoites, single bacilli moved without restriction throughout the cytoplasm outside any vacuoles (Fig. 1c). Trophozoite lysis with spillage of cytoplasmic contents occurred shortly afterwards. These phenomena were not observed in cocultures containing 2-h bacterial preparations or lacking stellate, acanthapodium-bearing trophozoites.
Coincubation with other Acanthamoeba species. Some of the early features of bacterial endocytosis by A. astronyxis were demonstrated by repetition of the coculture procedure under optimal conditions using A. castellani and A. polyphaga. However, as vacuolation was less pronounced in these species than in A. astronyxis, the sequence of events was more difficult to deduce. No bacterium-containing vacuoles or external bacillary tufts were observed in either species. Initial phagocytosis and production of bacillary tangles were all demonstrated more clearly with A. castellani and A. polyphaga when challenged with B. pseudomallei at 37°C.
Recovery of intracellular bacteria. A mean bacterial count of 13 CFU/ml (±6.7) was obtained from three replicates from an aliquot taken immediately after commencement of coculture. After 60 min, the mean intracellular bacterial count from three replicates was 507 CFU/ml (±76.9) (t = 6.39, df = 4, P = 0.0031). No growth was obtained from saline and sterile water controls. The mean viable count of B. pseudomallei in control coculture medium was 3.6 × 106 (±2.7 × 105) CFU/ml at t = 0 and 3.9 × 106 at t = 60 min (±1.1 × 106). There was no statistically significant difference between viable counts of controls at t = 0 and t = 60 min in coculture medium.
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DISCUSSION |
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Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
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An interaction between B. pseudomallei and Acanthamoeba spp. has not been described previously. In this investigation of a possible interaction between the two species, we observed coiling phagocytosis, the survival of B. pseudomallei in amebic vacuoles, and eventual bacterial escape from vacuoles into amebic cytoplasm and then into the surrounding medium. The optimal conditions for cellular interaction were dependent on the condition of both components of the coculture. A combination of rapidly motile bacilli and acanthapodium-bearing amebae was required to obtain a full range of cellular interactions.
The ability of B. pseudomallei to cause relapsing and late-onset infection up to 26 years after initial exposure has been attributed to an ability of this species to survive in macrophages (17, 24, 27). Intramacrophage survival may be helped by an ability to tolerate prolonged periods of nutrient deprivation and relatively acid conditions as is known to occur with B. pseudomallei (18, 32). It is therefore notable that we were able to recover viable bacteria from amebic trophozoites 1 h after endocytosis and were able to observe motile intracellular bacilli for up to 72 h after coculture began. Similar investigations into other facultative intracellular pathogens such as L. pneumophila have emphasized intracellular replication (2, 7). The formation of extracellular bacillary tufts and tangles provides a major methodological obstacle to using the conventional approach to determining whether or not B. pseudomallei is capable of intraamebic replication. Moreover, the acidic conditions expected in amebic vacuoles may have rendered intracellular bacilli viable but not immediately culturable, as proposed recently (16), further confounding quantitative bacteriology. If late-onset endamebic replication of B. pseudomallei occurs, as has been shown recently with the related species B. cepacia (21), its demonstration will depend on advanced laboratory methods such as those developed recently for investigation of M. avium-Acanthamoeba interactions (28). Nevertheless, the current lack of evidence for endamebic replication of B. pseudomallei does not detract from the analogy between in vitro survival in endamebic vacuoles and survival in mammalian macrophages.
The coexistence of many intact trophozoites with dense clusters, tangles, or mats of B. pseudomallei shows that these species are capable of a nondestructive relationship. Furthermore, the small numbers of intracellular bacteria recovered after 60 min of coincubation with many times that number of bacteria implies a degree of control over bacterial access to the interior of the trophozoite. The observation of coiling phagocytosis, a process noted to occur during endocytosis of L. pneumophila by both Acanthamoeba and human monocytes (3), shows another possible connection between amebic endocytosis and mammalian phagocytosis of B. pseudomallei. The asymmetric, coiling extension of phagocytic pseudopodia by human mononuclear cells was one of the first pieces of evidence linking amebic endocytosis of Legionella to phagocytic events during L. pneumophila infection (14). Coiling phagocytosis of B. pseudomallei by mammalian macrophages has yet to be reported. If the analogy with L. pneumophila infection holds, the recent observation that prior intra-amebic bacterial infection enhances monocyte entry and virulence provides a possible experimental approach to the early stages of cellular interactions (6). The vacuolar and amebic escape of bacilli observed during the later stages of coculture further demonstrates the viability of intracellular bacteria. However, its relevance to human B. pseudomallei infection is speculative since the escape of sequestered bacilli from macrophages in vivo can only be inferred from bacterial replication that must occur in late onset or relapsing infections.
The cellular events that span the interaction between B. pseudomallei and amebic trophozoites bear some resemblance to phenomena previously observed in Legionella-Acanthamoeba cocultures (2, 3, 26). The ecological relationship between these two genera has been described as an endosymbiosis. A commensal relationship between B. pseudomallei and Acanthamoeba is possible in shared environmental habitats, but as we have yet to demonstrate their simultaneous occurrence in vivo or show a mutual codependency, it is not possible to describe the interaction as a true symbiosis. By analogy with L. pneumophila (1-3, 6, 7, 11, 26), it is possible that the capacity of B. pseudomallei to enter, survive within, and exit free-living amebae confers an ability to invade mammalian phagocytic cells, persist within, and escape from them as is thought to occur in relapsing and late-onset melioidosis. By the same token, it is also possible that there are key differences between the mechanisms used during amebic cytoinvasion compared with penetration of human macrophages (12, 13).
The observation of rapid rotation of the entire bacillus while anchored to a fixed point on the external trophozoite surface implies flagellar adhesion. It is of note that mutagenesis and animal model experiments highlight a possible role for the flagellum in the pathogenesis of B. pseudomallei infection (10). Other aspects of the initial interaction between B. pseudomallei and the amebic trophozoite that warrant further attention include expression of acid phosphatase, an enzyme produced by B. pseudomallei under acid conditions and thought to be a virulence factor for intracellular bacterial pathogens (9). This enzyme has been identified as a tyrosine phosphatase (19). It is therefore of note that tyrosine phosphatase activity has been implicated recently in attachment of L. pneumophila to another free-living protozoan, Hartmanella vermiformis (30).
Exposure to B. pseudomallei-contaminated soil or water in the region where the organism is endemic is thought to be the principal means of exposure to infection (5, 15, 29). A detailed appraisal of the possible ecological significance of a B. pseudomallei-Acanthamoeba interaction in vivo is beyond the scope of this investigation. Nevertheless, the remarkable ability of B. pseudomallei to persist in hostile environmental niches could be explained in part by an ability to survive in free-living amebae whose normal habitat lies within the vadosphere and in particular at air/water interfaces in the soil (25). It is possible that detailed study of the microbial ecology of the region where the organism is endemic may provide evidence for a putative role for free-living phagocytic protozoa in the pathogenesis of melioidosis. The observation that Acanthamoeba species such as A. astronyxis can be recovered from the human nasal mucosa (22) make inhalation of endamebic B. pseudomallei a little more plausible and provides a further avenue of investigation.
In conclusion, we have examined the interaction between cells of the facultative intracellular pathogen B. pseudomallei and several Acanthamoeba species. Nondestructive intracellular passage of bacteria and bacterial escape during trophozoite lysis were demonstrated. As yet it is unclear whether these are mutually exclusive or the extremes at either end of a range of outcomes. Whichever interpretation proves to be correct, these observations indicate that B. pseudomallei can adapt to a free-living intracellular habitat such as may be found in acanthamebae. The successful demonstration of an interaction between B. pseudomallei and A. astronyxis that includes coiling phagocytosis and nondestructive bacterial passage via trophozoites may help attempts to demonstrate similar phenomena in mammalian phagocytic cells. Endamebic passage of B. pseudomallei deserves further attention as a possible in vitro model of intracellular B. pseudomallei infection.
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ACKNOWLEDGMENT |
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The Biomedical Confocal Microscopy Research Centre is supported by the Lotteries Commission of Western Australia.
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FOOTNOTES |
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* Corresponding author. Mailing address: Division of Microbiology and Infectious Diseases, PathCentre, Locked Bag 2009, Nedlands, WA 6009, Australia. Phone: 618 9 346 3461. Fax: 618 9381 7139. E-mail: tim.inglis{at}health.wa.gov.au.
Editor: D. L. Burns
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Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
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