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Applied and Environmental Microbiology, June 2000, p. 2613-2619, Vol. 66, No. 6
Department of Laboratory
Medicine1 and School of
Fisheries,3 University of Washington, Seattle,
Washington 98195; Lehrstuhl für Mikrobiologie, Technische
Universität München, 85350 Freising,
Germany2; and Washington State
Public Health Laboratory, Seattle, Washington
981554
Received 22 September 1999/Accepted 8 March 2000
The recently proposed reorganization of the order
Chlamydiales and description of new taxa are broadening our
perception of this once narrowly defined taxon. We have recovered four
strains of gram-negative cocci endosymbiotic in
Acanthamoeba spp., representing 5% of the
Acanthamoeba sp. isolates examined, which displayed developmental life cycles typical of members of the
Chlamydiales. One of these endosymbiont strains was found
stably infecting an amoebic isolate recovered from a case of amoebic
keratitis in North America, with three others found in acanthamoebae
recovered from environmental sources in North America (two isolates)
and Europe (one isolate). Analyses of nearly full-length 16S rRNA gene
sequences of these isolates by neighbor joining, parsimony, and
distance matrix methods revealed their clustering with other members of
the Chlamydiales but in a lineage separate from those of
the genera Chlamydia, Chlamydophila,
Simkania, and Waddlia (sequence similarities,
<88%) and including the recently described species
Parachlamydia acanthamoebae (sequence similarities, 91.2 to
93.1%). With sequence similarities to each other of 91.4 to 99.4%,
these four isolates of intra-amoebal endosymbionts may represent three
distinct species and, perhaps, new genera within the recently proposed
family Parachlamydiaceae. Fluorescently labeled
oligonucleotide probes targeted to 16S rRNA signature regions were able
to readily differentiate two groups of intra-amoebal endosymbionts
which corresponded to two phylogenetic lineages. These results reveal
significant phylogenetic diversity occurring among the
Chlamydiales in nontraditional host species and supports the existence of a large environmental reservoir of related species. Considering that all described species of Chlamydiales are
known to be pathogenic, further investigation of intra-amoebal
parachlamydiae as disease-producing agents is warranted.
All members of the order
Chlamydiales are recognized pathogens of mammals,
marsupials, or birds. The ability to produce respiratory disease, among
other clinical presentations, is a feature of most species within the
order and is especially characteristic of infections produced by
Chlamydophila pneumoniae, Chlamydophila psittaci, certain serovars of Chlamydia trachomatis, and the recently
described species Simkania negevensis (20, 21,
23). The recent finding of evidence for seroconversion to antigen
of Parachlamydia acanthamoebae, an intra-amoebal
Chlamydia-like bacterium, in a small number of humans
experiencing community-acquired pneumonia, along with the findings of
novel Parachlamydia-related 16S rRNA sequences in respiratory specimens, peripheral blood, and aortic tissue, suggests that this and related species of protozoal endosymbionts may also be of
clinical significance, warranting further investigation (2, 5,
30).
Previously, we have reported on the common occurrence of uncultured
bacterial endosymbionts in protozoa of the genus
Acanthamoeba (13). While 20% of axenically
growing Acanthamoeba isolates recovered from clinical and
environmental sources were found to be host to gram-negative rod
endosymbionts, 5% were host to gram-negative coccus endosymbionts;
none could be cultured by standard microbiological techniques.
Phylogenetic analyses of the gram-negative rod endosymbionts to date
have included two lineages of alpha-Proteobacteria: one containing Rickettsiales-affiliated isolates and the other
containing isolates related to the Paramecium caudatum
symbiont Caedibacter caryophilus (14, 18).
Subsequent ultrastructural studies of the coccoid endosymbionts
demonstrated the presence of a developmental cycle suggestive of a
relationship to members of the Chlamydiales (R. Gautom, R. Herwig, and T. R. Fritsche, Abstr. 96th Gen. Meet. Am. Soc.
Microbiol., abstr. R-29, p. 474).
In this paper, we present further morphologic and phylogenetic analyses
of four isolates of Chlamydiales endosymbionts found naturally infecting Acanthamoeba sp. trophozoites, three of
which were recovered from environmental amoebic isolates originating in
North America (two isolates) and Europe (one isolate), and one which
was from amoebae infecting the corneal tissues of a patient in North
America. Because these bacterial isolates could not be cultivated by
standard microbiological techniques, we applied the culture-independent
ribosomal RNA (rRNA) approach to determine phylogenetic relatedness to
each other and to other strains for which sequence data are available.
Fluorescence in situ hybridization (FISH) with oligonucleotide probes
designed to target ribosomal signature regions was used to verify the
origin of the retrieved sequences and further assist with
characterization of these endosymbionts. The finding of a potentially
large environmental reservoir of intracellular
Chlamydia-like organisms has implications for the evolution
of Chlamydiales and their preadaptation and ultimate recruitment to higher animals.
(Portions of this work were presented as an abstract at the 96th
General Meeting of the American Society for Microbiology in New
Orleans, La., 1996.)
Isolation and maintenance of Acanthamoeba
strains.
The techniques used for recovery and maintenance of
acanthamoebae from clinical and environmental sources have been
described in detail elsewhere (13, 39). Four isolates of
Acanthamoeba spp. were included in this study and were
recovered from infected corneal tissues (UWC22), soil samples from
western Washington State (UWE1 and UWE25), and municipal sewage sludge
from Munich, Germany (TUME1). All isolates were found to have
gram-negative cocci occurring as endosymbionts within the cytoplasm
which could be readily demonstrated using Giemsa, Hemacolor (Harleco,
Gibstown, N.Y.), and other appropriate bacterial stains. The general
phenotypic characteristics of three of these endosymbiont strains
(UWE1, UWE25, and UWC22) have been described previously
(13).
Electron microscopy.
Amoebic isolates containing
endosymbionts were examined by electron microscopy using a variation of
published methods (16). Briefly, aliquots of amoebae in
broth were fixed with 2% glutaraldehyde in 0.1 M cacodylate. Fixed
amoebae were then pelleted in agar and embedded. Thin sections were
stained with uranyl acetate and lead citrate and examined with a
Phillips CM-10 electron microscope.
DNA isolation and PCR amplification of nearly full-length 16S
rDNA.
UWC22, UWE1, and UWE25 amoebae in log-phase growth were
pelleted, washed three times with cold (4°C) sterile distilled water, and freeze-thawed three times followed by aspiration and expulsion through a 24-gauge needle. The cell slurry was resuspended in 5 ml of
cold (4°C) sterile physiologic (0.15 M) saline and gently centrifuged
(5 min at 120 × g). The supernatant was filtered
through a 5-µm syringe filter, and the bacteria were pelleted by
centrifugation (10 min at 3,000 × g). Extraction of
bacterial DNA was performed by standard procedures (34).
0099-2240/00/$04.00+0
Copyright © 2000, American Society for Microbiology. All rights reserved.
Phylogenetic Diversity among Geographically
Dispersed Chlamydiales Endosymbionts Recovered from Clinical
and Environmental Isolates of Acanthamoeba spp.
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
2 to 18) and 5'-TGTCGACAAAGGAGGTGATCCA-3'
(E. coli 16S rDNA positions 1554 to 1537),
respectively. Amplification reactions were performed in a reaction
volume of 50 µl in a thermal capillary cycler with reaction mixtures,
including a 20 mM MgCl2 reaction buffer, prepared as
recommended by the manufacturer (Idaho Technology, Idaho Falls, Idaho)
with Taq DNA polymerase (Promega, Madison, Wis.). Thermal cycling consisted of an initial denaturation step at 94°C for 30 s, followed by 30 cycles of denaturation at 94°C for 20 s, annealing at 50°C for 15 s, and elongation at 72°C for 30 s, with a final elongation step at 72°C for 1 min. Positive controls
containing purified DNA from E. coli were included along
with negative controls (no DNA added).
The presence and size of all amplification products were determined by
agarose gel electrophoresis and ethidium bromide staining.
Cloning and sequence analysis. Amplified DNA from UWC22, UWE1, and UWE25 was purified by electrophoresis in low-melting-point agarose and ligated into the cloning vector Bluescript II (Stratagene, La Jolla, Calif.), while amplified DNA from TUME1 was ligated directly into the cloning vector pCR2.1 (Invitrogen, Carlsbad, Calif.), with subsequent transformation of E. coli by each vector. The nucleotide sequences of the cloned DNA fragments were determined by automated dideoxynucleotide methods with the Taq Dye Deoxy Terminator cycle sequencing kit (Applied Biosystems, Foster City, Calif.) for UWC22, UWE1, and UWE25 and the Thermo Sequenase cycle sequencing kit (Amersham Life Science, Little Chalfont, England) for TUME1.
Phylogenetic analysis. Obtained sequences were added to the 16S rRNA sequence database maintained at the Technische Universität München (encompassing about 16,000 published and unpublished homologous small-subunit rRNA primary structures). Alignment of the new sequences was performed using the program package ARB and its automated alignment tool (O. Strunk and W. Ludwig, www.biol.chemie.tu-muenchen.de/pub/ARB/), with refinement of positioning by visual inspection and by secondary-structure analysis. The ARB parsimony, distance matrix, and maximum-likelihood treeing methods, combined with and without use of filters which exclude highly variable regions, were applied to different data sets.
Oligonucleotide probes and FISH.
Oligonucleotide probes
Bn9658 (a probe described previously [2]),
specific for P. acanthamoebae and targeting E. coli positions 658 to 675) and C22658 (designed from
sequence data derived in this study from the endosymbiont infecting
Acanthamoeba sp. isolate UWC22) were used to differentiate
between two groups of endosymbionts (Table
1). Probe C22658 is
designated S-*-ParaC-0658-a-A-18 according to the standard proposed by
Alm et al. (1). Both probes were synthesized and directly
labeled with the hydrophilic sulfoindocyanine dye Cy3 or Cy5
(Interactiva, Ulm, Germany).
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Nucleotide sequence accession numbers. The recovered 16S rDNA sequences have been deposited in GenBank under accession numbers AF083616 (endosymbiont of Acanthamoeba sp. UWC22), AF083614 (endosymbiont of Acanthamoeba sp. UWE1), AF083615 (endosymbiont of Acanthamoeba sp. UWE25), and AF098330 (endosymbiont of Acanthamoeba sp. TUME1).
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RESULTS |
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Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
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Morphologic analyses.
As seen by light microscopy, the
organisms are coccoid in appearance and stain gram negative. Use of
Giemsa and related stains revealed that they are dispersed throughout
the cytoplasm and do not form discrete inclusions, although small
clusters and morulae are occasionally noted (Fig.
1A). By electron microscopy, large numbers of cocci are seen in each amoebic trophozoite and display a
developmental life cycle typical of Chlamydiales, consisting of smaller electron-dense forms (elementary bodies) and larger dividing
forms (reticulate bodies) (Fig. 1B and C). Rather than growing as
discrete intravacuolar inclusions, the bacteria are found dispersed
throughout the cytoplasm but appear to be surrounded by vacuolar
membranes which insinuate themselves around each bacterial cell (Fig.
1C). Some bacteria are found in food vacuoles but appear to be in
various stages of disintegration; these forms may have been nonviable
bacteria released from other amoebae previously and phagocytosed. When
present in amoebic cysts, the bacteria appear to form inclusions and
lack evidence of a developmental cycle, suggesting that they are in a
resting state (Fig. 1D).
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Comparative sequence results and phylogenetic analysis.
Nearly
full-length 16S rDNA sequences from the four endosymbiont strains were
amplified, cloned, and sequenced. Comparative sequence analysis using
parsimony, distance matrix, and maximum-likelihood treeing methods
revealed that the four clustered unequivocally with other members of
the Chlamydiales but formed a lineage with P. acanthamoebae and, more distantly, S. negevensis and
Waddlia chondrophila that is distinct from
Chlamydia and Chlamydophila spp. (Fig.
2). Further analysis of tree topology
revealed the presence of two lineages of intra-amoebal endosymbionts:
one contains the closely related isolates UWC22 and TUME1 (99.1%
sequence similarity), and a second includes UWE1, UWE25, and P. acanthamoebae (sequence similarities of 91.5 to 93.2%). All four
isolates warrant inclusion, along with P. acanthamoebae, in
the recently proposed family Parachlamydiaceae and may
represent up to three new genera and species (Table 1) (11,
36). With sequence similarities of 83.5 to 88.2% and 84.2 to
87.9% to all other Chlamydiales, S. negevensis
and W. chondrophila, respectively, would appear to reside in
separate families, as has been proposed (11).
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FISH analysis.
The oligonucleotide probe Bn9658,
specific for P. acanthamoebae, fully matched the
corresponding sequence in endosymbionts from Acanthamoeba
sp. strains UWE1 and UWE25, whereas probe C22658 was
designed to recognize the endosymbionts present in
Acanthamoeba sp. strains UWC22 and TUME1 (Fig.
3). These probes displayed two mismatches
between each other and at least three mismatches to other members of
the Chlamydiales and other bacteria.
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DISCUSSION |
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Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
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Phylogenetically, members of the Chlamydiales comprise a unique bacterial assemblage which is highly divergent from other groups within the domain Bacteria in the 16S rDNA-based universal tree. Among the Bacteria, rDNA sequence analysis has revealed their closest relatives to be members of the Planctomycetales, a group of free-living, mostly aquatic bacteria which also display developmental cycles (10).
While little is known about the evolution of traditional Chlamydiales species, the recent description of the Parachlamydiaceae occurring as endosymbionts in protozoa, specifically Acanthamoeba spp., has measurably broadened the possible evolutionary origins of the group (2, 11). Of the six isolates of Parachlamydiaceae described in the literature for which partial or nearly full-length 16S rDNA sequencing has been performed, including the four presented here, sequence dissimilarities suggest the existence of up to four genera and species; two of the isolates studied previously appear to be highly (>99% sequence similarity) related (2, 5). The finding of additional related endosymbionts in these and other protozoa would therefore not be surprising, given the species diversity and geographic range noted to date, and suggests that a large environmental reservoir for them may exist. We conclude that the phylogenetic diversity of the Chlamydiales is extensive but is underrepresented in the literature as a result of bias towards the study of species recognized as being medically and economically important.
Considering that all described species of Chlamydiales are known to be pathogenic and capable of producing respiratory tract disease, among other clinical presentations, analysis of the intra-amoebal forms for pathogenic potential should be stressed as well. Recently, seroepidemiologic evidence has been presented that "Hall's coccus," an endosymbiont of acanthamoebae which appears to be identical to P. acanthamoebae, may be responsible for some cases of community-acquired pneumonia (5).
Other evidence comes from the amplification of Chlamydiales 16S rDNA fragments (216 to 224 bp) in respiratory tract specimens from humans with pulmonary disease, an aortic specimen, and peripheral blood mononuclear cells using broad-range primers designed to detect any member of the order (29). While sequences characteristic of Chlamydophila pneumoniae (three specimens) and Chlamydia trachomatis (one specimen) were recovered from the 42 respiratory specimens examined, four sequences (two respiratory, one blood, and one aortic tissue) were also detected which group in the second major Chlamydiales lineage, which includes the Simkaniaceae and Parachlamydiaceae. Because S. negevensis (Fig. 2) has been reported to cause both community-acquired pneumonia in adults and acute bronchiolitis in infants (20, 23), further analysis of the Parachlamydiaceae as disease-producing agents should be undertaken.
Acanthamoebae and other free-living protozoa are uniquely positioned ecologically to support the dissemination of environmental respiratory pathogens. Able to colonize water supply, cooling, and humidification systems, they also serve as amplification vehicles and possibly reservoirs for Mycobacterium avium and a variety of Legionella spp.; even C. pneumoniae has been shown to survive and replicate within acanthamoebae (8, 9, 12, 27, 29, 33, 37). Such intraprotozoal multiplication followed by aerosolization and inhalation of bacterium-laden vesicles has been proposed as a mechanism to explain the epidemiology of legionellosis and the apparent lack of case-to-case spread (4, 6). The ubiquity of acanthamoebae in the environment, the presence of a resistant cyst stage, and their ability to support growth of a variety of intracellular pathogens make them prime suspects in the epidemiology of respiratory disease caused by other organisms as well.
Many free-living protozoa mimic the role of professional phagocytes in their abilities to ingest and destroy large numbers of bacteria. Such bacterium-protozoan interactions have undoubtedly provided selective pressure resulting in the emergence of environmental species capable of escaping intracellular destruction, which are known to include Legionella spp., Burkholderia pickettii, Listeria monocytogenes, Vibrio cholerae, and M. avium, among others (3, 7, 24, 26, 37, 38). The adaptation of legionellae and M. avium to an intracellular existence in free-living protozoa may also have been a driving force in the evolution of virulence mechanisms which permit their survival within pulmonary macrophages (3, 22, 37).
While many species of facultative bacteria develop in and ultimately lyse their protozoal host, a variety of obligate endosymbionts of ciliates and amoebae are well known to stably infect their host, suggesting the occurrence of longer-term coevolution (15, 17, 31). The Parachlamydia-like endosymbionts described here also appear to be well adapted to their natural amoebic hosts and have been maintained in continuous cultivation for several years. The presence of these bacteria in amoebic cysts in an apparent nondividing resting stage (Fig. 1D) further supports the stable symbiotic nature of the relationship.
Considering that, until recently, all Chlamydiales were considered to be pathogenic, the finding of a potentially large environmental and geographically dispersed reservoir of related organisms which are adapted to stable intracellular growth in specific hosts provides an important link in the evolution of the order. The existence of a pool of protozoal symbionts stably adapted to the intracellular milieu may have provided the genetic material from which recruitment to vertebrates accidentally occurred, with the attendant appearance of pathogenic properties. Such recruitment would be facilitated by the frequent occurrence of these endosymbionts in Acanthamoeba spp. (and perhaps other protists), the ubiquity of acanthamoebae in the environment, and the ease with which these amoebae transiently colonize the respiratory tract of humans and animals (28).
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ACKNOWLEDGMENTS |
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We are grateful for the assistance of Daniel E. Possin of the University of Washington for preparation of the electron micrographs.
This study was supported in part by Public Health Service grant F06 TW02279-01 to T.R.F. and by Deutsche Forschungsgemeinschaft grant WA 1027/2-1 to M.W. and K.-H.S.
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FOOTNOTES |
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* Corresponding author. Mailing address: Department of Laboratory Medicine, University of Washington, 1959 N.E. Pacific St., Seattle, WA 98195-7110. Phone: (206) 598-6131. Fax: (206) 598-6189. E-mail: fritsche{at}u.washington.edu.
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Top
Abstract
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Materials and Methods
Results
Discussion
References
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