Neochlamydia hartmannellae gen. nov., sp. nov. (Parachlamydiaceae), an endoparasite of the amoeba Hartmannella vermiformis

Matthias Horn1, Michael Wagner1, Karl-Dieter Müller2, Ernst N. Schmid2, Thomas R. Fritsche3, Karl-Heinz Schleifer1 and Rolf Michel2

Lehrstuhl für Mikrobiologie, Technische Universität München, Am Hochanger 4, D-83530 Freising, Germany1
Central Institute of the Federal Armed Forces Medical Service, D-56065 Koblenz, Germany2
Department of Laboratory Medicine, University of Washington, Seattle, WA 98195, USA3

Author for correspondence: Michael Wagner. Tel: +49 8161 715444. Fax: +49 8161 715475. e-mail: wagner{at}mikro.biologie.tu-muenchen.de


   ABSTRACT
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ABSTRACT
INTRODUCTION
METHODS
RESULTS AND DISCUSSION
REFERENCES
 
Free-living amoebae are increasingly being recognized to serve as vehicles of dispersal for various bacterial human pathogens and as hosts for a variety of obligate bacterial endocytobionts. Several Chlamydia-like Acanthamoeba endocytobionts constituting the recently proposed family Parachlamydiaceae are of special interest as potential human pathogens. In this study coccoid bacterial endocytobionts of a Hartmannella vermiformis isolate were analysed. Infection of H. vermiformis with these bacteria resulted in prevention of cyst formation and subsequent host-cell lysis. Transfection experiments demonstrated that the parasites were not capable of propagating within other closely related free-living amoebae but were able to infect the distantly related species Dictyostelium discoideum. Electron microscopy of the parasites revealed typical morphological characteristics of the Chlamydiales, including the existence of a Chlamydia-like life-cycle, but indicated that these endocytobionts, in contrast to Chlamydia species, do not reside within a vacuole. Comparative 16S rRNA sequence analysis showed that the endocytobiont of H. vermiformis, classified as Neochlamydia hartmannellae gen. nov., sp. nov., is affiliated to the family Parachlamydiaceae. Confocal laser scanning microscopy in combination with fluorescence in situ hybridization using rRNA-targeted oligonucleotide probes confirmed the intracellular localization of the parasites and demonstrated the absence of other bacterial species within the Hartmannella host. These findings extend our knowledge of the phylogenetic diversity of the Parachlamydiaceae and demonstrate for the first time that these endocytobionts can naturally develop within amoebae of the genus Hartmannella.

Keywords: Hartmannella, endoparasite, Parachlamydiaceae, Neochlamydia hartmannellae, Chlamydia

Abbreviations: EB, elementary body; FLA, free-living amoebae; RB, reticulate body

The GenBank accession number for the sequence reported in this paper is AF177275.


   INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS AND DISCUSSION
REFERENCES
 
Free-living amoebae (FLA), such as Hartmannella spp., Acanthamoeba spp., Naegleria spp. and Vahlkampfia spp., are an important component of soil and water ecosystems, acting as important predators controlling bacterial populations (Rodriguez-Zaragoza, 1994 ). They are cosmopolitan in distribution, and can be found in fresh water, in marine waters, in soil, on plants and animals, and inside vertebrates, feeding on bacteria, fungi, yeasts, algae and other protozoa. In addition to their environmental significance, some FLA have been identified as human pathogens, causing the diseases amoebic keratitis and meningoencephalitis, and systemic infections (Visvesvara, 1995 ).

A significant fraction of environmental and clinical FLA isolates harbour, like many other protozoa (Heckmann & Görtz, 1992 ; Preer & Preer, 1984 ), bacterial endocytobionts (Fritsche et al., 1993 ; Michel et al., 1995 ). Recent studies have begun to elucidate the phylogenetic diversity of FLA-associated endocytobionts by applying the rRNA approach. The majority of the endocytobionts identified thus far are related to bacterial genera currently recognized as important human pathogens. For example, Legionella-related, Rickettsia-related and Chlamydia-related organisms are known to occur in FLA (Amann et al., 1997 ; Birtles et al., 1996 ; Fritsche et al., 1999 ). In addition, several endocytobionts which group phylogenetically with the Paramecium caudatum symbiont Caedibacter caryophilus (Springer et al., 1993 ) are known to proliferate within Acanthamoeba spp. (Horn et al., 1999 ). Whereas the relationship between hosts and endocytobionts remains largely unexplored, there is increasing evidence that some FLA endocytobionts are of medical importance. Endocytobiont-mediated increase of Acanthamoeba cytopathogenicity in tissue culture suggests that these intracellular bacteria enhance FLA virulence (Fritsche et al., 1998 ). Furthermore, some of the endocytobionts have been implicated as causative agents for disease, as indicated by the presence of specific antibodies against Chlamydia-related endocytobionts of Acanthamoeba in blood from respiratory-disease patients, and by the detection of Parachlamydia-like 16S rDNA sequences in specimens from bronchitis patients (Birtles et al., 1997 ; Ossewaarde & Meijer, 1999 ).

This report describes the investigation of coccoid bacterial endocytobionts of Hartmannella vermiformis strain A1Hsp isolated from the water conduit system of a dental unit, by (i) transfection experiments, (ii) electron microscopy, and (iii) the rRNA approach including comparative 16S rRNA sequence analysis and fluorescence in situ hybridization using 16S rRNA-targeted oligonucleotide probes and confocal laser scanning microscopy. Portions of this work were presented as an abstract at the 99th General Meeting of the American Society for Microbiology in Chicago, IL, 1999.


   METHODS
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ABSTRACT
INTRODUCTION
METHODS
RESULTS AND DISCUSSION
REFERENCES
 
Isolation and maintenance of Hartmannella vermiformis.
The original amoebal host strain H. vermiformis strain A1Hsp was isolated from the water conduit system of a dental unit in Lahnstein, near Koblenz, Germany, by filtering a 100 ml water sample obtained from the source water as described elsewhere (Michel et al., 1995 ). Coccoid endocytobionts recovered from this isolate were transferred to H. vermiformis strain OS101 on NN-agar plates (non-nutrient agar; Page, 1988 ) seeded with Enterobacter cloacae. The infected H. vermiformis strain OS101 could subsequently be axenized in fluid SCGYE medium (De Jonckheere, 1977 ) by temporary addition of penicillin and streptomycin (0·2 mg ml-1 each). Cultures were incubated at 30 °C and fresh medium was applied every 5–10 d. To investigate the capability of the original amoebal host strain H. vermiformis A1Hsp to form cysts, amoebae were cured of their endocytobionts by treatment with rifampicin as described by Michel et al. (1994) .

Extracellular growth of H. vermiformis endoparasites.
Attempts to culture the Hartmannella endocytobionts extracellularly included cultivation on blood agar (Becton Dickinson), Casiton-agar (Biotest-Heipha) and SCGYE (De Jonckheere, 1977 ) at incubation temperatures of 20 and 30 °C. Both whole amoeba cells and filter-purified endocytobionts from lysed amoeba cells were transferred to the respective media. If no growth was observed after 14 d incubation, cultures were considered negative.

Transfection experiments.
Following lysis of endocytobiont-infected H. vermiformis cells from 4–5-d-old cultures by freeze–thawing, the coccoid bacterial endocytobionts were filter-purified (1·2 µm membrane filter). An aliquot of 80 µl of the resulting suspension was added to strains of different species of FLA, growing either in SCGYE medium or on NN-agar plates covered with a lawn of Enterobacter cloacae. The host range of the endocytobiont was investigated by transfection experiments with 14 different strains of FLA (Table 1), and one strain of Dictyostelium discoideum isolated from human nasal mucosa. Infection of each host species was monitored by phase-contrast microscopy. After 21 d incubation at 30 or 20 °C the host was considered resistant to infection if no infected cells nor any marked reduction in amoebal numbers (which may have resulted from parasitic activity of the endocytobiont) were observed.


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Table 1. Host spectrum of the endoparasite of H. vermiformis strain A1Hsp (Neochlamydia hartmannellae)

 
Electron microscopy.
For electron microscopical studies, the heavily infected trophozoites from 4–5-d-old cultures were harvested and prefixed in 2·5% glutaraldehyde in cacodylate buffer (pH 7·2) for 2 h. After prefixation the specimens were fixed in 1% osmium tetroxide followed by 2% uranyl acetate in aqueous solution. Subsequently specimens were dehydrated in alcohol and embedded in epoxy resin. Thin sections were stained with 1% lead citrate and examined with a Zeiss EM 910 electron microscope.

DNA isolation, amplification of 16S rDNA, cloning and sequencing.
Simultaneous isolation of DNA from the amoebae and their endocytobionts was performed using a modified UNSET procedure (Hugo et al., 1992 ). Amoebae and their endocytobionts were harvested from axenic cultures by centrifugation (2000 g, 3 min), washed twice with double-distilled water, resuspended in 500 µl UNSET lysis buffer (8 M urea, 0·15 M NaCl, 2% SDS, 0·001 M EDTA and 0·1 M Tris/HCl at pH 7·5) and incubated at 60 °C for 5 min. Lysates were extracted twice with phenol/chloroform (Roth) and DNA was precipitated for 3 h at -20 °C with 2 vols absolute ethanol. After centrifugation (10000 g, 10 min) at 4 °C the ethanol was removed and the pellet was washed twice with 80% ice-cold ethanol to remove residual salts. The pellet was air-dried and resuspended in 30 µl double-distilled water.

Oligonucleotide primers targeting 16S rDNA signature regions which are conserved within the Chlamydiales were used for PCR to obtain near-full-length bacterial 16S rRNA gene fragments. The nucleotide sequences of the forward and reverse primers used for amplification were 5'-CGGATCCTGAGAATTTGATC-3' and 5'-TGTCGACAAAGGAGGTGATCCA-3' (Pudjiatmoko et al., 1997 ). 16S rDNA amplification reactions were performed in a thermal capillary cycler (Idaho Technology) using reaction mixtures including 15 pM of each primer, 0·25 µg BSA ml-1, 2 mM MgCl2 reaction buffer and 2·5 IU Taq DNA polymerase (Promega). Thermal cycling was carried out as follows: an initial denaturation step at 94 °C for 30 s followed by 30 cycles of denaturation at 94 °C for 15 s, annealing at 52 °C for 20 s, and elongation at 72 °C for 30 s. Cycling was completed by a final elongation step at 72 °C for 5 min. A negative control was performed using a reaction mixture without added DNA. Amplified products were directly ligated into the cloning vector pCRII-TOPO and transformed into competent Escherichia coli (TOP10 cells) according to the instructions of the manufacturer (Invitrogen). Nucleotide sequences of the cloned DNA fragments were determined by the dideoxynucleotide method (Sanger et al., 1977 ) by cycle sequencing of purified plasmid preparations (Qiagen) with a Thermo Sequenase Cycle Sequencing Kit (Amersham Life Science) and an automated DNA sequencer (Li-Cor) under conditions recommended by the manufacturers. Dye-labelled vector-specific primers M13/pUC V (5'-GTAAAACGACGGCCAGT-3') and M13/pUC R (5'-GAAACAGCTATGACCATG-3') were applied.

Phylogenetic analysis.
The 16S rDNA sequences obtained were added to the rDNA sequence database of the Technische Universität München (encompassing more than 16000 published and unpublished homologous small-subunit rDNA primary structures) by use of the program package ARB (O. Strunk and others, unpublished; program available through the homepage of the Technische Universität München: http://www.mikro.biologie.tu-muenchen.de). Alignment of the new rDNA sequences was performed by using the ARB automated alignment tool (version 2.0). The alignments were refined by visual inspection and by secondary-structure analysis. Phylogenetic analyses were performed by applying the ARB parsimony, distance matrix and maximum-likelihood methods to different data sets. To determine the robustness of the phylogenetic trees, analyses were performed with and without the application of various filtersets to exclude highly variable positions.

Fluorescence in situ hybridization and confocal laser scanning microscopy.
The following oligonucleotide probes were used: (i) EUB338 (5'-GCTGCCTCCCGTAGGAGT-3'), targeting most, but not all, members of the domain Bacteria (Amann et al., 1990 ; Daims et al., 1999 ), and (ii) S-S-ParaC-0658-a-A-18 (5'-TCCATTTTCTCCGTCTAC-3'), previously designed as complementary to a signature region of the 16S rRNA of the Parachlamydia-related endosymbionts of Acanthamoeba spp. strains UWC22 and TUME1 (T. R. Fritsche and others, unpublished; probe designation according to the standard proposed by Alm et al., 1996 ). Oligonucleotides were synthesized and directly 5'-labelled with 5(6)-carboxyfluorescein-N-hydroxysuccinimide ester (FLUOS), or the hydrophilic sulphoindocyanine fluorescent dye Cy3 (Interactiva).

For in situ hybridization, Hartmannella cells were harvested from 4 ml liquid broth culture by centrifugation (2000 g, 3 min), washed twice with double-distilled water, and resuspended in 0·05% agarose. Twenty microlitres of this suspension was spotted on a glass slide, air-dried and subsequently dehydrated in 80% ethanol for 10 s. Hybridization was performed using the hybridization buffer (including 30% formamide) and the buffer washing (containing 112 mM NaCl, without SDS) described by Manz et al. (1992) . Slides were examined using a confocal laser scanning microscope (LSM 510, Carl Zeiss) in combination with a helium-neon laser (543 nm) and an argon-krypton laser (488 nm). Image analysis processing was performed with the standard software package delivered with the instrument (version 2.01).


   RESULTS AND DISCUSSION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS AND DISCUSSION
REFERENCES
 
Coccoid endocytobionts block cyst formation in H. vermiformis
The original amoebal host strain A1Hsp, containing coccoid prokaryotic endocytobionts, was isolated from the water conduit system of a dental unit. Phase-contrast microscopic observation of the amoebae revealed morphological characteristics typical for the genera Hartmannella and Cashia (Page, 1988 ). Since no cysts could be observed, the amoebae were provisionally identified as members of the genus Cashia. Attempts to grow the amoebal isolate axenically failed. Consequently, bacterial endocytobionts were transferred from the original host strain A1Hsp into H. vermiformis strain OS101, which was subsequently axenized in order to facilitate the following investigations, including evaluation of host spectrum, electron microscopy, and 16S rDNA sequencing. Interestingly, infection with the coccoid endocytobionts prevented cyst formation of H. vermiformis strain OS101, a phenomenon which has been previously reported for Acanthamoeba sp. Bn9 and Berg17 and Acanthamoeba castellanii strain C3 after infection with Parachlamydia acanthamoebae (Amann et al., 1997 ; Michel et al., 1994 ). Since the ability to form cysts discriminates the genera Cashia and Hartmannella, this observation forced us to re-evaluate the capability of the original amoebal host strain A1Hsp to form cysts in the absence of endocytobionts and thus its identification as member of the genus Cashia. For this purpose, strain A1Hsp was cured of its endocytobionts by rifampicin treatment. Despite its toxicity for most amoebae a few trophozoites survived rifampicin treatment and multiplied after transfer to a fresh NN-agar plate containing no rifampicin. Since microscopic observation demonstrated that these endocytobiont-free amoebae were now able to form cysts, strain A1Hsp was assigned to the genus Hartmannella as H. vermiformis.

At present, we can only speculate on the ecological significance of the suppression of FLA cyst formation by some endocytobionts. A possible clue might be provided by the observation that Escherichia coli (Steinert et al., 1998 ) is eradicated from artificially infected Acanthamoeba cells during cyst formation. Thus, prevention of cyst formation might be a protection mechanism for parasitic endocytobionts, which would be negatively affected by the differentiation of FLA into resting forms. This strategy would contrast with the one used by Legionella pneumophila (Steinert et al., 1998 ; Kilvington & Price, 1990 ) and several other obligate Acanthamoeba endocytobionts (Fritsche et al., 1999 ; Horn et al., 1999 ), which survive host-cell cyst formation and thus directly benefit from cyst-mediated host resistance against unfavourable environmental conditions.

Chlamydia-like life cycle and parasitic behaviour of the H. vermiformis endocytobiont
Electron micrographs revealed a Chlamydia-like morphology and developmental cycle of the endocytobionts (Fig. 1). Stages showing binary fission resembling those of reticulate bodies (RBs, 0·4–0·6 µm in diameter) of Chlamydia could be observed. Additionally, the highly condensed coccoid stages (0·5–0·6 µm in diameter) are similar to elementary bodies (EBs) of Chlamydia. While RBs of the H. vermiformis endocytobiont clearly possess a Gram-negative type cell wall, results of electron microscopic analysis of its EBs are ambiguous. However, since the EBs of the Hartmannella endocytobiont showed an outer membrane, we consider them as Gram-negative. This is in noticeable contrast to the Gram-positive type of cell wall which has been observed for the Chlamydia-related endoparasite of Acanthamoeba sp. strain BN9 (P. acanthamoebae; Amann et al., 1997 ). In contrast to Chlamydia species and Parachlamydia-related endocytobionts of Acanthamoeba (Amann et al., 1997 ; T. R. Fritsche and others, unpublished), RBs and EBs of the Hartmannella endocytobiont were not surrounded by vacuoles and were thus located directly within the cytoplasm of the host cell, indicating that the endocytobionts possess an escape mechanism from the phagosomes.



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Fig. 1. (A) H. vermiformis trophozoites harbouring the Chlamydia-related endocytobiont (arrows). Free EBs can also be seen outside the eukaryotic cells, some of which are attached to the amoebal cell membrane (arrowheads). (B) Different stages of the endocytobiont of H. vermiformis A1Hsp within the cytoplasm of the amoebal host cell: RBs and EBs can be observed simultaneously and do not reside within vacuoles. Arrows indicate constrictions of RBs undergoing binary fission. In contrast to Rickettsia- and Caedibacter-related Acanthamoeba endosymbionts (Horn et al., 1999 ; Fritsche et al., 1999 ) no electron-translucent layers surrounding the intracellularly located bacteria could be observed. (C) Adhesion and phagocytosis of EBs of the endocytobiont by a trophozoite of H. vermiformis: adhesion is mediated by fine fibrous material (arrowheads) discernible at the amoebal cellular membrane (glycocalyx) and also at the surface of the EBs. One EB of exceptional size has already been partially engulfed by the amoeba, forming a characteristic foodcup (fc). Within the cytoplasm a constricted stage (arrow) of an RB can be seen. N, nucleus of the host cell; mi, mitochondrion; L, lipid granules; er, endoplasmic reticulum. Bars, 1 µm.

 
Electron microscopic inspection of amoebal cells at different time points showed that massive amounts of mature EBs occurred 3–5 d after infection, and subsequently led to rupture or lysis of heavily infected trophozoites. Shedding of single mature EBs into the environment, not accompanied by host-cell destruction, was, however, already observed at earlier stages of infection (Fig. 1). Since ultimately all infected Hartmannella trophozoites are killed by the coccoid endocytobionts, they are considered by us to be intracellular parasites.

Natural stability of this host–parasite association would require an amoebal generation time shorter than the period between parasite infection and host cell lysis. The aggressive parasitic behaviour of the endocytobiont within its Hartmannella host suggests a limited adaptation of host and parasite caused by a relatively short evolutionary relationship. Hartmannella species may have only recently been infected by these parasites, suggesting their origin from another protist species. Limited adaptation of the endoparasite to the H. vermiformis host is also suggested by the suppression of cyst formation, which might protect the parasites from eradication but which may decrease the fitness of the association against environmental stress.

Host range of the obligate endoparasite of H. vermiformis
Standard cultivation techniques failed to support extracellular growth of the Hartmannella endoparasites, suggesting that Hartmannella cells are necessary for its growth. This finding is in accordance with the obligate intracellular growth of other endocytobionts of FLA (Amann et al., 1997 ; Fritsche et al., 1993 ).

The host range of the Hartmannella endoparasite was determined by transfection experiments encompassing a recently isolated D. discoideum strain and 14 different strains of FLA belonging to the genera Hartmannella, Acanthamoeba, Naegleria and Willaertia (Table 1). Except for the original host H. vermiformis A1Hsp and two more H. vermiformis strains, only D. discoideum strain Berg25 could be successfully infected. Whereas the extent of parasitic behaviour of the investigated endocytobionts varied slightly between the different H. vermiformis host strains, aggregation and stalk and fruiting body formation of D. discoideum were not disturbed by the endocytobionts and endoparasite-free spores were formed.

There are remarkable differences in host range between the investigated Hartmannella endoparasite and the Acanthamoeba endoparasite P. acanthamoebae. The Hartmannella endoparasite is not able to infect the tested Acanthamoeba strains, including A. castellanii C3, which is a suitable host for P. acanthamoebae (Amann et al., 1997 ). Conversely, P. acanthamoebae is unable to infect H. vermiformis strains, which serve as host for the investigated Hartmannella endoparasite (R. Michel, personal communication). Future studies are required to elucidate the molecular mechanism of a specific recognition system that may mediate specificity of infection.

Phylogeny and in situ identification of the H. vermiformis endoparasite
Near-full-length 16S rDNA amplicons (1529 bp) retrieved from mixed genomic DNA of amoebal hosts and bacterial endoparasites were successfully cloned and sequenced. Comparative sequence analysis revealed that the retrieved 16S rRNA sequence displayed highest similarity values with 16S rRNA sequences of members of the Chlamydiales (Table 2). In particular, the investigated Hartmannella endoparasites are moderately related to the Acanthamoeba parasite P. acanthamoebae strain Bn9 (92%), the only validly described member of the new family Parachlamydiaceae (Everett et al., 1999 ). It should be noted that even higher sequence similarities of between 96·5% and 97·1% to the Hartmannella parasite were calculated for 16S rRNA sequences of two recently investigated Parachlamydia-related endosymbionts of Acanthamoeba (Table 2; T. R. Fritsche et al., unpublished).


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Table 2. Overall sequence similarities for the retrieved 16S rRNA sequence of the endoparasite of H. vermiformis strain A1Hsp (Neochlamydia hartmannellae) and representative members of the Chlamydiales

 
Phylogenetic analysis using distance matrix, parsimony and maximum-likelihood treeing methods provided consistent evidence for an affiliation of the endoparasites of H. vermiformis A1Hsp with the Parachlamydiaceae. Within this family the retrieved sequence forms a monophyletic grouping with the two above-mentioned Acanthamoeba endosymbionts (strains UWC22 and TUME1; Fig. 2).



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Fig. 2. 16S rRNA-based phylogenetic tree reflecting the affiliation of the endoparasite of H. vermiformis strain A1Hsp (Neochlamydia hartmannellae). The tree was obtained using the neighbour-joining method. To exclude highly variable and thus phylogenetically non-informative sequence positions, only those sequence positions which are conserved in at least 50% of the deposited 16S rRNA sequences of the Chlamydiales were used for treeing. Nomenclature according to revised taxonomy of the Chlamydiales by Everett et al. (1999) . Bar indicates 10% estimated evolutionary distance.

 
Sequence analysis of 16S rDNA of the investigated endoparasites of H. vermiformis revealed the presence of the target site for probe S-S-ParaC-0658-a-A-18, specifically designed previously for the related Parachlamydia-like endosymbionts of Acanthamoeba sp. TUME1 and UWC22 (T. R. Fritsche et al., unpublished). Simultaneous fluorescence in situ hybridization of fixed Hartmannella cells with probe S-S-ParaC-0658-a-A-18 and the bacterial probe EUB338 demonstrated that all bacteria detectable by in situ hybridization also hybridized with the endocytobiont-specific probe, suggesting the absence of additional, phylogenetically different bacteria within the amoebal host. Confocal laser scanning microscopic analysis confirmed the intracellular localization of the endoparasites of H. vermiformis A1Hsp (Fig. 3).



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Fig. 3. In situ detection of N. hartmannellae (endoparasite of H. vermiformis strain A1Hsp) using bacterial probe EUB338 labelled with FLUOS (B), and endocytobiont-specific probe S-S-ParaC-0658-a-A-18 labelled with Cy3 (C). A phase-contrast image is shown in (A). Bar, 10 µm.

 
The genetic data described herein, and the morphological similarity, are consistent with a close relationship between the endoparasite of H. vermiformis A1Hsp and P. acanthamoebae. With a 16S rRNA sequence similarity of 92% with P. acanthamoebae, a Chlamydia-like development cycle, and the ability to multiply and survive within free living amoeba, the endoparasite of H. vermiformis A1Hsp meets the main requirements for inclusion within the family Parachlamydiaceae (Everett et al., 1999 ). However, keeping in mind that 16S rRNA sequence similarities between two bacteria of less than 95% are indicative of their affiliation with two different genera (Ludwig et al., 1998 ), the Hartmannella endoparasite most likely represents a new species of a new genus, since its 16S rRNA similarity to the closest validly described relative, P. acanthamoebae, is 92%. In this regard, it should be noted that 16S rRNA sequence similarities of the Hartmannella endoparasite with two recently discovered Parachlamydia-related endosymbionts of Acanthamoeba (T. R. Fritsche and others, unpublished) are higher than 95% but below 97·1%. Nevertheless, we believe that the Hartmannella and the Acanthamoeba endocytobionts should be assigned to different genera due to their profound differences in host spectra (see above). Consequently, we propose that the endoparasite of H. vermiformis A1Hsp be classified as Neochlamydia hartmannellae gen. nov., sp. nov.

Description of Neochlamydia gen. nov.
Neochlamydia (Ne.o.chla.my’di.a L. fem. n.; Neochlamydia referring to the modest phylogenetic relationship to the Chlamydiaceae).

Phylogenetic position: order Chlamydiales, family Parachlamydiaceae. Members of the genus Neochlamydia should have a 16S rDNA that is >95% identical to the 16S rDNA of the type species, Neochlamydia hartmannellae strain A1Hsp.

Description of Neochlamydia hartmannellae sp. nov.
Neochlamydia hartmannellae (hart’mann.el.lae. L. gen. sing. n. of Hartmannella, taxonomic name of a genus of Hartmannellidae; pertaining to the name of the host amoeba, Hartmannella vermiformis strain A1Hsp, in which the organism was first discovered).

Gram-negative reticulate bodies and Gram-negative elementary bodies; coccoid morphology; 0·4–0·6 µm in diameter. Basis of assignment: 16S rDNA sequence accession number AF177275, nucleotide probe S-S-ParaC-0658-a-A-18 (5'-TCCATTTTCTCCGTCTAC-3'). Not cultivated on cell-free media; obligate intracytoplasmatic parasite of H. vermiformis strain A1Hsp and other H. vermiformis strains, therein preventing cyst formation. Host range: able to multiply in D. discoidum, but not in Acanthamoeba spp.; mesophilic (20–30 °C). Isolated from the water conduit system of a dental unit (Lahnstein, Germany). Type strain, A1Hsp (=ATCC 50802).

Diversity within the Chlamydiales and clinical aspects of N. hartmannellae
In a more general perspective, our results and the recent identification of four Parachlamydia-related acanthamoebal endocytobionts (T. R. Fritsche and others, unpublished), a Chlamydia-like bovine intracellular organism (Waddlia chondrophila; Rurangirwa et al., 1999 ) and a Chlamydia-related organism observed within tissue culture (Simkania negevensis; Kahane et al., 1999 ) demonstrate a previously unrecognized diversity within the Chlamydiales. Interestingly, the order Chlamydiales still exclusively comprises obligate intracellular bacteria, some of which have developed mechanisms to survive and exploit uptake by protozoa. The adaptation to intracellular growth in the ubiquitously distributed FLA could have functioned as a preadaptation of Chlamydia-like ancestors to survival within other host cells of higher eukaryotes, including humans; this raises the question of the clinical significance of members of the family Parachlamydiaceae. Few studies have addressed this important issue. Among these, Birtles et al. (1997) screened for the presence of specific antibodies against Parachlamydia-like endocytobionts of Acanthamoeba sp. (‘Hall’s coccus’, displaying more than 99% 16S rDNA similarity to P. acanthamoebae) in blood sera from patients with pneumonia of undetermined cause, and found positively reacting sera that did not react with Chlamydia psittaci, C. trachomatis or C. pneumoniae. These researchers therefore suggested that ‘Hall’s coccus’ be considered potentially pathogenic for humans. Another remarkable finding was the recovery of novel Chlamydia-like 16S rDNA sequence fragments from specimens of respiratory-disease patients, recently reported by Ossewaarde & Meijer (1999) . The sequence similarities of these sequences and N. hartmannellae range between 72% and 84%. Reliable phylogenetic analysis of the Chlamydia-like sequences and the 16S rRNA sequence of N. hartmannellae could not be performed, due to the short length of the Chlamydia-like sequence fragments (approx. 220 bp). A stable tree topology could not be obtained by applying different treeing methods and data sets. Further research is needed to clarify whether the FLA endocytobionts of the family Parachlamydiaceae are indeed able to infect humans.

Concluding remarks
In conclusion, we have identified an obligate endoparasite of H. vermiformis, provisionally classified as Neochlamydia hartmannellae, as a new member of the family Parachlamydiaceae. These findings broaden our knowledge of the phylogenetic diversity within the Chlamydiales. Although it is too early to draw conclusions on the clinical significance of these bacteria, the detection of these organisms in FLA suggests that FLA may act as a general reservoir for Chlamydia-like organisms. More detailed knowledge is needed on the natural habitats, diversity, physiology and virulence of members of the family Parachlamydiaceae.


   ACKNOWLEDGEMENTS
 
This study was supported by Deutsche Forschungsgemeinschaft (DFG) grant WA 1027/2-1 to M.W. and K.-H.S. We are indebted to Gerhild Gmeiner for preparation of fixed material for electronmicroscopy and taking some of the micrographs (Head of the Department for Electron Microscopy: Dr Bärbel Hauröder), and to H. Burghardt for providing water samples in the course of a microbiological inspection.


   REFERENCES
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS AND DISCUSSION
REFERENCES
 
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Received 24 September 1999; revised 22 December 1999; accepted 24 January 2000.