Department of Virology, Faculty of Health Sciences, Ben Gurion University of the Negev, PO Box 653, Beer Sheva, Israel84105-IL1
Author for correspondence: Maureen G. Friedman. Tel: +972 8 6403857. Fax: +972 8 6276215. e-mail: maureen{at}bgumail.bgu.ac.il
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ABSTRACT |
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Keywords: Chlamydia-like micro-organism, infectivity, Simkania
Abbreviations: EB, elementary body; EM, electron microscopy; IFU, infectious-centre-forming unit; p.i., post-infection; PIPA, plate immunoperoxidase assay; RB, reticulate body
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INTRODUCTION |
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It has been proposed that Simkania negevensis be assigned to the new family Simkaniaceae, in the order Chlamydiales, based on its intracellular bimorphic parasitic growth in cultured cells and on rDNA sequence comparisons (Kahane et al., 1999 ; Everett et al., 1999
). Its genome length is 1·7 Mbp, compared to 1·01·2 Mbp for members of the family Chlamydiaceae (Kahane et al., 1999
); its full-length 16S and 23S rDNA sequences are each 8087% identical to those of members of the Chlamydiaceae, whereas all members of the Chlamydiaceae have >90% identity with each other. [Members of the genus Chlamydia have >95% identity with each other; members of the genus Chlamydophila have
95% identity with each other (Everett et al., 1999
).]
Parachlamydiaceae, Chlamydiaceae, Simkania negevensis and other Chlamydia-like bacteria placed in the order Chlamydiales exhibit some variation in their developmental morphology. In Chlamydiaceae, the infectious elementary body (EB) is an electron-dense, coccoid body, 0·20·35 µm in size (Rake, 1957 ). It changes into a replicating form that is 0·8 µm in size and can be seen 79 h after infection of a host cell (Rake, 1957
). This irregularly shaped replicative form is called the reticulate body (RB), because it is composed of amorphous or reticulated material with moderate density (Higashi et al., 1962
; Higashi, 1965
). For reasons that are not well-understood, the Chlamydiaceae RB is not infective and does not survive long outside of the host cell. One to three days after infection [depending on the strain and the multiplicity of infection (m.o.i.)], most RBs have reorganized back into EBs and are released either by host-cell lysis or by fusion of the phagocytic inclusion with the cell wall. A method that has been invaluable in elucidating the replication cycle and in characterizing the developmental forms in Chlamydiaceae is the purification of EBs and RBs using Renografin density gradients (Caldwell et al., 1981
; Barbour et al., 1982
). Pleomorphic Simkania negevensis forms, some of which are reticulated and large (0·30·7 µm) and some of which have electron-dense and electron-lucent areas, can be selectively fractionated on density gradients in much the same way as Chlamydiaceae spp. bodies (Kahane et al., 1999
). The purpose of this study was to describe the Simkania negevensis growth cycle in vitro and compare it with that of its close relatives, the Chlamydiaceae. In keeping with the degree of rDNA sequence divergence observed between members of the two families, it was expected that both similarities and differences would be found.
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METHODS |
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Fixation and staining of infected cell monolayers for observation under light microscopy.
Vero cell cultures were grown in Nunclon SlideFlasks and infected with Simkania negevensis (the 44/52% Urografin gradient fraction) at an m.o.i. of 0·3. At various times post-infection (p.i.) monolayers were washed with PBS (NaCl, 0·137 M; KCl, 0·0027 M; Na2PO4, 0·008 M; KH2PO4, 0·00147 M; pH 7·2), fixed for 3 min in MayGrünwald solution (MG-500, Sigma), washed three times with distilled water and further stained for 25 min with Giemsa stain (GS-500, Sigma) diluted to 27% (v/v) with distilled water. After washing with tap water, the Nunclon flasks were taken apart, and the slide portion was examined by light microscopy and the image photographed.
Infectivity assays for Simkania negevensis.
For growth curves, monolayers of Vero cells in replicate 25 cm2 flasks were inoculated with Simkania negevensis. At various times after infection cells were scraped into the medium with glass beads; the suspensions were frozen at -70 °C with 50% fetal calf serum. The number of infectious-centre-forming units (IFUs) for each sample was determined by 10-fold dilution and infection of Vero cells cultured in 96-well plates. Three days p.i. the plates were fixed in 95% ethanol for 10 min at room temperature and examined for IFUs by the microtitre plate immunoperoxidase assay (PIPA), performed as described previously (Kahane et al., 1998 ). Briefly, after fixation, plates were incubated for 1 h at 37 °C with Simkania negevensis-specific antisera raised in rabbits, washed and re-incubated (1 h, 37 °C) with swine anti-rabbit horseradish-peroxidase-conjugated antibodies, and after another wash, stained with diaminobenzidine as substrate. Infectious centres were counted under a magnification of x200 with an inverted microscope, and the counts for three wells were averaged; the IFU ml-1 count for the original sample was then calculated.
Titration of infectivity.
Simkania negevensis particles that banded at the 40/44% interface and at the 44/52% interface of a Urografin density gradient upon centrifugation were directly examined in two ways: (1) by titration on Vero cells to determine the number of IFUs in each fraction, and (2) by enumeration of aliquots mixed with known concentrations of polystyrene latex beads by negative-stain electron microscopy (EM). Replicative forms and electron-dense forms of the bacteria present in both fractions were determined by photography of random EM thin-section fields and by counting samples of several hundred particles. The relative infectivity of the constituents of each fraction was determined.
EM
Negative staining.
Purified Simkania negevensis particles suspended in 25 mM HEPES saline buffer were homogeneously mixed with polystyrene latex particles (mean diameter 0·175 µm, at 3·51x1011 particles ml-1; Agar Scientific) and applied to a 400-mesh copper Formvar/carbon-coated grid which was fixed and stained as described by Glauert (1975) . Briefly, the grids were dipped for 15 s in paraformaldehyde/glutaraldehyde fixative and then three times in distilled water, followed by staining in 2% ammonium molybdate solution, pH 7·4. Negative staining was used to count total particle numbers, without discrimination as to the type of the particle.
Thin sections of cultured cells or gradient-fractionated bacteria.
Infected cells were scraped from flasks and pelleted, or bacteria were fractionated on discontinuous Urografin gradients. These preparations were fixed, embedded in Araldite, and stained for EM as described by Biberfeld (1971) . The larger morphological forms, 0·30·7 µm, were designated replicative forms for the following reasons: (i) they were the first forms to appear after infection; (ii) they multiplied rapidly and were often captured apparently in the process of binary division; and (iii) they contained reticulated, homogeneously stained material and no white, unstained or electron-dense areas. The smaller particles, 0·20·3 µm, were designated electron-dense forms because they contained at least some electron-dense material with or without an electron-lucent area and no reticulated material (Kahane et al., 1993
). Typically, 810 randomly chosen EM fields of sectioned infected cells were photographed, and several hundred particles were counted from the photographs of each Urografin fraction to determine the relative proportion of the morphological forms.
Chlamydia trachomatis EB and RB infectivity.
The infectivity of Chlamydia trachomatis EBs and RBs prepared on discontinuous Urografin gradients was assessed as a control for the Simkania negevensis experiments. Chlamydia trachomatis-infected cells were identified in the PIPA by using polyclonal rabbit antisera raised against Chlamydia trachomatis (Gonen et al., 1993 ).
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RESULTS |
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DISCUSSION |
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In the present study, we extended our original observations that the phenotypic replication cycle of Simkania negevensis takes about 1215 days, while the number of IFUs produced increases exponentially for only 24 days, after which time a plateau is reached (Kahane et al., 1999 ). The photomicrographs of fixed and stained infected cells obtained in this study (Fig. 1
) clearly show the accumulation of bacterial particles with time in the infected cells, as well as the relative lack of spread of the infection to neighbouring uninfected cells. Growth curves obtained at lower m.o.i. values (Fig. 3
) also suggested that the infection does not spread to neighbouring cells, and that infectious particles are not released in significant amounts into the culture medium. The significance of the long plateau stage of the growth cycle during which cytopathic effects increased, but the organisms appeared to be trapped in their host cells, is not understood. We found that four sequential rapid passages of organisms present at 34 days p.i. did not result in a decrease of infectious yields, as compared to four long sequential passages of 10 days duration (data not shown). This would seem to indicate that at least under in vitro conditions, there is no need for maturation, which might be postulated to be taking place during the long plateau period. However, the stationary phase may result in some survival advantage for the organisms during transmission in nature. Cells infected with Chlamydia trachomatis L2 detach from the monolayer or lyse by 2 days after infection; uninfected neighbouring cells are rapidly infected (data not shown). On the other hand, cells infected with the more distantly related intracellular bacterium Coxiella burnetii show minimal cytopathic effects and it has been suggested that this organism may lack an efficient membranolytic system (Heinzen et al., 1999
); however, infectious forms can be mechanically released from infected cells early in the developmental cycle (Howe & Mallavia, 2000
).
The first hint that Simkania negevensis replicative forms may be infectious was the similar kinetics of growth of particles from the 44/52% and 40/44% Urografin fractions (Fig. 4). This possibility was lent more credibility by the fact that in growth curves, progeny infectivity increased not simultaneously with the appearance of electron-dense forms in EM thin sections, but earlier (Fig. 5
), and received further support from the results of infectivity experiments done with purified 44/52% and 40/44% fractions (Table 1
). In control experiments, using Chlamydia trachomatis, EBs totally accounted for the infectivity found in the 40/44% fraction, but this was not the case for Simkania negevensis. The infectivity of particles appearing to be in transition between the larger replicative form and the more compact electron-dense form is not known. For the calculations, transition particles showing either a dense area or an unstained area were counted as dense forms and presumed to be infectious. Counting these particles as dense forms may have resulted in an overestimation of the level of such forms in the 40/44% fraction and, therefore, may have led to an overestimation of their relative contribution to the infectivity in the 40/44% fraction. Nevertheless, the data indicate that in the case of Simkania negevensis, infectivity was apparently not limited to the dense forms.
Since the genome of Simkania negevensis is nearly twice the size of the Chlamydia trachomatis genome (Kahane et al., 1999 ), it is possible that additional genes permitting infectivity of all morphological forms are present in Simkania negevensis. If Simkania negevensis replicative forms are indeed infectious it may be predicted that these particles might be characterized by a physical stability comparable to that of the electron-dense forms, in contrast to Chlamydiaceae RBs which are osmotically fragile and incapable of infecting cells (Moulder, 1985
; Tamura et al., 1967
). Preliminary evidence suggests that Simkania negevensis replicative forms have a survival curve similar to that of electron-dense forms after exposure to various temperatures and to osmotic shock (unpublished data). More extensive studies comparing the morphological forms of Simkania negevensis are necessary; examination of the similarities and differences in membrane composition is of special interest.
It is notable that the growth cycle of Coxiella burnetii, which belongs to the order Rickettsiales (family Rickettsiaceae), includes two forms, a small cell variant and a large cell variant, both of which are infectious (Wiebe et al., 1972 ). Our results raise questions as to the role played by electron-dense bodies and larger bodies in the growth cycles of newly described Chlamydia-like micro-organisms, some of which have not yet been grown in cell culture (Fritsche et al., 2000
; Ossewarde & Meijer, 1999
), including members of the Parachlamydiaceae and Waddliaceae, in the order Chlamydiales (Everett et al., 1999
). Also, more general questions are raised as to the potential significance of Simkania negevensis morphological forms in the pathogenesis or epidemiology of infection by this novel micro-organism.
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ACKNOWLEDGEMENTS |
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Received 23 February 2001;
revised 26 June 2001;
accepted 22 October 2001.