Vol. 58, No. 11
INFECTION AND IMMUNITY, Nov. 1990, p. 3816-3818 0019-9567/90/113816-03$02.00/0 Copyright © 1990, American Society for Microbiology
Growth of Chlamydia trachomatis in Enucleated Cells EVE PERARA,1 T. S. BENEDICT YEN,2'3 AND DON GANEMl.3* Departments of Microbiology and Immunology,' Pathology,2 and Medicine,3 School of Medicine, University of California, San Francisco, California 94143 Received 7 May 1990/Accepted 8 August 1990
Chlamydia trachomatis is an obligate intracellular parasite of eucaryotic cells. Little is known about the role of the host in supporting chlamydial replication beyond the facts that host cells provide ATP and that de novo host protein synthesis is not required for bacterial growth. To further explore potential contributions of host nuclear function to chlamydial development, we questioned whether murine C. trachomatis could grow in mouse L cells that had been enucleated with cytochalasin B. Following enucleation, cells were infected with chlamydiae and analyzed morphologically and biochemically. Late in infection, substantial numbers of chlamydiae of all developmental stages were seen within large cytoplasmic inclusions that were indistinguishable from those seen in infected intact cells. Normal numbers of infectious progeny particles were produced from enucleated cultures. We conclude that active host cell nuclear function is not required to support the growth of chlamydiae.
with nuclei (1). To resolve this controversy and to extend these studies with more detailed morphological and biochemical analyses, we have reexamined the ability of chlamydiae to infect and develop within enucleated cells. Mouse L-cell fibroblasts were grown in Nunc 25-cm2 tissue culture flasks to 90 to 95% confluence and enucleated with cytochalasin B as described previously (10). Briefly, monolayers were treated by filling flasks to the neck with 15 ,g of cytochalasin B per ml in serum-free medium and incubating at 37°C for 30 min. Flasks were subjected to centrifugation at 12,500 rpm (25,000 x g) at 28°C for 70 min in a Sorvall GSA rotor whose wells had been filled with 100 ml of water. The medium, along with pelleted nucleoplasts and cellular debris, was removed, and cytoplasts, which remained adherent to the flasks, were returned to normal medium (Dulbecco modified Eagle medium plus 10% fetal calf serum) for 1 to 2 h to recover prior to infection. In general, this procedure resulted in the loss of 50 to 75% of the cells originally present. Typically, 90 to 95% of the remaining cells were enucleate, as judged by Hoechst staining. Control cells were treated with cytochalasin B but were not subjected to centrifugation and therefore remained nucleated. The preparations were then infected with equal titers of the mouse pneumonitis (MoPn) strain of C. trachomatis. Twenty-two hours postinfection, cells were removed from the flask with trypsin, fixed in 2% cacodylate-buffered glutaraldehyde, and prepared for electron microscopy. Samples were postfixed with osmium tetroxide, dehydrated in a graded series of alcohols, and embedded in Epon. Thin sections were stained with uranyl acetate-lead citrate and examined with a JEOL 100S electron microscope. Infection of enucleated cells by chlamydia was quite efficient (Fig. 1); between 10 and 30% of the scorable cytoplasts on the monolayer developed inclusions. In fact, the efficiency of infection in these cells was not significantly different from that seen in intact cells (data not shown). Following infection of enucleated cells, EBs differentiated efficiently into the replicative RBs, as determined by electron microscopy. Figure 2 shows an infected cytoplast in which a large vacuole containing mostly RBs is readily apparent. Since only the EB is capable of initiating infection (2, 11), these RBs must represent developmental intermediates in the chlamydial life cycle. Their presence demon-
Chlamydia trachomatis is a gram-negative bacterium which is an obligate intracellular parasite of eucaryotic cells (1). Its intricate life cycle has been described well morphologically but is otherwise poorly understood. Chlamydiae exist in two serially alternating forms: the elementary body (EB) and the reticulate body (RB). The small, sporelike EB is the metabolically inactive, infectious form of the organism. EBs enter the host cell within cytoplasmic membranebound vacuoles that are able to avoid phagosome-lysosome fusion (2, 5, 14). Within these vacuoles the EBs differentiate into metabolically active, replicative RBs, which then proliferate. Late in the developmental cycle, the RBs differentiate back into EBs, which are then released from the cell to reinitiate new rounds of infection. The interactions of chlamydiae with their eucaryotic hosts are poorly understood. It is presumed that ATP and other high-energy compounds are supplied by the host (7, 12), but the precise factors required by chlamydiae from the host cell remain to be determined. It is known that chlamydiae do not require the de novo synthesis of cellular proteins for their life cycle since they grow readily in the presence of inhibitors of eucaryotic protein synthesis (1). Although sometimes interpreted to mean that nuclear function is not required for chlamydial growth, this finding does not entirely rule out plausible nuclear contributions to bacterial development. Recent work in other systems has shown that in addition to proteins, nuclei can provide a variety of other important macromolecules, notably RNAs, to cytoplasmic organelles. For example, the mitochondrial endoribonuclease RNase MRP contains an essential RNA component which appears to be encoded in the nucleus (3). Mitochondrial DNA replication appears to depend upon the use of a small RNA of nuclear origin that serves as a primer (13). In addition, some mitochondrial tRNAs are of nuclear origin (8). One powerful way to evaluate the role of nuclear contributions to cytoplasmic events is to examine cells whose nuclei have been removed by biophysical methods. Some investigators have observed the development of sizeable chlamydial inclusions in enucleated cytoplasts (4, 6), while others have reported that inclusions developed only in cells *
Corresponding author. 3816
VOL. 58, 1990
FIG 2 Electron micrograph of a section through an enucleated L cell. An inclusion body is shown, containing primarily RBs.
Original magnification, x 11,000.
Calif.), to stain and count inclusion-bearing cells. The yield FIG. 1. Electron micrograph of a section of enucleated L ccDlls at 22 h postinfection with C. trachomatis MoPn. Inclusions cc )ntain chlamydiae at various stages in the developmental cycle, both iEBs and RBs. Original magnification, x3,000.
strates that ongoing host nuclear function is not require d for the initial morphological change from EBs to metaboliically active RBs. Consistent with this, metabolic labeling stiudies with [35S]methionine revealed that infected cytoplasts synthesized a complex array of chlamydial polypeptides siimilar to those found in infected nucleated cells (data not sho wn). At 22 h postinfection, the chlamydiae in most cytoplasts were in various stages of their developmental cycle, with many having differentiated from RBs back to EBs. Ex(amination of serial sections of infected cytoplasts confirmedI that in these cells no cytoplasmic bridge remained intact betP ween nucleus and cytoplast. The large size of the inclusions a,t this stage (and the large number of chlamydiae seen within t hem) indicates that multiple rounds of replication and divisioi n had occurred. Importantly, chlamydia-laden vacuoles in (cytoplasts were morphologically indistinguishable from tho)se in control intact cells (compare Fig. 3 and 4). These re sults indicate that the later events in the chlamydial replic ative cycle also proceed efficiently in the absence of onj going nuclear activity. Finally, we assessed the infectivity of the resulting 1progeny EBs produced from monolayers of infected cytop] lasts. EBs were harvested and prepared from cytoplasts and control cells as described previously (9). Infectivity was assayed by endpoint dilution with a fluorescein isothIiocyanate-coupled anti-chlamydial lipopolysaccharide monioclonal antibody, 8G3 (Diagnostic Products Corp., Los Ang;eles,
of infectious progeny from the enucleated culture was indis-
tinguishable from that produced by the nucleated control culture: 2.7 x i0' and 2.9 x iO' infectious units per ml, respectively. Since fewer than 3% of the cells in the enucleated culture remained nucleated, and since fewer than 2% of
FIG. 3. Electron micrograph of a section through an enucleated L cell at 22 h postinfection, showing a large, chlamydia-laden vacuole. Most chlamydiae are in the EB phase. Original magnifica-
our findings give encouragement that such a system may well be possible. We thank Jon Pollack and Joanne Engel for helpful comments on the manuscript. We also thank the National Institutes of Health for support of this work.
FIG. 4. Electron micrograph through a section of an intact L cell at 22 h postinfection, showing a large vacuole containing predomi-
nantly EBs. Original magnification,
all inclusions were observed in those intact cells, it is highly unlikely that this result could be due to release of chlamydiae from the remaining intact cells alone. Thus, enucleated cells appear capable of supporting the entire chlamydial life cycle; no ongoing production of nuclear products is required. While we cannot rule out the possibility that preformed RNAs or other preformed molecules of nuclear origin might still be used by replicating chlamydiae, this seems improbable. These results suggest that cytoplasmic extracts might be able to sustain the replication of chlamydiae in vitro under the appropriate conditions. Although previous attempts to achieve cell-free growth of chlamydiae have met with limited success thus far,
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