Molecular organization of the kinetoplast DNA of Trypanosoma cruzi treated with berenil, a DNA interacting drug

© 1972 by Academic Press, Inc.

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J. ULTRASTRUCTURE RESEARCH

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Molecular Organization of the Kinetoplast DNA of Trypanosoma cruz/Treated with Berenil, a DNA Interacting Drug CH. BRACK,1 t3. DELAIN,2 G. RIotJ, and B. F~STF

Unit~ de Pharmacologie MolOculaire and Unit~ de Microscopic Electronique (Laboratoire associ~ du CNRS, n ° 147, Pharmacologic Mol~eulaire) Institute Gustave Roussy, 94-V17lejuif, France Received November 12, 1971 The trypanocidal diamidine berenil has selective binding properties for the kinetoplast DNA (K-DNA) of Trypanosoma cruzi. It induces characteristic ultrastructural modifications in the kinetoplast which are similar to those observed after hydroxystilbamidine treatment. The purified K-DNA of bereniltreated T. cruzi contains long "lampbrush-like" structures, which are linked to complex associations of circular molecules. The "lampbrushes" are compared to the alterations observed in thin sections. Many replicating circular molecules are present in the K-DNA of berenil-treated trypanosomes. The possible relation between a drug-specific blocking of K-DNA replication and the formation of the "lampbrush-structures" is discussed.

The kinetoplast D N A (K-DNA) of trypanosomes is specifically modified by many trypanocidal drugs, e.g., acridines (30, 32), phenanthridines (8), and diamidines (6). When trypanosomes (Trypanosoma cruzi) were grown in the presence of hydroxystilbamidine (OHSA), a progressive reorganization of the K - D N A was observed. The most characteristic alteration was the appearance of cylindrical fibrillar "sausage-like bodies" (6). The trypanocide berenil (13, 18, 35) is a diamidine compound like OHSA. It has analogous D N A binding properties in vitro (9-12, 21, 33), and interacts preferentially with K - D N A in vivo (18, 22, 35). Both drugs induce very similar ultrastruct u r n alterations in the kinetoplast of T. cruzi. In this paper, we present some results on the molecular structure of the characteristic associations induced in the K - D N A by berenil. 1 Present address: Department of Microbiology, Biozentrum, University of Basel, CH-4056 Basel, Switzerland. 2 To whom requests for reprints should be addressed: Laboratoire de Microscopie 61ectronique-Institut Gustave Roussy--94-Villejuif--France.

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M A T E R I A L A N D METHODS

Trypanosome cultures. Trypanosoma cruzi (Mexican strain Tehuantepec, from Pasteur Institute, Paris) were freshly isolated from mouse blood and grown at 28°C in a diphasic culture medium consisting of blood-agar (10% human blood) and the synthetic liquid culture medium IP 199 (Parker 199, modified by the Pasteur Institute). Penicillin (250 IU/ml) and streptomycin (50 ~g/ml) were added to prevent bacterial contamination. Berenil (4,4'-diazoaminodibenzamidine diaceturate. 4H20) was kindly provided by Dr L~ewe from Hoechst AG. Germany. A sterile solution was introduced into trypanosome cultures (2 × 105 cells/ml) to give a final concentration of 2 #g/ml. After 4 days, i.e., in the exponential phase of culture, the trypanosomes were harvested for D N A extraction. Extraetion and purification of kinetoplast DNA. K - D N A was prepared mainly with the techniques already described (28); i.e., extraction of total cell D N A according to the method of Marmur (19) and fractionation of K - D N A in a Hg~+-Cs~SO4 gradient according to Davidson et al. (4). Some D N A samples were treated with pronase (B grade, Calbiochem), 1/~g/~g D N A for 30 minutes at 37°C; the stock solution of the enzyme (1 mg/ml) had been preincubated at 37 °C for 1 hour. Preparation of K-D NA for electron microscopy. The purified K - D N A was spread according to the technique of Lang and Mitani (15), modified by Delain and Brack (5). Droplets (10-15 ~1) of a solution containing at final concentrations: D N A 0.5 ~g/ml, ammonium acetate 0.05 M, and cytochrome e 15/~g/ml, were put on a Teflon plate. In some experiments formamide (puriss pro analysi, Fluka) was added to the D N A solution to a final concentration of 50 %. A droplet of formaldehyde or osmium tetroxide was placed beside the sample, and both were covered with a petri dish. The vapors of these fixatives denature the cytochrome c at the air-solution interface and greatly improve the D N A spreading. After 30-60 minutes diffusion time, the protein film was picked up on carbon-coated copper grids, dried in 95 % ethanol, and shadowed with platinum while rotating (30 mg, 6 °, 10 cm). Specimens were observed with a Philips EM 300 electron microscope. RESULTS The K - D N A of untreated T. cruzi is composed of large molecular associations and a varying a m o u n t of free circular molecules (Figs. 1 and 2). L o n g chain-like structures can be discerned in these large complexes. They consist of m a n y links all of which have the same contour length as the free circular molecules, i.e., approximately 0.5 #. These links could be either a succession of circles attached to each other, or the result of the regular crossovers of linear molecules. A few of the circular molecules are free (~+); the others are associated with the chains, either being interlocked like catenanes (+-->), or f o r m i n g figure 8 structures (+->). The latter resemble the "fused" molecules, as defined by Clayton et al. for the circular mitochondrial dimers obtained by renaturation of single-stranded m o n o m e r s and dimers (3). The morphological aspect and the arrangement of the K - D N A molecules of normal trypanosomes m a y be different, depending on the age of the cultures. I n the K - D N A extracted f r o m exponentially growing T. eruzi (day 4 of culture), the molecules seem

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to be m u c h m o r e densely p a c k e d (Fig. l) t h a n in K - D N A of 5-week-old culture forms (Fig. 2). These p r e l i m i n a r y observations suggest t h a t all the K - D N A seems to consist actually of identical 0.5 # units. A m o r e detailed description and i n t e r p r e t a t i o n of n o r m a l K - D N A o r g a n i z a t i o n will be p u b l i s h e d separately. It will be easier to u n d e r s t a n d the actual a r r a n g e m e n t of the K - D N A molecules within the kinetoplast after having studied the u l t i a s t r u c t u r a l a n d m o l e c u l a r modifications i n d u c e d by different drugs. The K - D N A of berenil-treated t r y p a n o s o m e s shows characteristic modifications which will be c o m p a r e d to the u l t r a s t r u c t u r a l alterations previously described for O H S A (6). I n some D N A complexes, the long chainlike structures are modified: the links between the crossing points are multiplied; it is possible to see an increase in the n u m b e r of the p a i r e d segments. I n Figs. 3 a n d 4 the arrows ~ , ~ , a n d ~ m a r k 2, 4, a n d 8 o r m o r e segments, respectively. M a n y circular molecules are a t t a c h e d to the crossing p o i n t s a n d f o r m rosettes which are often aligned along the edge of one K - D N A association (Fig. 3). This a r r a n g e m e n t a n d its p e r i p h e r a l p o s i t i o n reminds of the elongated cylindrical fibrillar bodies, which a p p e a r at the p e r i p h e r y of the kinet o p l a s t shortly after O H S A o r berenil a d m i n i s t r a t i o n . F i g u r e 4 shows several rosettes within the complex D N A network. I n w h a t we suppose to be a later stage, the segments between the rosettes a p p e a r sometimes densely p a c k e d a n d can no longer be resolved into several filaments, although, at some places, the filamentous structure of the axis remains visible (-+Fig. 5). A great n u m b e r of circles are a t t a c h e d to this axis a n d are stretched out on either side p e r p e n d i c u l a r l y to it. A l l these molecules have the s a m e c o n t o u r length as the free circles, hence the regularity of these structures, which e v o k e the picture of " l a m p b r u s h e s . " In some places, single or associated circular molecules are linked to the " l a m p b r u s h . " Even after p r o n a s e treatment, the axis FIG. 1. Portion of a large association of kinetoplast DNA molecules extracted from Trypanosoma cruzi (4-day-old culture). The circular units are densely interlocked, forming at some places chain-like structures; all links have a contour length of 0.5/z. Some circular molecules are free (-+), others are apparently catenated (++) or fused (-~--~), x 35 000. FIG. 2. Similar detail of a K-DNA association of a 5-week-old Trypanosoma cruzi culture. The molecules are less densely packed. The links of the chain like-structures are much more extended in this preparation, giving an aspect of regularly crossing over linear molecules. The same types of associations can be observed: free circles (-+), catenated (++) and fused (-~--~)circles, x 35 000. FIGS. 3-8. K-DNA of Trypanosoma cruzi treated for 4 days with berenil (2 pg/ml). FIG. 3. At the edge of the K-DNA complex, the links of the chains are multiplied. In one chain, the number of paired segments increases from the left to the right: two, four, and eight segments are indicated by the single, double, and triple arrows, respectively, x 50 000. II II FIG. 4. The Sinks are also multiplied within the K-DNA network. The arrows indicate similarly the increase of the paired segments. Sometimes, many circular molecules are attached to crossing points, forming rosettes, x 50 000. Flo. 5. "Lampbrush" structure composed of a dense axis with many circular molecules attached to it. At some places, the axis contains still distinguishable filaments (-+). × 50 000. ~" FI~. 6. Large portion of a berenil-altered K-DNA complex. Four long "lampbrushes" (-+) linked together by chains or linear filaments, are surrounded by densely packed circular units, x 25 000.

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J . Ultraetructure Research

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of these structures has the same condensed aspect. Thus it seems rather unlikely that the '°lampbrushes" are artifacts consisting of molecules that are stuck together by residual proteins. The fact that all attached circular molecules are stiffly stretched on either side and perpendicularly to the axis is due to the spreading technique, which arranges all molecules two-dimensionally. The three-dimensional reconstruction of serial sections had shown that these bodies are actually cylindrical, and that their diameter is smaller (0.1-0.2 #) than in spread preparations (0.5 #). Figure 6 shows part of the D N A association of a kinetoplast with some localized modifications. Some long characteristic "lampbrushes" (-+) can easily be discerned among the complex of linear and circular molecules. Long, straightened chains of linked molecules attach the "lampbrushes" to each other. In Fig. 7a, a wholemount preparation of an altered kinetoplast shows many long, sinuous, branched "lampbrush" structures, which are again mainly situated at the periphery of the D N A mass. This large association of molecules has a diameter of approximately 8 ¢~. About twenty similar patches have been observed; they all have 7-20 # in diameter, and we assume that they represent most of the D N A content of individual kinetoplasts. Some of the circular molecules which are distinguishable at the periphery of the K - D N A are double-branch circles as defined by Schn6s and Inman (29), and are assumed to be replicating molecules, according to the model of Cairns (2) (Fig. 7a-+, and 7b, c). Similar forms have been found in a relatively high proportion (about 1%) among the free molecules (Fig. 7d). In some kinetoplasts, nearly all the K - D N A is arranged into °'lampbrush" structures (Fig. 8). Such a preparation permits the evaluation of the approximate number of circular molecules per kinetoplast (assuming that it consists of the D N A of only one single kinetoplast). We have counted 30-40 molecules attached to the axis segment between the two arrows (length 0.5 #). The total length of the "lampbrushes" of this completely modified K - D N A was measured to be about 1 mm. Thus we estimate one such large DNA-association to contain a minimal number of 60 000-80 000 circular molecules, counting only the circles attached to the "lampbrushes." DISCUSSION The initial preparation technique for the isolation of K - D N A led to the conception that the kinetoplast D N A consisted mainly of minicircles associated with linear molecules (24, 25, 27, 31). The interpretation has been discussed and progressively FIO. 7". (a) The whole-mount preparation of a modified kinetoplast shows the mainly peripheral localization of the branched, sinuous '"lampbrusbes." Replicating circles can be detected at the edge (-+b and -,c). (b and c) Enlarged details of these replicating molecules. (d) Two of the replicating forms, which are found in a relatively high proportion among the free circles. (a) x 15 000; (b-d) x 80 000.

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reevaluated along with the use of new preparation methods (16, 17, 23, 25). We actually think that the D N A of the kinetoplast consists of a large, complex, and coherent association of circular and "linear" molecules, and of free circles in variable proportions. Our results showed that it is possible to visualize such large D N A associations in chemically extracted and purified K - D N A . The K - D N A of untreated T. cruzi spreads to form large patches with a diameter of 10-20/~, which are similar in size to those obtained by osmotic lysis of isolated kinetoplasts of T. cruzi (unpublished results), as well as those described for T. mega (16). These results support the hypothesis that each of the large D N A complexes corresponds to most of the D N A content of one single kinetoplast. When we compare the size of such a large macromolecular association with the size of the kinetoplast (diameter 1-2 #) in thin sections, we note that the spreading technique enormously expands the K - D N A aggregation. The diffusion up to 60 minutes permits a slow adsorption and therefore a progressive expansion of the K - D N A . With the classical spreading technique of Kleinschmidt and Zahn (14) used in our previous description, we were not able to display accurately the large K - D N A complexes: only small associations had been described (26). Laurent et al. (17) have calculated that kinetoplasts of Crithidia luciliae contained 3.73 + 0.17 × 10-14 g of K - D N A . In preparations of osmotically lysed kinetoplasts of T. mega, the same authors observed large D N A patches with a diameter of 10 # (16), quite similar in size to the ones described here. In a previous report, we had estimated the kinetoplast D N A content in T. cruzi to be about 3.6 × 10 -1~ g per kinetoplast, corresponding to 24 000 circular molecules with a M W of 9 × 105 daltons; these results had been obtained from chemical analysis. There is a discrepancy between those first estimations and the ones mentioned in this paper (1 × 10 -13 g of D N A per kinetoplast) which were obtained by counting the molecules on the micrographs, assuming that one large D N A patch corresponds to the K - D N A of one kinetoplast. We did not include the free circles, a variable amount of which may even be lost during extraction and spreading. Furthermore, the D N A content of one species may even vary with the physiological state of the trypanosome, e.g., with the evolutionary stage of the parasite's life cycle. The trypanosomes used for the present study had been recently isolated from mouse blood and had only been subcultured for a few months (8 passages on culture medium). An accurate quantitative estimation is therefore difficult to make and the various K - D N A estimations of other authors must be compared very carefully. As seen in thin sections the kinetoplast of O H S A (6) and berenil treated trypanoFIG. 8. In this preparation, almost all K-DNA is arranged into "lampbrushes." The number of molecules between the two arrows has been counted for an estimation of the total number of attached circles, x 40 000.

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somes contains long fibrillar "sausage-like structures". Their axes consist of long, parallel filaments, which are surrounded by irregularly oriented short fibrils. In this paper we have described "lampbrush-like" molecular associations in the purified K-DNA of berenil treated T. cruzi. These characteristic structures can be compared relative to many features. They have a similar disposition within the K-DNA mass-at first peripheral and then invading the whole DNA. Both are composed of a fibrillar axis, surrounded by perpendicularly oriented filaments. So we think that the "sausages" and the "lampbrushes" are two different morphological aspects of the same thing, namely the OHSA or berenil induced modification of the paracrystalline organization of the DNA molecules in the kinetoplast. Even if not very precise, the quantitative data permit the evaluation of the in situ concentration of the K-DNA of T. cruzi to be several hundreds of milligrams per milliliter. Small identical units of DNA molecules in such a concentration could give to the K-DNA the particular physical properties of the liquid crystals. The morphological alterations induced by intercalating and nonintercalating drugs had been discussed in this respect (6). It has been shown that the trypanocide OHSA, which is a nonintercalating drug closely related to berenil (10, 33), inhibited the DNA polymerase activity of a crude rat liver extract in vitro (10). The incorporation of 5-bromouracil into K-DNA of T. mega is inhibited by berenil (21). These two diamidines inhibit selectively thymidine-all incorporation into T. cruzi K-DNA (unpublished results). Similar inhibitions have been observed with intercalating trypanocidal drugs, such as acridines (30) and phenanthridines (26). Moreover, these drugs have been shown to inhibit selectively the mitochondrial DNA polymerase of animal cells (20, 33), probably by a direct action on the enzyme. The unusually high proportion of replicating molecules found in the K-DNA of berenil treated T. cruzi (1) suggests that the drug may interfere with the replication process. The progressive transformation of the K-DNA into "lampbrush" structures has been interpreted to be the result of an accumulation of circular molecules which remain attached together within the complex K-DNA network. Until further results have explained the real organization of the K-DNA and the eventual relationship between the circular and "linear" units, it is difficult to understand the exact mechanism by which these "lampbrushes" are formed. These investigations were undertaken during the tenure of a Research Training Fellowship awarded by the International Agency for Research on Cancer to one of us (Ch. B.). REFERENCES

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