Rapid digestion of Staphylococcus aureus by Paramecium: An evaluation of the role of bacterial autolytic enzymes

Europ.J. Protistol. 26, 103-109 (1990) October 19, 1990

European Journal of

PROTISTOLOGY

Rapid Digestion of Staphylococcus aureus by Paramecium: An Evaluation of the Role of Bacterial Autolytic Enzymes IIka Mehlis and Klaus Hausmann Division of Protozoology, Institute of Zoology, Free University of Berlin, Berlin (West)

Jorg Wecke Robert Koch-Institute of the Federal Health Office, Berlin (West)

SUMMARY Paramecium fed exclusively with the human pathogenic bacterium Staphylococcus aureus can survive over a period of at least 2 months and is able to use the bacteria as substrate. In this study, Paramecium was fed with S. aureus for 45 min and the process of digestion of these Gram-positive bacteria and their remains was analysed. The degradation starts with very slight damage to the outside of the bacterial cell wall. During digestion, first the cytoplasm and later the membranes are degraded. The cell wall material is gradually broken down at the same time. After 45 min of phagocytosis, defecation competent digestive vacuoles (DVs) are present. There is no difference in the digestive process using bacterial cells from the logarithmic or stationary phase of growth. Cells with abnormally thick cell walls induced by treatment with erythromycin were digested similarly. By using thermally inactivated S. aureus cells, we can exclude the possibility that bacterial autolysines are responsible for the rapid destruction and degradation of the procaryotic cell wall. The Paramecium cells needed longer to digest the coagulated cytoplasm of heat-treated cells.

Introduction

Paramecia are filter feeders living mainly on bacteria, but also flagellates and algae can be digested. The food particles are concentrated from the surrounding medium and become enclosed into digestive vacuoles (DVs). In the process of cyclosis the DVs undergo a period of maturation during which digestion takes place. During this period structural and physiological changes of the DVs and their contents allow 4 stages to be identified [1]. The duration of the cyclosis varies in Paramecium from 15 min up to 2 or 3 h, depending on the kind of the food and the nutritional condition [3]. Beside protists there are other cell systems which are capable of degrading bacteria, such as the macrophages of the mammalian immune system. These are relatively ineffective digestive systems especially in degrading bacterial cell walls of numerous pathogenic bacteria. The cell © 1990 by Gustav Fischer Verlag, Stuttgart

wall of the pathogenic bacterium S. aureus is rather resistant to the lytic potential of the macrophages. Consequently, undegraded wall material can be detected even after 4 days of digestion within phagolysosomes of macrophages [10]. We had previously noted that Paramecium could completely degrade S. aureus within an hour (unpubl. observation). We wish to establish if this is a reflection of the digestive competence of Paramecium or if Paramecium simply activates S. aureus autolytic enzymes. Two such enzymes are needed to effect cell division in S. aureus. Activation of autocatalytic enzymes has been proposed to explain the very rapid digestion of blue green algae by Pseudomicrothorax dubius [8]. The purpose of this study is to investigate the degradation of S. aureus within the digestive system of Paramecium, and to find out if the autolytic system of staphylococci may play an important role in wall breakdown within the lysosomes of Paramecium. 0932-4739/90/0026-0103$3.50/0

104 . Mehlis et al.

Material and Methods

Results

Paramecium was grown in the dark in 100 ml cultures of carbonate acid free mineral water (Fa. Volvic). Every two or three days a hay-suspension with various Gram-negative bacteria was added. S. aureus strain SG 511 from the culture collection of the Robert-Koch-Institute, Berlin, was kept on 1.5% peptone-agar plates. After being transferred to flasks containing an aqueous solution of 2.5% peptone with 0.5% NaCl the bacteria were incubated in a lab-shaker at For experiments on phagocytosis S. aureus was treated in four different ways: cells from the logarithmic phase of growth were harvested after 2 h cultivation with shaking; cells from the stationary phase of growth after 22 h; staphylococci from the stationary phase of growth were boiled in a water bath for half an hour to kill them; or finally cells were incubated with 1 ug/ml erythromycin (Schering AG, Berlin) for 18 h. Before ingestion studies, both Paramecium and S. aureus were washed and the culture fluid was replaced with Volvic-water. The phagocytosis lasted for 45 min. For electron microscopy, Paramecium was fixed in 2.5% (v/v) glutaraldehyde in 0.05 M Na-cacodylate buffer (pH 7.2) at 4°C for 60 min or overnight. After washing with buffer the material was postfixed in 1.5% (v/v) osmium tetroxide in 0.05 M Na-cacodylate buffer at room temperature for 60 min. After another brief wash the organisms were embedded in 2% agar with 0.8% NaCI and cut into 1 rnrn ' sized cubes. Then poststaining followed with 1% uranyl acetate (60 min, room temperature). After dehydration in an increased series of ethanol, the material was embedded in L. R. White and polymerized at 55°C for 24 h. Thin sections were made with a Reichert Ultracut E. Finally, the sectioned material was stained with lead citrate for 3-5 min and examined in a Zeiss EM 10 or a Philips EM 400.

3rc.

Paramecium has been grown for more than 10 weeks using exclusively S. aureus as food. There was no indication of any depression of growth or cellular abnormalities during this period of time. Staphylococci of the stationary phase of growth are characteristically rounded (Fig. 1). The cross wall divides the cell into two equal sized parts (Fig. 2). The cell wall is about 30 nm thick. After ingestion by Paramecium the bacteria are enclosed in digestive vacuoles (DV according to [1]) (Fig. 3). Within DV-I and DV-II the staphylococci are electron dense globular structures and the cytoplasm looks normal. Degradation occurs in DV-III (Fig. 4). The bacteria still keep their characteristic roundish form, but in many cells the cytoplasm has been partially digested and loses its electron density. Membranous structures and cell walls or wall fragments persist. In the late DV-III stage an active retrieval of lysosomal membrane takes place, and this may continue in DV-IV (Fig. 5). By this stage only occasional wall fragments are seen and these are in late stages of digestion. Digestion of S. aureus is complete before defecation. The detailed sequence of degradation of S. aureus can be characterized as follows. Very slight etching of the outside surface of the cell wall is visible in DV-II (Fig. 6). All other components of the bacteria remain intact. With the onset of the enzymatic digestion in DV-III first the cytoplasm is lysed. Sometimes the two halves of S. aureus are in different states of degradation (Fig. 7). At this stage, the

Figs. 1-3. Staphylococcus aureus of the stationary phase of growth. - Fig. 1. S. aureus. - Fig. 2. Higher magnification of S. aureus showing an entirely closed cross wall (arrows). - Fig. 3. Food vacuoles of Paramecium filled with S. aureus and single rodlike Gram-negative bacteria (arrows). - Scale bar in Figs. 1 + 3 = 111m, in Fig. 2 = 0.2 11m.

Bacterial Digestion by Paramecium . 105

Figs. 4-12. Digestion stages of S. aureus in the logarithmic and the stationary pha se of growth. In these and the follow ing figures the classification of the food vacuolar stages is according to Allen & Staehelin [1]. - Fig. 4. DV-III. Remarkable changes in the app earance of staphylococci can be observed. Most bacteria reveal a degrad ation of cytoplasm but the cell wall is still preserved. - Fig. 5. DV-IV. Except for the cell wall remnants in the upper right corner of the food vacuole (arrow) all staphylococci are digested. In the vicinity of the vacuole small vesicles can be detected (arrowheads). - Fig. 6. Section of S. aureus from DV-II. Th e cytoplasm is unchanged whereas the cell wall shows an irregular surface (arrowheads). - Figs. 7- 12. S. aureus from DV-III. - Fig. 7. In parts the cell wall is dist inctly degraded. The cross wall segregates the bacterium into an electr on dense and an electron lucent part. The plasma membrane can be detected (arrowheads ). - Fig. 8. Plasma membrane and cell wall persist while the cytopla sm is digested. In the center there is a mesosome (M) . - Fig. 9. Several breaks in the cell wall can be seen (arrows). Inside the cell remnants of the plasma membr ane are still left (arrowhead). - Fig. 10. Th e peripheral cell wall is digested whereas the cross walls still persist. Note the gap between the cross walls. - Fig. 11. Remnants of the bacterial membranes (arrows). - Fig. 12. Cell wall fragments with the starting areas of the cross wall (arrow). - Scale bar in Figs. 4 + 5 = 1 urn, in Figs. 7- 12 = 0.2 urn,

106 . Mehlis et al.

Figs. 13-20. S. aureus after 18 h treatment with erythro mycin. - Fig. 13. S. aureus before ingestion. Huge amounts of wall material have been synthesized due to the dru g treatment. The bacterial surface is irregularly shaped. At the periphery a few nm thin electron dense primary wall was built witho ut the influence of erythromycin while the inner electron lucent layer was synthesized during the antibiotic tr eatm ent. - Fig. 14. Section of DV-IlI. Undigested bacteria and bacteria with pa rtly digested cytoplasm and wa ll mater ial are simultaneo usly present. - Fig. 15. S. aureus in DV-Il. The partial disintegration of the underlying erythro mycin wa ll has taken place (arrows). - Figs. 16-20. Different stages of degradation to S. aureus in DV-II1. - Fig. 16. The wall material on the left side (arrowheads) is about 130 nm thick, whereas the rest measure s 70 nm. The cytoplasm of the cell is still undigested. - Fig. 17. The cytoplasm of the cell is degrad ed. Wall disintegration from th e outside can be detected. - Fig. 18. Beside the destru ction from the outside (arrowheads) a doubl e layer structure becomes obvious. Th e two wall layers show the same electron density. Cytoplasm and most of the membrane materi al have been disintegraded. - Fig. 19. Wall material from the periphery is degraded first. Only little degradat ion has taken place at the cross wall. - Fig. 20. Fluffy material is surrounded by wa ll fragments. The thickness of th e bacterial wall is highly reduced; only in one place the wall is about 80 nm thick. - Scale bar in Fig. 14 = 1 urn, in Figs. 13, 15-20 = 0.2 urn.

Bacterial Digestion by Paramecium : 107

:

•

Figs. 2 1- 29. S. aureus thermally inacti vated. - Fig. 21. S. aureus before ingestion. - Fig. 22 . Lipids are con centrated in one place and translocated most probably durin g sectioning (asterisk). - Fig. 23. Section of DV-II. Th e vacuole is highly condensed. Berween the bacteria fluffy material is visible. - Fig. 24. S. aureus from DV-III. Th e cell wall has a slightly irregular shape, the cytop lasm is intact. Fig. 25 . DV-III. N umero us bacteria are seen in the process of being digested. - Fig. 26 . S. aureus from DV-III. Th e outer wall is distinctly attacked (arrows) and a gap appears between the two halves of the cross wa ll. Lar ge pa rts of the coagulated cytop lasm persist, others have been degraded. - Fig. 27. Sectio n of DV-III, late phase, conta ining coag ulated cytoplasm and only few wall fragments (arrow heads). - Fig. 28. S. aureus from DV-III, late phase. Coa gulated cytopl asm and wall fragments still persist. - Fig. 29 . Bacteria-like shaped coagulated cytopla sm with out remn ants ofthe cell wall. Scale bar in Figs. 21, 23, 25, 27 = 1 urn, in Figs. 22, 24, 26, 28, 29 = 0.2 urn.

surfa ce of the cell becomes irregularly shap ed and deform ed. As the process continues, wall materi al and remnants of membranes (e.g., the mesosome in the center of the cell) persist while the cytopl asm is digested (Fig. 8). After furth er degrad ation, only the remains of wall mat erial are detectable (Fig. 9).

Experiments using staph ylococci from the logarithmic pha se of growth have shown that cross walls persist longer than periph eral wall material (Fig. 10). However, even these walls are gone in late DV-I1I vacuoles and these cont ain onl y a few membr anou s structures (Fig. 11) and small fragment s of wall materia l (Fig. 12). All these

108 . Mehlis et al,

st ructures, however, are digested when the vacuole changes to DV-IV, the defecation stage. The wall of S. aureus is con siderably thick er after treatment with high do ses of erythromycin (10 ug/rnl) (Fig. 13). A double layer structure is visible within the wall. Th e thin primary wall built befor e erythromycin treatment differs from the thick secondary w all by its high er electron density. Digestion of th ese bacte ria resembles that of untreated log-ph ase S. aureus, with the first signs of degradation occurring in DV-II vacuoles and the process largel y complete in DV-III vacuoles. Initial wall damage is to its outer face (Fig. 15) before any changes occur in the cytoplasm (Fig. 16 ). The surface of the wall is irregularly shaped and a lysed gap within the secondary wall is visible, while the cytoplasm of the bacteria in DV-II seems to be intact (Fig. 15 ). In DV-III first of all the primary wall , whose degradation is clearly initiated from outside, becomes digested, whil e the cytoplasm is not yet attacked (Fig. 16 ). Whereas the degradation of the per iph eral wall continues, the digestion of the cytoplasm starts (Fig. 17). Some cells can be dete cted whose cytoplasm is to tally degraded ; the double wa ll, with a gap between the primary and the secondary wall, persists (Fig. 18). After further digestion, large fragments of the primary wall ma y be seen surro unding a th in layer of secondary wall material (Fig. 19). Before all remn ants of S. aureus are completely digested , thin fragm ent s of the secondary wall with little portions of primary wall material are visible (Fig. 20) . In order to establish if bacteri al autolytic enzymes are used in the degradation process, we studied the degradation of heatinactivat ed sta phylococci. The heat tre atment lead s to formation of lipid droplets and aggregates of coagulated cytoplasm in the sta phylococci (Fig. 21). In some bacteria the lipids seem to permeate the cross wall but we think that this is most probably a sectioning arti fact (Fig. 22). Staphylococci in DV-II, the most condensed vacuol ar sta ge, are embedded in a fluffy material (Fig. 23 ). The walls are slightly irregular (Fig. 24 ). Enzymatic degr adation takes place in DV-III (Fig. 25 ). Firstly the lipid s disappear and th e periph eral wall is attacked fro m the outside, while large parts of the coagulated cytopl asm persist. Gaps become visible in the cro ss wall s of dividing cells (Fig. 26 ). Late ph ase DV-III vacuoles contain bacteria-shaped remnants of coagulated cytoplasm and few wall fragments (Figs. 27-29). Discussion Th e experiments revealed that the Paramecium digestive system is very effective compared with that of verte bra te macrophages in taking 45 min to complete the process o f digestion of S. aureus. Ma cropha ges ma y fail to effect digestion even after several da ys. There are no basic differences in degr ad ation between these Gram-positive bacteria from the logarithmic and from the station ar y phase of growth. In all cases, the first changes in the

bacteria start in DV-II with very faint irre gularities o f the peripheral bacterial cell wa ll. Thi s may result from th e rapid decrease of the vacuo lar pH from 7 to 3 which take s place within the first 5 min after the DV is pinched off from th e cytopharyngeal membrane [1,4]. Fusion wi th lysosomes cause the DV-II to change to the DV-III stage. Th e DV-II is characterized by an increase in size and internal pH and is the only stage to contain active acid phosph atase [5,3]. In DV-III the cytoplasm is digested first, then th e membranes, while remn ants of the per ipheral wall mat erial of S. aureus persist. Membrane fragments and cell wall remnants may be found in the same DV-III vacu oles. Using bacterial cells from the logarithmic phase of growth, the cross wall may be seen to persist after the peripheral wall has been degraded. This phenomenon could po ssibly be due to different chemic al wall qualities or its location may render it inaccessible to digestive enzymes. In the late phase of DV-III a gap between th e tw o halves of the cross wall is visible. In fact, this is the plane where cell separation takes place. For the autolytic process during cell separ ation two bacterial enzyme systems ar e necessary: Th e cutting-system, which starts the cutting-thro ugh of the primar y wall above the new cross wall, and the splitting-system , which is responsible for splitti ng within the cro ss wa ll [6]. Since the primar y wa ll above the cro ss wa ll appear s completely intact, on e could conclude that the enzy mes of the splitt ing system, not th e enzymes of the cutting system, were acti ve. Thi s is not very likely. A gap in the cro ss wall occurs in cells whose cyto plasm and also peripheral walls are for the most part degraded. However, the cross walls themselves reveal smoo th sur faces. There is no evidence of degradation of the external pr ima ry wall nor of th e cro ss wall. We conclude that thi s separa tion does not reflect wall digestion, but rather passive separation following the loss of the structural integrity of the cell. The erythromycin treatment of the staphylococci caus es an abnormally thick cell wall in which two different layers were distinguished. Th e inner wall is characterized by an increased number of Ovacetyl groups on the Cg-atom of the muramic acid [2]. The increased number of O-acetyl groups provides resistanc e to digestion by macrophages

[7,9] . In contrast to the situa tio n in macrophages, erythro mycin-treated bact eria co uld be completely degr ad ed by Paramecium within 45 min. Th at the primar y and seco ndar y layers ofthe cell wa ll are dissolved differently prob abl y ind icate s different wall qualities and the secondary wa ll with its higher degree of Ovacerylation are mor e resistant to the degradation system of Paramecium. Th e experiment with heat -tr eat ed S. aureus sho ws that digestion is achieved by th e Paramecium as th e pr ocess occ urs unh indered in the bacteri a. The bacterial cell wa lls are destructed from the outside. Th e coagulated cytoplasm is relatively resistant to enzy matic degradation and reta ins its sha pe despite the lack of a peptidoglycan layer. 45 min after phagocytosis, the digestion of the coagulat ed cyto pla sm is not yet completed. The experiments show th at the autolytic wall system of S. aureus does not play an important role in wall disinre-

Bacterial Digestion by Paramecium . 109

grati on processes within the lysosomes of Parame cium. We may therefor e conclude that the Param ecium digestive system is a very potent one when compared to macroph ages but the basis of thi s potency is unknown. Acknowledgements Th e authors wish to th ank Mrs R. Hahmann and Mrs C. H an for excellent techn ical assistance. Th is study was suppo rted by the Deut sche Forschungsgemeinscha ft, Bonn - Bad God esberg, FRG.

References Allen R. D. and Sraehclin L. A. (1981): Digestive system membra nes: freeze-fracture evidence for differenti ati on and flow in Param ecium. J. Cell. BioI., 89, 9-20. 2 Burghau s P., Johann sen L., N aum ann D., Labischinski H. , Bradaczek H. and Giesbrecht P. (1983 ): The influen ce of different antibiotics on th e degree of O-acetylation o f sta phylococcal cell walls. In: Hakenbeck R., Holt je J.-V. and Labischinski H. (eds.): The target of penicillin, pp. 317-322. Walter de Gruyter, Berlin. 3 Fok A. K. and Allen R. D. (1988 ): Th e lysosomal system. In: Gortz H. D. (ed.): Paramecium , pp. 30 1-324. Springer Verlag, Berlin-Heidelberg.

4 Fok A. K., Lee Y. and Allen R. D. (1982): Th e corr elation of digestive vac uo le pH and size with the digestive cycle in Paramecium caudatum . J. Protozool., 29, 404 -4 14. 5 Fok A. K., M ur aoka J. H. and Allen R. D. (1984): Acid phosphatase in th e digestive vacuoles and lysosornes of Paramecium caudat um: a time study. J. Protozool., 31, 2 16- 22 0. 6 Giesbrecht P. and Wecke J. (1980): On th e structu re and function of autolytic wall system in Gram positive bacteria. Proc. 7th Eur. Congr. EM, Th e Hague, 2, 44 6-453. 7 Giesbrecht P. und Wecke J. (1987): Z ur Abba ubar keit bakteri eller Zcllwa nde und ihre Bedeutu ng fur die Induktion chro nisch-entzundlicher Prozesse. In: Holzmann H . (Hrsg.): Dermatol ogie und Rheuma, S. 78- 88. Spr inger Verlag, Berlin- Heidelberg. 8 Peck R. K. and Hausmann K. (1980) : Prim ar y lysosomes of the ciliate Pseudomicrothorax du bius: cytochemical identification and role in ph agocyto sis. J. Protozool., 27 (4), 401-409. 9 Wecke J. , Lah av M., Bliimel P. and Giesbrecht P. (1989 ): Reduced wa ll degr ad ation of staphylococci after pretreatment with bacteriostatic antibiotics. In: Peters G. an d Pulverer G. (eds.): Influ ence of antibiotics on the host-parasite relationship, pp . 63 -70. Springer Verlag, Berlin-Heidelberg-New York. 10 Wecke J. , Lah av M., Ginsburg 1., Kwa E. and Giesbrecht P. (1986): Inhibition of wall aut olysis of sta phylococci by sodium po lyanethole sulfona te " liquo id" . Arch. Mi crobiol., 144, 110-11 5.

Key w ords: Bacterial cell wall degradation - Bacteri al autolysines - Erythromycin treatm ent Thermally inactivated bacteria - Paramecium Klau s Hausmann , lnstitut fur Zoologie, Freie Universita r Berlin, Kon igin-Luise-Str . 1-3, 1000 Berlin 33 (West), FRG