A Lectin-like Molecule is Discharged from Mucocysts of Tetrahymena pyriformis in the Presence of...

J. Euk. Microbrol., 44(S), 1997 pp. 487-491 0 1997 by the Society of Protozoologists

A Lectin-like Molecule is Discharged from Mucocysts of Tetrahymena pyriformis in the Presence of Insulin

PETER KOVACS,* WERNER E. G. MULLER,~.** AND GYORGY CSABA* *Department of Biology, Semmelweis University of Medicine, Nagyva'rad tPr 4, H-1445 Budapest, Hungary, and

**Institut f i r Physiologische Chemie, Abteilung Angewandre Molekularbiologie. Gutenberg Universitat, Duesbergweg 6, 0-55099 Mainz, Germany

ABSTRACT. By use of a monoclonal antibody directed against purified lectin from the sponge Geodia cydonium it was demonstrated that the mucocysts of Tetrahymena pyriformis contain a substance immunologically similar to that found in G. cydonium. In extracts of T. pyrijiormis the monoclonal antibody recognizes a 36 kDa protein; binding could be abolished by adsorption of the antibody with (i) crude extract, (ii) purified lectin from G. cydonium and (iii) a 29 aa long peptide. In addition the data show that M of insulin causes first the release of mucocyst material, which reacts with the lectin antibody, and second its subsequent redistribution on the surface of the somatic cilia and the oral field.

Supplementary key words. Evolution, insulin, lectin, mucocyst discharge, receptors, Tetrahymena pyrifomis.

ECEPTORS are cell-surface molecules that recognize spe- R cific stimuli from the extracellular environment. They either comprise within their molecule a signal-amplification system or they are coupled noncovalently with a separate ampli- fying molecule. By those effector systems, receptors initiate signal transduction pathways, ultimately changing cell behavior and/or metabolism. In Metazoa the tuned and controlled inter- action among cells, and cells and extracellular molecules is essential for the development of an organism and the maintenance of the homeostasis in the cell metabolism. Hence, adhesion molecules and growth hormones, and their corresponding receptors are key molecules which had to be developed during evolution of unicellular eukaryotes to multicellular animals

In Metazoa an example for a hormone receptor molecule with an intrinsic amplifier is the insulin (tyrosine kinase) receptor; its existence was discovered already in the lowest metazoan phylum, the sponges (Porifera); it was cloned from Geodia cydonium [36] and found to carry in its extracellular part an im- munoglobulin-like domain [35]. Most hormones in Metazoa in- teract with seven-transmembrane receptor molecules which transmit the ligand (hormone)-caused conformational change to G-proteins, resulting in a modulation of ion channels or membrane-associated enzymes, e.g. adenylate cyclases or protein kinases C [38]. The prototype for this signal transduction was discovered in the sponges by cloning genes coding for serine kinases [18].

Ancestral adhesion molecules of Metazoa were also identified in sponges [33]; cloning and analysis of one adhesion molecule, an S-type lectin or galectin revealed high homology in its binding site molecule to those of higher vertebrates. Addi- tional data revealed that the divergence of the sponge lectin from those of other Metazoa occurred approximately 800 mil- lion years ago [30].

In T. pyriformis the presence of adhesion molecules seems to be meaningless, considering the unicellular life-form. Nev- ertheless, on the surface of cells from T. pyriformis such molecules could have a role different from that inherent in receptors from metazoans, e.g. in trapping specific molecules from the extracellular milieu which are required by the cell [17].

Also for Protozoa adhesion receptors [for review, see 241, and hormone receptor-like molecules [for review, see 5 , 7, 8, 161 have been described. An example for adhesion molecules in Trypanosoma cruzi a 60 kDa nonglycosylated heparan sulfate binding protein, penetrin, has a role in cell adhesion by binding to components of the extracellular matrix [12]. In T.

~ 7 1 .

' To whom correspondence should be addressed. Telephone: 6131- 395910; Fax: 6131-395243.

pyriformis many hormone receptors are present or can be pro- voked by imprinting [7, 81 and molecular biological evidence demonstrates their similarities to vertebrate respective receptors [44]. Furthermore, it has been reported that T. pyriformis syn- thesizes and releases insulin-like substances [22]. Taken togeth- er, these data indicate that Protozoa in general and Tetrahymena in particular are provided with structural elementseither ac- quired during the process of hormonal imprinting [7] or by horizontal gene transfer [42]-to respond immediately to sig- nals from the environment.

In T. pyriformis insulin not only influences sugar metabolism [9], but also regulates the chemotactic behavior [14] and the locomotion pattern [25]. In the present study we applied antibodies, directed against one adhesion molecule from the sponge G. cydonium, the lectin [29, 331. Monoclonal antibodies were used to minimize potential nonspecific cross-reactivity. We could demonstrate the presence of a lectin-like molecule in T. pyriformis mucocysts. Furthermore, we show that after incubation of T. pyriformis with insulin, the content of the mucocysts which reacts with the antibody against the sponge lectin, is released and distributed in an organized manner on the surface of somatic cilia of the organism.

MATERIALS AND METHODS Bacto tryptone medium and yeast extract were obtained from

Difco, MI; mouse serum, goat serum and secondary antibodies were obtained from Sigma, Deisenhofen, Germany; PVDF Im- mobilon from Millipore, Saint-Quentin, France and insulin Semilente MC, from Novo, Copenhagen, Denmark.

Cells and extract. Tetrahymena pyriformis GL strain was grown axenically in 0.1% yeast extract, containing 1% tryptone medium at 28" C [15]. Populations growing in the logarithmic phase were used for the experiments.

Extracts from T. pyriformis were prepared with a 50 mM Tris-HC1 buffer (pH 7.5; 2 mM EDTA, 100 mM NaCl and 0.2 mM phyenylmethanesulfonyl fluoride); 5 X lo6 cells were ex- tracted with 0.5 ml of buffer. After sonication the extract was centrifuged (15,000 X g; 10 min; 4" C) and the supernatant was collected.

Insulin treatment. Tetrahymena pyriformis was treated with M insulin for 60 min. Then the cells were washed with

fresh culture medium and resuspended to a density of approximately lo6 cells/ml.

Antibodies. Monoclonal antibodies [McAbs] directed against purified sponge lectin were prepared in female BALB/c AnHan mice as described [l]. After three injections, spleen cells were prepared and fused with P3X63-Ag 8.653 cells. The McAb against the lectin was selected by western blotting technique; for the present studies anti-lectin Ab-L93 was used.

In control studies 10 p1 of the anti-lectin Ab-L93 was ad-

487

488 J. EUK. MICROBIOL., VOL. 44, NO. 5, SEPTEMBER-OCTOBER 1997

sorbed with (i) 20 pg/ml of crude extract (membrane fraction) from G. cydonium [28], (ii) 5 p g / d of purified lectin from G. cydonium, or (iii) 10 pg/ml of the peptide HPKLDY- DKVRHITCKGLEHAVLEAIGVTY for 2 h at 4" C prior to the application for western blotting. This peptide was selected by analysis of the deduced aa sequence of the cDNA, coding for the G. cydonium lectin (EMBL accession no. X 93925).

Homology searches to select the peptide were performed via the Email servers at the European Bioinformatics Institute, Hinxton Hall, UK ([email protected]). The peptide was syn- thesized using an automated solid-phase peptide synthesizer.

Binding studies with anti-lectin antibody. Tetrahymena cells were fixed with 4% paraformaldehyde, containing phosphate-buffered saline [PBS] (pH 7.2) for 5 min. Then the cells were washed twice with washing buffer (0.1% bovine serum albumin and 0.059'0 Tween-20 in PBS), and subsequently concentrated by gentle centrifugation. After treatment with 1 ml of blocking buffer (1% mouse serum in PBS) for 30 min aliquots of 50 pl were incubated with 50 p1 anti-lectin Ab-L93 (dilution 1:lOO) for 60 min at room temperature. After incubation the samples were washed twice with washing buffer, and twice with 0.1% goat serum containing PBS. Finally the cells were incubated with FITC-labeled secondary antibody (goat anti-mouse IgG) for 30 min, and subsequently washed four times in washing buffer. Each experiment was repeated at least five times; the results of all of them are identical.

Photographs were taken (i) with an Olympus AHBT3 microscope, outfitted with a xenon fluorescent lamp, using a lOOX PlanApo objective, or (ii) with a confocal laser scanning microscope LSMlO [Zeiss] as described [37].

Western blotting. Gel electrophoresis of the protein extracts was performed in 15% polyacrylamide gels containing 0.1% NaDodSO, (PAGE) according to Laemmli [20]. Protein extracts from T. pyrifonnis were subjected to gel electrophoresis. Semi- dry electrotransfer was performed according to Kyhse-Ander- sen [ 191 onto PVDF-Immobilon. Membranes were processed [ l ] and incubated with McAb anti-lectin Ab-L93 (diluted 1: 500) for 90 min at room temperature. After blocking the membranes with 5% bovine serum albumin, the immune complexes were visualized by incubation with anti-mouse IgG (biotin con- jugated), followed by staining with streptavidin-alkaline phos- phatase conjugate/4-chloro- 1-naphthol.

Further analytical procedure. For protein determination the Fluoram method was used [41]; the standard was bovine serum albumin.

RESULTS

Identification of mucocyst material with monoclonal antibodies against G. cydonium lectin. Extract of T. pyrifonnis was subjected to gel electrophoresis followed by blot transfer. As shown in Fig. 1 (lane a) the anti-lectin Ab-L93 recognized one protein species in the extract with a M, of 36 kDa. Binding of the anti-lectin Ab-L93 to the 36 kDa-protein was almost completely suppressed after adsorption of the antibody with crude extract from G. cydonium (lane b) or with purified G. cydonium lectin (lane d).

As a further proof that the antibody recognizes specifically a component of T. pyrifomis which is related to the sponge lectin, homology searches using the deduced aa sequence of the cDNA coding for G. cydonium lectin [33] have been performed via the Email servers at the European Bioinformatics Institute, Hinxton Hall. As outlined in the discussion a peptide, corresponding to the sequence HPKLDYDKVRHITCKGLEHAV- LEAIGVTY, was chosen for the neutralization studies with the anti-lectin Ab-L93. It was found that after absorption the anti-

a b c d kDa

- 55.6

- 39.2

- 26.6 - 20.1

- + + + ce p lec

ad

Fig. 1. Identification of protein in the crude extract from T. pyriformis reacting with the anti-lectin Ab-L93. Protein extract (5 p g of protein per lane) was size-separated by PAGE using a 15% polyacrylamide gel; it was subsequently transferred to Immobilon sheets. Lane a: treatment with nonadsorbed (-ad) McAb anti-lectin Ab-L93. In separate experiments the blots were treated with anti-lectin Ab-L93 which had been pretreated with (+ad) crude extract from G. cydonium (ce; lane b), the peptide HPKLDYDKVRHITCKGLEHAVLEAIGVTY (p; lane c) or with purified G. cydonium lectin (lec; lane d). The immu- nocomplexes have been visualized by labelled secondary antibodies.

body reacted only slightly with the 36 kDa peptide (Fig. 1, lane c).

Induction of release and distribution of mucocyst material after insulin treatment. By use of G. cydonium anti-lectin antibody, anti-lectin Ab-L93, the mucocysts of T. pyrifomis showed a very bright punctuated fluorescence (Fig. 2A, B). The other cellular components, such as cilia and basal bodies gave only a background fluorescence. In a control experiment, the primary antibody [anti-lectin antibody] was omitted. Under this condition the secondary antibody failed to detect any structure of the cells (data not shown).

M insulin a drastic change of the staining pattern with this antibody was detectable. The cilia and the oral (cytopharingeal) area showed a very strong fluorescence (Fig. 2C, D).

The mucocyst material, which reacted with the antibody, was successfully identified also by confocal laser scanning micro- scopical analysis using the anti-lectin Ab-L93. In nontreated cells the material is located in punctuated pattern at the rim of the cells in the mucocysts (Fig. 3A). In a control experiment the G. cydonium antibody was adsorbed with G. cydonium crude extract. Under these conditions only background fluorescence could be visualized. (Fig. 3B). The same result-no staining of cell structures-was found when the antibody was adsorbed with purified lectin (data not shown). It should be stressed that the crude sponge extract contains >50% of lectin 1311.

However, after treatment of the cells for 60 min with

DISCUSSION In the present study it is demonstrated that the anti-lectin

Ab-L93 is bound to mucocysts of T. pyriformis, implying that this protozoa is provided with a protein in these organelles,

KOVACS, MULLER & CSABA-SPONGE LECTIN-LIKE MOLECULE IN TETRAHYMENA PYRZFORMZS 489

Fig. 2. Effect of insulin on the distribution of the mucocyst material in T. pyriformis. The cells were incubated with insulin and after fixa- tion, treated with the anti-lectin Ab-L93 as described under Materials and Methods. A and B. Cells not treated with insulin; the focus has been adjusted to the plain of the cells (A), or to the surface (B). C and D. Cells treated with insulin. The arrow points to the oral (cytopharingeal) area. Magnifications: A, X850; B, X950; C, X1,700; and B, X1.000.

which reacts immunologically like the G. cydonium lectin. Western blotting studies revealed that the anti-lectin Ab-L93 recognizes a 36-kDa protein. In contrast, this antibody recognizes in G. cydonium lectin(s) of M, 13 to 18 kDa [31]. The binding of the anti-lectin Ab-L93 to the 36-kDa protein from T. pyrifomis is specific and can be prevented by adsorbing the antibody both to crude extract and purified lectin from G. cydonium. It is established that the sponge lectin belongs to the class of metazoan galactose-specific lectins, the galectins, which contain besides =15 kDa large proteins also molecules of sizes 32-36 kDa [13]. Therefore, we can postulate that galectin-like molecules exist in T. pyrifomis as well.

The finding, that the anti-lectin Ab-L93 which was raised against a sponge protein binds to a peptide from T. pyrifomis, was unexpected. Until now, a lectin from T. pyriformis has not yet been cloned. Therefore, we had to search for a peptide be present both in the sponge lectin and in protozoan polypeptides. Comparison of the deduced aa sequence from the cDNA of the

sponge lectin with other sequences from the data banks revealed that the peptide of 29 aa, HPKLDYDKVRHITCKGLEHAV- LEAIGVTY, shares especially between aa P and V (these aa are marked) high homology to a series of binding proteins, as found e.g. in the bacterium Neisseria meningitidis (transfemn- binding protein) [23], in Plasmodium falciparum (integral membrane protein Al ) [32] and in human (leukocyte adhesion glyoprotein) [2 11. To neutralize monoclonal antibodies, aa stretches of 10- to 15-aa residues are known to be sufficient [4]. The experiments revealed that this peptide of 29 aa in length abolished binding of the antibody t o the T. pyriformis 36-kDa protein.

The G. cydonium lectin-like material was discharged from the mucocysts under the influence of insulin and was subsequently distributed on the somatic cilia and on the oral field. These results indicate, that (i) insulin can influence mucocyst extrusion, and (ii) the adhesion molecule-like substance can be translocated to the cell surface.

490 J. EUK. MICROBIOL., VOL. 44, NO. 5 , SEPTEMBER-OCTOBER 1997

Fig. 3. A. Immunolocalization of lectin-like material in mucocysts of T. pyriformis using the anti-lectin Ab-L93. B. Reaction of the anti-lectin Ab-L93 which had been adsorbed with membranes from G. cvdonium. The cells have been inspected using laser scanning microscopy. Magnifi- cation: X850.

The function of the extrudable mucocyst material is not fully understood. It can be assumed that the material released from mucocysts facilitates (i) food uptake (in molecular or particle form), (ii) recognition of signal molecules, or (iii) defense against harmful environmental changes (e.g. osmotic, pH or toxic changes) [ l l ] . Furthermore, it has been described that bacteria-fed Tetrahymena express at their surface and excrete into the medium a glycoconjugate which is absent in axenically grown cells [Z]. This observation indicates that the surface- adsorbed extrusome (mucocyst, trychocyst) material might promote the uptake of food, or avoid the toxic effect of bacterial molecules. Insulin treatment of Tetrahymena populations-and especially after the second treatment, after imprinting-resulted in a higher growth rate [6]. Considering these data and the fact that the mucocyst material is released and subsequently adsorbed onto the surface of insulin treated cells, it can be assumed that this released mucocyst material promotes food uptake.

Growth promoting substances are secreted into the culture medium by the ciliate Paramecium [39] and by Tetrahymena [3]. Presumably, these growth-factor like substances act as autocrine proliferation factors-in a receptor-like m a n n e r d n the surface of these protozoa. Similarly, insulin also displays growth factor-like effects in T. pyriformis [6]. It can be sup- posed, that the surface coat originating from the mucocysts promotes the binding of these factors onto the cell membrane.

Several lysosomal acidic hydrolases of Tetrahymena were found to be released into the culture medium [26]. This process of secretion is affected e.g. by addition of nutritive substrates [34]. Hydrolytic activities have also been observed on the surface of Tetrahymena [43]. These enzymes are glycoproteins [401; hence it can be suggested that the lectin-like material from mucocysts binds to these molecules. Previously it has been described that the secreted enzymes increase the phagocytic activity of T. pyrifornzis, alter lectin binding capacity and promote imprinting in response to insulin [ 171.

Tetrahymena cells synthesize, contain and secrete insulin-like molecules 1223. The secretion of this hormone seems to be meaningless on the first view, considering the aqueous milieu around this protozoan and the high dilution of the hormone in

this medium. However, Tetrahymena is provided with receptors, which can be activated by low concentrations of hormones; e.g.

M of insulin, are enough to induce response [6, 141; after imprinting even lower concentrations are required [ 101. Based on these data it can be postulated that insulin-like material, produced by T. pyriyormis, is able to induce extrusion in mucocysts in an intracrine or autocrine manner.

ACKNOWLEDGMENTS Supported by grants from the Deutsche Forschungsgemein-

schaft [348/12- 11, the International Human Frontier Science Program [RG-333/96-M] and the National Research Fund (OTKA) [T-0133551, Hungary.

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Received 4-11-96, 10-16-96, 4-2-97; accepted 6-2-97

1. Euk. MicrobroL. 44(5). 1997 pp. 492-496 0 1997 by the Society of Protozoologists

Detection of Penicillin-binding Proteins in the Endosymbiont of the Trypanosomatid Crithidia deanei

MARIA CRISTINA M. MOTTA,* LUIS HENRIQUE M. LEAL,*l** WANDERLEY DE SOUZA,*.***.' DARCY F. DE ALMEIDA**** and LUIS CARLOS S. FERREIRA****

*Laboratdrio de Ultraestrutura Celular Hertha Meyer, Instituto de Biofisica Carlos Chagas Filho, Universidade Federal d o Rio de Janeiro - CCS, Rio de Janeiro, RJ, 21941-590, B r a d

**Departamento de Histologia e Embriologia. Insrituto de Biologia, Universidade Estadual do Rio de Janeiro, Av. Manoel de Abreu, 48 - Maracana-, Rio de Janeiro, RJ, 20550-170, Brasil

* * * Laboratdrio de Biologia Celular e Tecidual. Centro de BiociCncias e Biotecnologia, Universidade Estadual do Norte Fluminense, Avenida Albert0 Lamego 2000, Campos, RJ, 28015-620, B r a d

****Laboratdrio de Fisiologia Celular, Instituto de Biojisica Carlos Chagas Filho, Universidade Federal do Rio de Janeiro - CCS, Rio de Janeiro, RJ, 21941-590, B r a d

ABSTRACT. Growth by serial transfers of the trypanosomatid Crithidia deanei in culture medium containing 1 mg/ml of the p-lactam antibiotics ampicillin or cephalexin resulted in shape distortion of its endosymbiont. The endosymbiont first appeared as filamentous structures with restricted areas of membrane damage. An increase of electron lucid areas was also observed in the endosymbiont matrix. The continuous treatment with p-lactam antibiotics, resulted in endosymbiont membranes fragmentation; and later on the space previously occupied by the symbiont was identified as an electron lucid area in the host cytoplasm. The putative targets of p-lactam antibiotic were two membrane-bound penicillin-binding proteins (PBPs) detected in the Sarkosyl-soluble fraction of purified symbionts labeled with [3H]-benzylpenicillin. The apparent molecular weight of the proteins were 90 kDa (PBP1) and 45 kDa (PBP2). PBP2 represented 85% of the total PBP content in the membrane fraction of the endosymbionts. Competition experiments using the tested antibiotics and [3H]-benzylpenicillin showed that ampicillin and cephalexin have half saturating concentrations considerably higher than ['HI-benzylpenicillin and indicated that PBPl is the probable lethal target of the antibiotics tested. These results suggest that a physiologically active PBP is present in the cell envelope of C. deanei endosymbionts and may play important roles in the control of processes such as cell division and shape determination.

Supplementary key words. p-lactam antibiotics, electron microscopy analysis, ['H]-benzylpenicillin, peptidoglycan layer.

OME trypanosomatids of the Crithidia genus harbor self- S replicating endosymbionts which supply their host cells with essential nutrients as hemin, purines, amino acids and vi- tamins [2, 5, 16, 201. The endosymbionts divide synchronously with host cells and their presence result in morphological alterations of the protozoa [7]. The bacterial nature of the symbionts has been suggested by biochemical data based on the definition of biosynthetic pathways and composition of their ribosomes and DNA [6, 9, 12, 181. Other evidence of the prokaryotic origin of these structures is their susceptibility to chloramphenicol, an efficient curing agent [3, 151.

Ultrathin sections of trypanosomatids analyzed by transmission electron microscopy reveal that the endosymbiont presents a matrix with an electron-dense domain corresponding to the ribosomes in the cytoplasm and an electron lucid one, the nu- cleoid. The symbionts are surrounded by two unit membranes similar to the gram-negative bacterial cell envelope, with an outer membrane and an inner cytoplasmic membrane, but no evidence for a component equivalent to a cell wall or peptidoglycan layer could be obtained [3]. Several evidences indicate that an organized peptidoglycan layer does not exist in the cell envelope of the trypanosomatid endosymbionts: (i) no structure similar to a bacterium septum is formed during the division process [3]; (ii) isolated symbionts are sensitive to osmotic shock once isolated from the host cell [17]; and (iii) morphological alterations are rarely observed at high concentrations of penicillin [3, 151.

The peptidoglycan or murein layer, the major component of the bacterial cell wall, is a macromolecular structure located in between the cytoplasmic and the outer cell membrane [14, 19, 241. In Escherichia coli the peptidoglycan is composed by in- terconnected polysacharide strands and tetrapeptide side chains [ 101. The cross-linking of peptides from neighbor strands con- fers the mechanical strength that protects the cell against lysis. Moreover, the peptidoglycan layer has important roles in cell shape definition and cell division control [14, 191. Several an-

' To whom correspondence should be addressed.

tibiotics, including the p-lactams, exert their lethal action on bacterial cells by inhibition of essential steps of the peptidoglycan metabolism. The targets of p-lactam antibiotics are murein- synthesizing enzymes which, due to their ability to bind cova- lently these antibiotics, are called PBPs [8, 22, 23, 261.

In an attempt to evaluate the effects of p-lactam antibiotics on the stability and morphology of the trypanosomatid endosymbionts we observed that ampicillin and cephalexin bring about rather drastic morphological alterations in the C. deanei endosymbionts. Labeling experiments with [3H]-benzylpenicillin demonstrated that the symbionts possess PBPs in their membranes. These results suggest that PBPs might play important roles in the stability and/or synthesis of a cell-wall like component of the C. deanei endosymbiont.

MATERlALS AND METHODS Organisms and growth conditions. The C. deanei (ATCC

30255) strain used in this work was from our laboratory strain collection. An endosymbiont-free derivative of C. deanei was kindly supplied by Celuta Alviano (Microbiology Institute, Fed- eral University of Rio de Janeiro). C. deanei was grown for 1 day at 28" C in Warren's medium supplemented with 10% fetal bovine serum [25].

Ampicillin or cephalexin was added to the culture medium to a final concentration of 1 mg/ml, and the medium filtered through 0.22 Frn pore-size Millipore disposable filter before inoculation with the protozoan cells. New antibiotic-containing cultures were started every week with a 1% inoculum from a grown culture, incubated for one day at 28" C. Part of the culture was saved and kept at low temperature (4" C) for one week and, finally, used for another transfer. Such procedure was repeated for a total of 41 transfers.

Purification of the endosymbionts. C. deanei cultures (500 ml) were sedimented at 2,000 X g for 10 min, washed twice with 0.1 M phosphate buffer containing 0.25 M sucrose, pH 7.2, resuspended in 10 ml of the same buffer and submitted to a series of sonication pulses under cooling using a model W-380 ultrasonic disruptor (Heat Systems, Inc., Farmingdale, USA). Cell disruption was monitored using a phase contrast

492

MOTTA ET AL.-PENICILLIN-BINDING PROTEINS IN ENDOSYMBIONTS 493

Fig. 1. Effects of p-lactam antibiotics on the symbionts of C. dennei observed by electron microscopy. A shows a control cell cultivated in the absence of antibiotics (X34,650). B-F show C. deanei cells cultivated in the presence of ampicillin (1 mg/ml) in a progressive shape distortion process.

microscope and care was taken to avoid rupture of the endosymbionts. The whole-cell homogenate was sedimented at 270 X g for 10 min, and the pellet was discarded. The supernatant was centrifuged at 12,000 X g for 10 min at 4" C, the pellet was resuspended in 3 ml of sucrose-phosphate buffer, carefully layered (0.5 ml) on top of a four-step Percoll gradient (18%, 24%, 4570, and 53%) in 0. I M phosphate buffer containing 0.25 M sucrose, and centrifuged at 5,700 X g for 30 min at 4" C. The endosymbiont-enriched fraction, visible as a band at the 45-53% layer interface, was pooled, diluted in sucrose-phos-

phate buffer, centrifuged at 12,000 X g for 10 min at 4" C and finally resuspended in sucrose-phosphate buffer containing leupeptin and aprotinin at final concentrations of 1 kg/ml. The purity of the endosymbiont preparations was assessed by electron microscopy.

Electron microscopy analysis. Cells or endosymbiont-enriched fractions were washed twice (3,000 X g for 10 min) with 0.1 M cacodylate buffer, pH 7.2, fixed for 1 h in 2.5% glutar- aldehyde in the same buffer at room temperature, washed twice, post-fixed for 1 h in 1% OsO, in 0.1 M cacodylate buffer con-


k 6

.p

Fig. 2. A pure fraction of C. deanei endosymbionts obtained by fractionation in Percoll gradients. Amplification, X9.800.

taining 0.8% potassium ferrycianide, dehydrated in acetone and embedded in Epon. Ultrathin sections were stained with uranyl acetate and lead citrate and observed in a model 900 Zeiss transmission electron microscope.

Labeling of endosymbionts with [3H]-benzylpenicillin. The endosymbionts membrane fraction was obtained after sonication of the purified endosymbionts and centrifugation at 40,000 X g for 45 min at 4" C. The pellet was resuspended to a final protein concentration of approximately 2 mg/ml in 50 mh4 phosphate buffer, pH 7.2, containing 1 pg/ml of leupeptin and aprotinin. Membrane samples (20-40 pg total protein) were labeled as described [22] using 10 pg/ml of [3H]-benzylpenicillin for 20 min at 28" C. Labeling of trypanosomatids ho- mogenates was performed with 200-300 pg of the crude sonic extract following a similar procedure. Labeled samples were mixed with concentrated electrophoresis sample buffer, boiled for 3 min and loaded on polyacrylamide gels.

Affinity of endosymbiont PBP to the p-lactam antibiotics. Membrane samples (20 pg of protein) were labeled using 5 p1 of [3H]-benzylpenicillin (final concentrations of penicillin rang- ing from 0.3 1-20 pg/ml). Affinity of ampicillin and cephalexin to the endosymbiont PBP was determined by competition experiments as described [22].

Gel electrophoresis and detection of PBPs. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) was performed as described [22]. Fluorography was carried out with 0.5 M sodium salicylate containing 0.5% glycerol for 30 min at room temperature. Gels were dried and exposed to Ko- dak X-OMAT AR film at -80" C for two to five weeks. De- veloped films were analyzed in a Quick Scan Jr. densitometer (Helena Laboratories, Beaumont, USA) coupled to a Varian 4290 integrator (Varian, Walnut Creek, USA).

Chemicals. [3H]-benzylpenicillin (2 1 CVmmol) was pur- chased from Amersham. Ampicillin and cephalexin were from Bristol Laboratories and Eli Lilly and Co., respectively. Acry- lamide, sodium dodecyl sulphate and all other chemicals used in electrophoresis were from BioRad. Leupeptin, Aprotinin, Sarkosyl and Percoll were from Sigma.

RESULTS Morphological alterations of the C. deanei endosymbiont

in the presence of ampicillin and cephalexin. Electron microscopic analysis of C. deanei cells incubated in the presence of 1 mg/ml of ampicillin or cephalexin showed that endosymbionts suffer a progressive shape distortion process, leading to

PBP 1-

PBP2 4

1 2 3 4 M W (kDa)

- 90 - 45

Fig. 3. Identification of C. deanei endosymbiont PBPs with ['HI- benzylpenicillin. Whole cell extracts of C. deanei and of a syrnbiont- free strain were labeled with [3H]-benzylpeniciliin and applied on tracks 1 and 2, respectively. Endosymbiont-enriched fractions from wild type C. deanei and the symbiont-free derivative were collected from Percoll gradients and applied on tracks 3 and 4. Molecular weights and location of the PBPs are indicated on the sides of the figure.

the appearance of pleomorphic forms (Fig. 1A-E). Both antibiotics affected symbionts in a similar way, but alterations induced by ampicillin were detected earlier than those due to treatment with cephalexin. The symbionts first appeared more filamentous than usually observed (Fig. IB). Next, membranes seemed to have suffered some disruption at some portions of the endosymbiont envelope (Fig. lC, D, arrows). There was also an increase in the electron lucid areas in the matrix (Figs. IE, arrows). Later on, membranes fragments could be observed in the host cell cytoplasm (Fig. 1E arrows). This process seems to be completed when the space previously occupied by the symbiont is identified as an electron lucid area. The whole process was not synchronous and different shape distortions could be found simultaneously in the same sample.

A symbiont-free strain could never be obtained after treatment with these antibiotics. Transfer of the treated cultures to antibiotic-free medium resulted in the accumulation of cells with normal shaped symbionts (data not shown). Symbionts were never observed inside host cell vacuoles, as usually occurs during autolysis. The ultrastructure of nuclei, mitochondria or other organelles in antibiotic-treated protozoa was indistin- guishable from those observed in control cultures without antibiotics. Similar changes affecting the endosymbionts were observed in C. oncopelti treated with ampicillin or cephalexin (data not shown).

Isolation of C. deanei endosymbionts. Endosymbionts were purified after centrifugation in Percoll density gradients. The endosymbiont fraction was composed almost exclusively of well preserved endosymbionts, as evaluated by transmission electron microscopy (Fig. 2). Residual contamination was due mainly to membrane fragments and kinetoplasts.

Binding of [3H]-benzylpenicillin to the endosymbionts PBPs. Labeling with [3H]-benzylpenicillin revealed the presence of two PBPs with apparent molecular weights of 90 kDa (PBP1) and 45 kDa (PBP2) (Fig. 3). Both proteins were satu- rated at 5 pg/ml of [3H]-benzylpenicillin and could be detected at concentrations as low as 0.62 p g / d of radioactive penicillin (data not shown). At saturating concentrations of [3H]-benzylpenicillin, PBP2 represents 85-90% of the total radioactivity in the Sarkosyl-soluble fraction of the endosymbiont labeled membranes. No PBP was detected in whole-cell extracts or pooled

MOlTA ET AL.-PENICILLIN-BINDING PROTEINS IN ENDOSYMBIONTS

0 loa 10' lo2 lo3 o loo 10' lo2 lo3

f-- PBP 1 - PBP2

195

Fig. 4. Competition assays for cephalexin (CPL ) or ampicillin (AMP) of the PBPs of C. deunei endosymbionts. Endosymbiont membrane fractions were incubated with different concentrations of the nonradioactive antibiotics before labeling with [3H]-benzylpenicillin as described in Materials and Methods. The concentrations of each antibiotic (pglml) are indicated on the top of the figure.

Percoll fractions recovered from an endosymbiont-free C. deanei isolate (Fig. 3). PBPs with similar molecular weights were also detected in C. desouzai labeled with [H3]-benzilpenicillin (data not shown).

Affinities of cephalexin and ampicillin to endosymbiont PBPs. Cephalexin at 1 mg/ml reduced the binding of [3H]-benzylpenicillin to PBPl by approximately 70%. No reduction in the binding of PBP2 to [3H]-benzylpenicillin was detected even after incubation with 1 m g / d of the cold antibiotic (Fig. 4). Ampicillin at 100 pg/ml reduced the binding of [3H]-benzylpenicillin to PBPl and PBP2 to less than 10% and 40%, respectively, when compared to samples incubated only with [3H]-benzylpenicillin (Fig. 4).

DISCUSSION Previous attempts to induce morphological changes in the C.

oncopelti endosymbiont with high Concentrations of penicillin were unsuccesfull, even at concentrations as high as 24 mg/ml during a five week treatment [3]. On the other hand, pleomorphic endosymbionts could be observed in the cytoplasm of similarly treated Blastocrithidia culicis [3]. The differences between the results reported previously and those described here might be attributed to different permeability rates of different hosts membranes to penicillin.

Cured strains of trypanosomatids are usually obtained after growth in the presence of chloramphenicol [4, 151. In our experiments, even though the p-lactams treatment has led to se- rious impairment of the endosymbiont ultrastructure, a cured strain was never obtained. Antibiotics may have blocked only partially the binary fission process of the symbionts, otherwise

a faster and efficient cure of the symbiont would be expected. In contrast to peptidoglycan-containing bacteria, cell division in the Crithidia endosymbionts seems to be only partially affected by the binding of (3-lactams to the PBPs. This might indicate that endosymbiont PBPs perform functions physiologically distinct from those found in bacteria.

An interesting parallel exists between the properties of bacterial Chlamydia cells and the Crithidia endosymbionts. Chla- mydia are intracellular gram-negative pathogens with a complex growth cycle during which the infectious form penetrates a sus- ceptible cell and is converted into a larger developmental form which multiplies by fissicn before undergoing maturation [ 11. Although no peptidoglycan could be detected in Chlamydia cells, either by biochemical or microscopic methods, penicillin treatment of C. psittuci-infected cells inhibits division and dif- ferentiation of the developmental form and aborts the infective process [13]. Moreover, three PBPs with apparent molecular weights of 88 kD, 61 kD and 36 kD were found in the Sarkosyl- soluble fraction of C. trachomatis [l]. Although the biochemical composition of the Crithidia endosymbionts envelope has not yet been reported, our attempts to identify peptidoglycan- specific sugars in the envelope of C. deanei by cytochemistry and electron microscopy have failed (unpublished observa- tions).

Competition assays suggest that the PBPl of the C. deanei endosymbiont represents the main physiological target of these antibiotics since no significant binding of cephalexin to PBP2 could be detected at concentrations able to produce morphological alterations. The higher affinity for PBPl could explain the


faster shape modification process induced by ampicillin on the symbiont of C. deanei. Although direct evidence is limited, trypanosomatid endosymbionts may represent an intermediary step in an evolutionary adaptation of some bacteria to the intracellular environment. Reduction in the number of PBPs could represent important steps in such adaptation process.

Some authors have postulated that symbionts are surrounded by an envelope similar to that found in gram-negative bacteria [ll] while others have claimed that a vestigial, reduced or chemically modified cell wall could exist in endosymbionts [ 3 , 9, 211. This work shows for the first time the presence of a typical prokaryotic molecule, the PBP, in the envelope of C. deanei endosymbionts. Such protein constitutes a target to p-lactam antibiotics, which induce morphological changes in these microorganisms, indicating that PBP may play important physiological roles in the cell division process and/or in shape maintenance.

ACKNOWLEDGMENTS The authors thank Renato DaMatta, Dr. Jayme Angluster and

Dr. Celuta S . Alviano for all assistance. This work was supported by CNPg Capes, FINEP and Fenorte.

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Received 7-29-96, 12-27-96. 4-21-97; accepted 6-2-97

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