Zbl. Bakt. 272, 210-215 (1989)

Adherence of Mycoplasma mycoides Subspecies mycoides to Cultured Endothelial Cells

ALFONSO VALDIVIESO-GARCIA, S0REN ROSENDAL, and SHELLEY SEREBRIN

Department of Veterinary Microbiology and Immunology, University of Guelph, Guelph, Ontario, Canada N1G 2W1

With 2 Figures· Received February 24, 1989 . Accepted in revised form August 29, 1989

Abstract

The LC- and SC-type strains of Mycoplasma mycoides subspecies mycoides were examined for adherence to guinea pig erythrocytes and bovine and caprine endothelial cells. The LC-type strains but not the SC-type strains adsorbed guinea pig erythrocytes and caprine endothelial cells. Difference in cytoadherence was observed between strains of the LC-type. The interaction between the most adherent LC-type strain and caprine endothelial cells was examined by transmission and scanning electron microscopy.

Zusammenfassung

Die LC- und SC-Typ-Stamme von Mycoplasma mycoides subsp. mycoides wurden auf Adharenz an Meerschweinchen-Erythrozyten und Endothelzellen von Ziege und von Rind untersucht. Nur LC-Typ-Stamme, jedoch nicht SC-Typ-Stamme adsorbierten an Meerschweinchen-Erythrozyten und Ziegen-Endothelzellen. Verschiedene LC-Typ-Stamme unterschieden sich in der Zelladharenz. Die Wechselwirkungen zwischen dem am starksten adharenten LC-Stamm und Ziegen-Endothelzellen wurde mit Scanning- und Transmissions­Elektronenmikroskopie untersucht.

Introduction

Members of the genus Mycoplasma are strongly host adapted and typically colonize mucosal surfaces of the upper respiratory tract and lower genital tract. Studies with animal mycoplasmas suggest they possess adhesins with specificity for their natural hosts (9).

The purpose of this study was to examine attachment of Mycoplasma mycoides subspecies mycoides to cultured endothelial cells in order to understand the role of adherence in the process which leads to the vasculitis and the coagulation disturbances seen in natural disease caused by these mycoplasmas.

Adherence of Mycoplasma mycoides 211

Materials and Methods

Cell cultures. Isolation and characterization of bovine and caprine endothelial cells have been described (12). The cell cultures were ascertained to be mycoplasma free by the fluoro­chrome Hoechst 33258 staining method (7).

Mycoplasma strains. The strains used in this study are listed in Table 1; their origin and cultural conditions have been described (13).

The number of colony forming units per ml (CFU/ml) of the mycoplasma suspensions was determined by standard plate count procedures (4).

Hemadsorption and endothelial cell adsorption. The method described by Manchee and Taylor-Robinson (6) was modified for endothelial cells. Briefly, mycoplasma agar in 90 X 15 mm Petri dishes was divided into 3 sections and 3 drops of 25 III each from dilutions 10-5, 10--6 and 10-7 of the mycoplasma suspensions were inoculated in each sector in order to obtain an area with 20-40 separated colonies. Plates were incubated for 48 and 72 h for the large colony (LC) and small colony (SC) type strains, respectively. A 2 ml suspension of endothelial cells at a concentration of 2.1 x 106 cells/ml was prepared in mGHFC medium (12) and added to each plate and incubated for 30 min at 37°C in an incubator with 5% CO2, Every 5 to 7 min the plates were swirled very gently. Excess endothelial cells were removed by washing twice with 2.5 ml of cold medium swirled very gently 10 to 15 times over the mycoplasma colonies. Adsorption of cells to the colonies was observed using a stereomicroscope l . The same procedure was used for hemadsorption with fresh guinea pig red blood cells at a concentration of 0.5%. One set of plates was incubated for 30 min, washed and examined. Another set was incubated overnight with an additional 20 ml of mGHFC medium in order to prevent dehydration.

DNA fluorochrome staining of mycoplasmas adhering to endothelial cells. The technique has been described by McGarrity et al. (7). Briefly, 2 ml of endothelial cell suspension at a concentration of 1. 7 X 105 cells/ml were allowed to attach for 24 h to coverslips in Leighton tubes. The endothelial cell cultures were then infected with 1 ml of a mycoplasma sus­pension containing 2.8 X 109 to 3.1 X 1010 colony forming units. After 1.5 h incubation at 37°C, unbound mycoplasmas were removed by washing three times in phosphate buffered saline, pH 7.4 (PBS). Three cycles of fixation were performed, with 1 ml of a mixture of glacial acetic acid and methanol (1: 3) added for 5 min at room temperature. One ml of a working solution of Hoechst stain No. 33258 (Bisbenzimide) was added, incubated for 30 min in the dark, and rinsed 3 times with double distilled water. Preparations were mounted in citric acid-glycerol solution and examined using an epifluorescent microscope2•

Transmission electron microscopy. Cellulose culture plate inserts3 were inoculated with 300 III of 1. 7 X 105 goat endothelial cells (GEC-2)/ml. Cells were allowed to adhere for 24 h in a 5% CO2 incubator. Cell culture medium was removed and 300 III was added. Infected cells were incubated at 37°C in 5% CO2 atmosphere for 60 min. Samples were rinsed three times with PBS, and fixed with 2.5% glutaraldehyde in PBS at room temperature for 60 min. Monolayers were rinsed for 15 min in PBS and fixed in 1 % osmium tetroxide in PBS for 60 min at room temperature, and subsequently dehydrated by rinsing with solutions with gradually increasing concentrations of ethanol, for 15 min each. Samples were embedded in epon, sectioned, stained with lead citrate and uranyl acetate and examined by transmission electron microscopl.

Scanning electron microscopy. Leighton tubes containing glass coverslips were inoculated with 1.5 ml of a GEC-2 suspension of 1. 7 X 105 cells/ml. Cells were allowed to attach for 24 h prior to inoculation. Mycoplasma strains suspended in mGHFC medium at concen-

I Stereo microscope Zeiss, Model DRC. 2 Microscope Zeiss, Model RA. 3 Millicell-HA 0.45 flm. Culture plate Insert. 12 mm diameter. Millipore Division, Bed­

ford, Ma. 4 JEM 100B JEOL, Tokyo, Japan.

212 A. Valdivieso-Garcia, S. Rosendal, and S. Serebrin

trations described above were added to the cells and incubated for 60 min. Infected cells and uninfected control cells were washed with PBS (320 mmol/kg) followed by a 2 h fixation with 2.5% glutaraldehyde in PBS. Coverslips were rinsed 3 times in PBS with subsequent fixation in fresh 1 % osmium tetroxide in PBS for 2 h at room temperature. Samples were dehydrated using ethanol as described for transmission electron microscopy, critical point­dried with CO2, and metal coated using gold-palladium deposition (30 nm)5. Scanning of the samples by electron microscopy6 was performed at 15 kV.

Results

Results of hemadsorption and endothelial cell adsorption of all mycoplasma strains are shown in Table 1. The LC-type strains but not the SC strains adsorbed guinea pig erythrocytes when incubated overnight (Fig. 1) whereas none of the mycoplasmas hemadsorbed when incubated for only 30 minutes.

Cytoadsorption of the LC-type strains occurred avidly when endothelial cells from goats were used, particularly with strain 74/2488. Only strain 74/2488 was able to adsorb bovine endothelial cells. The SC-type strains did not adsorb bovine or caprine endothelial cells.

Strains G17S178 and D44 were found in previous experiments to represent the most cytotoxic strains of the SC and LC-types respectively (13). However, instead of using the most cytotoxic strains for further experiments it was decided to use strain 74/2488 because of its higher binding capability. Binding experiments using the DNA fluoro­chrome staining method showed that strains D44 and 74/2488 bound equally well to bovine and caprine endothelial cells. Strain G17S178 did not bind either cell type. Because no binding was observed with strain G17S178 and any of the endothelial cell types used, this strain was not included for electron microscopy studies. Goat endothe­lial cells were used with strains D44 and 74/2488 because they showed higher binding

Table 1. Hemadsorption and adsorption of bovine (BEC-6) and caprine (GEC-2) endothe-lial cells to mycoplasma colonies

Myco- Hemad- Hemad- Myco-plasma sorption sorption BEC-6 GEC-2 plasma Strain 30 min overnight Group

G175178 SCT JB481 SCT O-goat SCT P-goat SCT 1915 SCT D44 +++ + LCT LH280 +++ + LCT 74/2488 +++ + +++ LCT Y3343 ++ ++ LCT

+ = 25% colonies or more, ++ = 50% colonies or more, +++ = 75% colonies or more

5 Anatech Hummer VII Sputter Coater. Anatech LTD. Alexandria, VA. 6 Hitachi, 5-570. Tokyo, Japan.

Adherence of Mycoplasma mycoides 213

Fig. 1. Cytoadsorption of guinea pig erythrocytes on colonies of strain 74/2488 (x 80).

of mycoplasmas. Strain D44 adhered to goat endothelial cells and because of its strong cytotoxic activity the cells were markedly injured. Strain 74/2488 was clearly seen to adhere to caprine endothelial cells (Fig. 2a and 2b).

Discussion

The subtypes of Mycoplasma mycoides subspecies mycoides differ in several biochemical and morphological characteristics (2), but the main difference resides in their pathogenic potential (3, 10). Endotheliopathic activities of both subtypes have been described previously (13), but the mechanism of cytopathogenicity remains unin­vestigated.

Hemadsorption properties usually reflect the adherence capability of mycoplasmas, and in Mycoplasma pneumoniae (9) this has been associated with virulence. Results obtained with the Mycoplasma mycoides subspecies mycoides strains indicated no attachment under the experimental conditions described by Manchee and Taylor­Robinson (6). In our studies, overnight incubation using the same procedure resulted in hemadsorption of all LC-type strains tested, but no adsorption with the SC-types, although these mycoplasmas were previously demonstrated to be cytotoxic (13).

Although adherence was measured only semiquantitatively, it was clear that strain 74/2488 had high binding capability. This strain (5) does not differ in any particular feature from other LC-type strains, except that it is less cytotoxic than strains D44 and LH280. On the other hand the most cytotoxic LC strain, strain D44, did not show higher cytoadsorption for endothelial cells in suspension. It seems that attachment and cytotoxicity are two different mechanisms which may be or may not be related. Similar observations have been reported for Staphylococcus aureus (8, 11).

The intimate interaction between the LC-type strains and endothelial cells demons­trated by electron microscopy has also been observed with other mycoplasmas with

214 A. Valdivieso-Garcia, S. Rosendal, and S. Serebrin

Fig. 2a. Scanning electron microscopy of unexposed caprine endothelial cells. Bar equals 6 !-tm.

Fig. 2b. Scanning electron microscopy of caprine endothelial cells exposed to strain 741 2488. Bar equals 3 !-tm.

binding properties (1, 9). The adherence of the LC-type strains, but not the SC-type strains indicates an important difference in their interaction with endothelial cells. Further research is needed to provide information about the nature of the receptor site as well as the cytoadhesin involved in the attachment of the LC-type strains.

Acknowledgements. The technical assistance of Mr. Paul Huber and Ms. Sandra Smith with the scanning and transmission electron microscope is acknowledged. This study was supported by the Natural Sciences and Engineering Research Council of Canada. Dr. A. Valdivieso-Garcia was a recipient of Government of Canada Awards administered by World University Service of Canada.

Adherence of Mycoplasma mycoides 215

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Dr. A. Valdivieso-Garcia, Health of Animals Laboratory, 110 Stone Road West, Guelph, Ontario, Canada NIG 3W4