EXPERIMENTAL

PARASITOLOGY

Nuegleria

41,

290-306

( 1977)

fowleri: Fine Phosphatase MARK

Department Medical

of Tropical Medicine Center, 1542 Tulane

(Accepted

Structural Localization and Heme Proteins

of Acid

R. FELDMAN

and Medical Parasitology, Avenue, New Orleans,

for publication

Louisiana State Louisiana 70112,

University

U.S.A.

12 May 1976)

FELDMAN, M. R. 1977. Naegleria fowteri: fine structural localization of acid phosphatase and heme proteins. Experimental Parasitology 41, 290-306. Cytochemical assays, utilizing acid phosphatase (EC 3.1.3.2) and auto-oxidized 3,3’-diaminobenzidine (DAB), were carried out on in vivo trophozoites in order to identify the extent to which lysosomes and heme proteins contribute to the cytoarchitecture of Naegleria fowled. A full spectrum of lysosomal activity is present. Hydrolytic enzymes may be utilized extracellularly to enhance the amoebic phagocytic capacity. Intracellularly, a multivesicular organelle appears to function as a processing center Hhich may sequester and concentrate materials. DAB analyses reveal that heme proteins constitute major structural elements in the N. fowleri cell, Strongly positive electron-dense globular bodies appear to be cytoplasmic storage sites for lysosomal hydrolases and/or catalase. It is proposed that N. fowleri actively utilize host erythrocytes as their major source of nutriment. These data suggest that this pathogen’s rapid invasive behavior is the physiologic result of the overwhelming availability of erythrocytes in the host inflammatory reaction, and the combined mechanisms of enhanced extracellular destruction with subsequent phagocytosis. INDEX DESCRIPTORS : Naegleria fowleri; Amoeba; Protozoa; Primary amebic meningoencephalitis; Fine structure; Acid phosphatase (EC 3.1.3.2) activity; Lysosomal enzymes; Diaminobenzidine; Heme proteins; Invasive behavior; Extracellular erythrocytic destruction; Storage organelles; Phagocytosis.

bertson, Ensminger, and Overton (1968) postulated that the ability of Naegkria to penetrate, destroy, and lyse host Lysosomal enzymes have been impli- fowl& neural tissue may be accomplished through cated by various authors, using different the aid of an elaborated cytolytic subbiological systems, as the extracellular stance. The findings of Chang (1971, 1972) means by which cells effect digestion and/ or invasion (deDuve 1963; Allison 1967; and Visvesvara and Callaway (1974) lend Glauert, Fell, and Dingle 1969). The in- credence to the previous statement, in that the pathogenicity of N. fowbri is associated vasive capacity of Entamoeba histolytica is enhanced by the extracellular activity of with the presence of a cytotoxic or cytolytic lysosomal enzymes which are released via substance in cell cultures. The present study was initiated to confirm these obserspecialized secretory processes (Eaton, Meerovitch, and Costerton 1969; Eaton, vations about cytolysis relative to the rapid invasive behavior of N. fowleri and to Meerovitch, and Costerton 1970; Miller, Gillman, and Villarejos 1972; Lushbaugh identify a preferred amoebic nutritional 1973; Lushbaugh and Miller 1974). Cul- source. 290 Copyright AU rights

0 1977 by Academic Press, Inc. of reproduction in any form reserved.

ISSN

0014

4894

NaegZeria MATERIALS

fOWk?‘i:

ACID PHOSPHATASE

AND METHODS

Biological Trophozoites of the WM-1 strain of fowZeri (Duma, Rosenblum, Mcgehee, and Jones 1971) were cultivated axenically in a culture medium consisting of dehydrated liver extract, dextrose, and inactivated calf’s serum in Page’s saline (Nelson, 1971). Weanling Swiss white mice weighing 8-9 g were infected by lightly anesthetizing them individually with nasally administered ethyl ether. Each mouse then was hand-held in the supine and slightly dependent position while l-2 drops of saline-washed inoculum ( 10F logphase trophozoites/ml) from a Pasteur pipet were instilled at the external nares. Within 6 to 8 days postinfection, moribund mice were anesthetized with inhalant ethyl ether and pinned in the supine position to a restraining board. The chest wall was quickly opened and resected in order to expose the myocardium and associated vasculature. Perfusion fixation was accomplished by clamping the great descending vessels and injecting the left cardiac ventricle with redistilled, ice-cold 2% glutaraldehyde in 0.1 M sodium cacodylateHCl buffer (pH 7.2). After cessation of cardiac activity and observation of generalized rigor mortis, the carcass was decapitated. The bony cranial vault was exposed by skinning the head. An incision was made basally from the foramen magnum to the middle of the orbit and continued anteriorly to the roof of the nasal cavity. By following such a route of incision bilaterally, the cranium could be opened and peeled off easily; the cerebral lobes and olfactory bulbs and tracts were exposed adequately for thorough in situ fixation with the same ice-cold fixative.

Naegleria

Microstechnical Axenic trophozoites were harvested and concentrated from culture media by several

AND HEME

PROTEINS

291

successive washes with saline and centrifugation. Hyperemic and necrotic portions of the brain (usually the olfactory tracts and bulbs, and the rostroventral aspects of the cerebral lobes) were excised, cut into tissue strips measuring 1 mm X 1 mm x 1 cm, and fixed for 2 hr in the same ice-cold fixative. The tissue then was washed in several changes of the same buffer containing 7% sucrose and cut into nonfrozcn sections (2040 pm), according to the methods of Smith and Farquhar (1966). Cut sections were rinsed additionally overnight in several changes of the buffer with sucrose to remove traces of the fixative. Washed tissue sections were resuspended in the appropriate buffers and reaction mixtures, as cited below. After postfixation in cacodylate-buffered osmium solutions, tissues were washed with deionized water and dehydrated through a graded series of ethanols to propylene oxide, embedded in Spur low viscosity embedding medium in Beem capsules, and polymerized in a 70 C oven overnight. Thin sections were cut with glass knives on an LKB Ultratome I, collected on naked 200- or 300-mesh copper grids, and viewed unstained in a Hitachi HU-11A electron microscope at an operational power of 50 kV.

Cytochemical Acid phosphotase (EC 3.1.3.2) activity of WM-1 amoebae in central nervous system lesions was assayed according to the methods of Gomori, as modified by Barka and Anderson (1962). The 40-pm washed tissue sections were incubated for 30 min at 25 C in the complete medium (pH 5.0). Control specimens were incubated in either? the complete medium plus 10 mM sodium fluoride, or the medium minus the lead substrate. In uiuo trophozoites were assayed for the presence of heme compounds by the methods of Hirai (1971). The 20-pm washed tissue sections were incubated for 6 hr at 30 C in a medium of auto-oxidized

292

MARK R. FELDMAN

DAB l and Tris-HCl buffer ( pH 7.2). The

demonstrable also within host tissue (Figs. control medium consisted of the same com- 6 and 7). ponents plus a catalase inhibitor. FollowSome sections reveal reaction product ing incubation in both procedures, the sec- localized within a system of vesicles (Fig. tions were washed in three 5-min changes 7). These vesicles are similar in morpholof 0.1 M cacodylate-HCl buffer containing ogy to those present in the cytoplasmic 7% sucrose ( pH 7.2). All stained materials matrix and extruding from the amoebic were postfixed in 1% 0~0~ in 0.25 A4 caco- plasmalemma. dylate-HCl (pH 7.2). Very frequently, trophozoites may be observed in contact with, or in the process of, RESULTS phagocytosing host erythrocytes. These Acid Phosphatase areas display reaction product (Fig. 8). Reaction product, in the in viva trophozoite, is found in the nucleus, vacuolar system, granular endoplasmic reticulum and its cisternal dilatations, globular bodies, and along the membranes of the host-parasite interface (Figs. 1 and 2). The reaction in the nucleus is considered to be nonspecific, and that at the host-parasite interface may be adsorbed reaction product. The globular bodies react uniformly more densely than any other cellular organelle. These bodies appear to be membrane-bound (Figs. 4 and 11). Those bodies whose borders appear to be diffuse (Figs. 2 and 3) probably result from section artifact. The globular bodies are present in many sizes, shapes, and configurations and exist either singularly or in groups. They seem to display some preference for a peripheral cytoplasmic distributiorl (Fig. 1). It is not unusual to visualize many of these bodies in close association with vacuoles, mitochondria, and what may be primary lysosomes (Figs. 2 and 3). At the parasite surface the particulate reaction product is localized within membranous fronds (Figs. 2, 4, and 5). It is 1 Since DAB was used in this study as a general indicator of heme proteins, there is no applicable Enzyme Commission identification number.

DAB Analysis for Hemes Heme proteins are major constituents of the Naegleria fowled trophozoite. Reaction product is localized heavily along the membranes of the mitochondrial cristae, the entire membranous system (nucleus, endoplasmic reticulum (ER ), and plasmalemma), and the globular bodies (Figs. 9, 10). Less dense staining reaction occurs within the vacuolar system, the mitochondrial matrices, and throughout the general cell cytoplasm. The DAB stain has a strong affinity for the globular bodies, in that the majority of them stain densely and homogeneously. A greater number of these structures are visible with the DAB technique than was demonstrated previously by other chemical methods. These bodies occasionally appear as cisternal dilatations of the ER (Fig. 11). Smaller varieties of these bodies, which appear to be of the same density as the large globular bodies, also are found as constituents of the plasmalemma, and in areas of amoeba-erythrocyte contact (Figs. 12-15). These smaller bodies appear to form via apparent pinocytotic capture of erythrocytic material (Figs. 13 and 14). Similar structures are visible also in close

FIGS. l-15. Abbreviations used: BM, body membrane; C, core; ER, endoplasmic reticulum; CB, globular body; HA, hydrolytic activity; HT, host neural tissue; M, mitochondrion; MF, membranous frond; MVB, multivesicular body; N, nucleus; NU, nucleolus; PL, primary lysosome; PV, pinocytotic vesicle; RB, residual body; RBC, host erythrocyte. FIGS. l-8. Naegleriu fowkri trophozoites assayed for acid phosphatase.

i%ZC$&Z

fOWk?Ti:

ACID

PHOSPHATASE

AND

HEME

PROTEINS

FIG. 1. General distribution of reaction product. Note peripheral orientation of globular bodies and the association of vesicles with the ER and globular bodies (double arrows).

293

294

MARK

R.

FELDMAN

FIG. 2. Micrograph illustrates the concentration of reaction product origin. Note the presence of hydrolytic activity in host neural tissue.

in fronds of amoebic

ih?&?i~

fderi:

ACID

PHOSPHATASE

AND

HEME

PROTEINS

FIG. 3. Deposition of reaction product in the globular bodies. Note the diffuse appearance of the globular body borders. FIGS. 4-5. Micrographs illustrate the most frequently encountered types of frond formation at the amoebic surface.

295

296

MARK

R.

FELDMAN

FIG. 6. Hydrolytic activity is evident within appears to have extruded its contents ( * ). This a closely associated ER-globular body complex.

host tissue. Note process of enzyme

the ghost vesicle which release usually maintains

~&?&??%l

fOWhi:

ACID PHOSPHATASE AND HEME PROTEINS

FIG. 7. The multivesicular body is an organelle which seems to function as a processing center. Reaction product is most heavily concentrated at the structure’s membranous border. Little, if any, reaction product may be visualized within the vesicles.

297

298

MARK

FIG. 8. Micrograph the amorphous-appearing between the globular

R.

FELDMAN

illustrates hydrolytic activity at the amoeba-erythrocyte border of the globular body and the areas bodies and the red cell border.

of

contact. Note reaction product

h+Zeg~eriU

fowled:

ACID

PHOSPHATASE

relation to the nuclear membrane (Fig. 12). Various sections indicate that these bodies may have an internal lamellar core or nucleoid (Fig. 10). The staining characteristics of the erythrocytes and the globular bodies are similar. Throughout the course of this investigation whole, or portions of, host erythrocytes have been observed closely proximal to, and within, the amoebae. Erythrocytes in proximity to the amoebae are degenerating, presumably due to enzyme activity (Figs. 8, 12, 13, and 14).

AND

HEME

PROTEINS

299

vesicles and heavily deposited at the membranous borders of the organelle, it is logical to assume that the first and last possibilities are more plausible. The MVB appears to be a site of sequestration and concentration. The fate of these vesicles remains undetermined. It appears that fronds with hydrolytic activity are released from the amoebic plasmalemma and contact host tissue (Figs. 1, 2, 6, and 7). Fronds in contact with host cells then may initiate hydrolytic activity against the membranes. This mechanism would facilitate invasive activity and provide for increased nutriment. Additionally, DISCUSSION the increase in amoebic phagocytosis, as suggested by other investigators (Martinez, The acid phosphatase technique reveals a full spectrum of lysosomal activity in Nelson, Jones, and Duma 1971; Visvesvara and Callaway 1974) and confirmed by the NaegEeria fotcleri. Enzyme concentration, present study, would complement this however, appears to occur along a route different from the classically described ER process. The heavy localization of oxidized DAB to Golgi to membrane-bound packet reaction product in this investigation indischeme. cates that the heme proteins constitute A Golgi apparatus has not been identielements within the N. fied in this investigation, nor has one been major structural cell. Therefore, the amoebae must reported by other investigators. This ap- fowleri assimilate and utilize heme compounds. parent deficit and interpretation of the acid The degree to which this conversion takes phosphatase assays suggest that lysosomal by the availability of enzymes are synthesized on the system of place is rate-limited a heme source, the amoebic ability to inmembrane-associated polysomes and changest that nutriment, and the processing neled via the ER to various cellular locations. At these cytoplasmic sites, through a capacity of the metabolic schema. mechanism of cisternal dilatation and budIt is proposed that N. fowleri actively ding, membrane-bound vesicles are formed utilize host erythrocytes as a major nutriwhich contain one or more potentially ac- tional source. The erythrocytes are present tive hydrolases. in great quantity because of the host inThe multivesicular body (MVB) con- flammatory reaction. The combined mechatains vesicles which are similar to those in nisms of the observed extracellular destruction of erythrocytes with subsequent phathe amoebic cytoplasm (Fig. 7), outside gocytosis provides the amoeba with a the amoebic plasmalemma (Figs. 2, 5), constant supply of heme material. and within host cells (Figs. 6, 7). It is The globular bodies of N. fowkri, as possible that this organelle functions either in the incorporation of pinocytosed vesicles, demonstrated in this study, have been in the augmentation of the ER’s capacity labelled as lipid globules by other investigators (Martinez, Nelson, Jones, and Duma to form lysosomal packets, or in the trans1971; Visvesvara and Callaway 1974). Refer, concentration, and activation of materials stored in the globular bodies. Since sults of the present cytochemical analyses show these structures to be more complex. reaction product is lightly deposited in the

300

MARK

R. FELDMAN

These bodies, which contain a core or nucleoid and dominate the amoebic cytoarchitecture, appear to represent storage organeIles. They may be cytoplasmic depositories of inactive lysosomal hydrolases and/or catalase. The latter enzyme would indicate that these globular bodies are peroxisomes. However, it is probable that they participate in both Iysosomal and peroxisomal functions. Upon metabolic demand, stored materials may be activated and directed toward various cellular activities. These include structural and metabolic functions, as well as those which facilitate phagocytosis and invasive behavior. Further studies of these unique organelles are necessary to evaluate their function more definitively. ACKNOWLEDGMENTS The author wishes to express his appreciation to Drs. Joseph H. Miller and J. Clyde Swartzwelder for their continued interest, guidance, and criticism throughout the course of this investigation. Gratitude also is extended to Dr. E. Clifford Nelson, Medical College of Virginia, for providing the WM-1 strain of Naegleria fowl& This study is part of a dissertation submitted to the Graduate School of the Louisiana State University Medical Center in partial fulfillment of the requirements for the degree of Doctor of Philosophy. It was supported in part by U.S. Public Health Service Research Grant AI-02347 from NIH, the Schleider Educational Foundation Training Grant, and NSF Grant GZ-845. REFERENCES ALLISON, A. 1967. Lysosomes tific American 217, 62-73. BAFXA, T., AND ANDERSON,

and disease. Scien-

P. J. 1962. Histochemical methods for acid phosphatase using hexazonium pararosanilin as coupler. Journal of Histochemisty

and

Cytochemistry

10, 741-753.

S. L. 1971. Small, free-living amebas: cultivation, quantitation, identification, classification, pathogenesis, and resistance. Current

GANG,

Topics in Comparative Pathobio!ogy 1, 201-254. CHANG, S. L. 1972. Personal communication. CULBERTSON, C. G., ENSMINGER, P. W., AND OVERTON, W. H. 1968. Pathogenic Naegleria sp.: Study of a strain isolated from human cerebrospinal fluid. Journal of Protozoology 15, 353-363. DE DUVE, C. 1963. The lysosome. Scientific American 208, 64-72. DUMA, R. J., ROSENBLU~I, W. I., MCGEHEE, R. F., JONES, M. M., AND NELSON, E. C. 1971. Pri-

mary

amebic

meningoencephalitis

caused

by

Naegleria. Annals of Internal Medicine 74, 923931. EATON, R. D. P., MEEROVITCH, E., AND COSTERTON, J. W. 1969. A surface active lysosome in. Entamoeba histolytica. Transactions of the Royal Society of Medicine and Hygiene 63, 67& 680. EATON, R. D. P., MEEROVITCH, E., AND COSTERTON, J. W. 1970. The functional morphology of pathogenicity in Entamoeba histolytica. Annals of Tropical Medicine and Parasitology 64, 299304. GLAUEHT, A. M., FELL, H. B., AND DING=, J. T.

1969. Endocytosis of sugars in embryonic skeletal tissues in organ culture. II. Effect of sucrose on cellular fine structure. Journal of Cell Science 4, 105-131. HIRAI, K.

1971. Comparison between 3,3’-diaminobenzidine in the cytochemical demonstration of oxidative enzymes. Journal of Hi&o-

chemistry LUSHBAUGH,

and

Cytochemistry

19, 434442.

W. B. 1973. Comparative cytochemia cal characterization of the surface fine structure of Entamoeba histolytica Schaudinn, 1903.

Ph.D. dissertation, Louisiana Medical Center, New Orleans, LUSHBAUGH, W. B., AND MILLER,

structural topochemistry Eytica Schaudinn, 1903.

State University La. J. H. 1974. Fine of Entamoeba histo: Journal of Parasitology

60, 421-433. MARTINEZ, A. J., NELSON, E. C., JONES, M. DUMA, R. J., AND ROSENBLUM, W. I. 1971.

FIGS. 9-14. Naegleria fowleri trophozoites assayed for heme FIG. 9. Strongly positive response of the globular bodies, ER,

protein. mitochondrial

cristae,

and

the

entire membranous system. Note the cisternal dilatation of the ER. FIG. IO. Globular bodies maintain internal cores or nucleoids which may display chatter artifact. Globular bodies, ER, and a mitochondrion are apparent in a frequently encountered association.

M., Exr

h%Zk+3iU

fOWh+:

ACID

PHOSPHATASE

AND

HEME

PROTEINS

301

302

MARK

FIG.

11. Globular

bodies

originate

from

R. FELDMAN

the cisternal

dilatations

of the ER

(double

arrows).

hk?&&Z

fOWtt??‘i: ACID PHOSPHATASE

AND HEME

PROTEINS

FIG. 12. Micrograph illustrates an erythrocyte being phagocytosed. Globuk~r bodies are evident at points of parasitic contact, along the exterior of the nuclear membrane, and in association with various accumulations of ER. Note their variation in size.

303

304

MAX

FIG. 13. Apparent pinocytotic globular bodies. FIG. 14. Higher magnification Note the amorphous appearance

capture

R. FEZDMAN

of erythrocytic

material

of globular body formation of the erythrocyte border.

and incorporation

at amoeba-erythrocyte

to form contact.

Naeglefia

fowleri:

FIG. 15. Acid phosphatase DAB control.

ACID ~HOSPHATASE

control.

Naegleria

fothri

AND

HEhIE

specimen

305

PROTEINS

is similar

in appearance

to

306

MARK

R. FELDMAN

perimental Naegler~a meningoencephalitis~ An electron microscope study. Laboratory lnvestig&ion 25, 465475. MILLER, J, H., GILLMAN, FL H., AND VILLAREJOS, V. M. 1972. The mode of action of the surface lysosome of Entamoeba histolytica in human infections. In “Proceedings of the Thirtieth Annual Electron Microscopy Society of America Meeting” (C. J. Arcenaux, ed. ), pp. 152-153. Claitor’s, Baton Rouge, La.

NELSON, E. C. 1971. Personal communication. SMITH, R. E., AND FARQUHAR, M. G. 1966. Lyso-

some function in the regulation of secretory processes in cells of the anterior pituitary gland. Journal of Cell Biology 31, 319-347. VISVESVARA, G. S., AND CALLAWAY, C. S. 1974. Light and electron microscopic observations on the pathogenesis of Naegleria fowleri in mouse brain and tissue culture. Journal of Protozoology 21, 239-250.