Neuraminidase Activity in Acanthamoeba Species Trophozoites and Cysts

Transcription

1 Investigative Ophthalmology & Visual Siene, Vol. 32, No. 12, November 1991 Copyright Assoiation for Researh in Vision and Ophthalmology Neuraminidase Ativity in Aanthamoeba Speies Trophozoites and Cysts Jean-Lu J. Pellegrin, Eduardo Orrega-Barria, Mihael Barza, Jules Baum, and Mierio E. A. Pereira Aanthamoeba speies, a widely distributed group of free-living amoeba, an infet humans and spread hematogenously after diret interation with the muosal surfaes. The mehanism underlying Aanthamoeba damage to the target ell is unknown. The authors report that trophozoites and ysts of Aanthamoeba speies exhibit a neuraminidase ativity that is membrane assoiated and released into the ulture medium at the start of the logarithmi phase of growth. The enzyme ativity is optimal at ph 5 and at C. Live parasites release siali aid from human ells. Therefore, the neuraminidase of Aanthamoeba speies ould be relevant in the olonization and damage of the siali aid-rih orneal epithelium and in the alterations of glyolipids assoiated with meningoenephalitis. Invest Ophthalmol Vis Si 32: ,1991 Members of the genus Aanthamoeba are widely distributed in nature and have been isolated from soil, water, air, and the upper respiratory trat of healthy humans. 1-2 At least seven speies of Aanthamoeba are pathogeni for humans, namely A. astellanii, A. polyphaga, A. ulbertsoni, A. astronyxis, A. hathetti, A. palestinensis, and A rhysodes. 1 The organism may exist in the form of trophozoites and ysts, and both developmental forms have been deteted within affeted tissues. 3 The infetious proess involves attahment to the surfae of the epithelial ell by means of the ventral surfae of the parasite, 4 whih may beome established as part of the loal flora. By mehanisms not well understood, Aanthamoeba is able to invade through epithelial defets or through the intat epithelium to produe progressive tissue destrution. In immunosuppressed patients, suh as those with aquired immune defiieny syndrome (AIDS), or those reeiving steroids or hemotherapy for aner, there may be hematogenous spread of Aanthamoeba from the nasal sinuses, lungs, or skin lesions to produe granulomatous amoebi enephalitis In the normal host, Aanthamoeba represents an inreasing reognized ause of keratitis, partiularly in ontat lens wearers, probably as a result of mehanial or hypoxi trauma. 7 Despite the report of Aantha- From the Division of Geographi Mediine and Infetious Diseases, New England Medial Center Hospitals, Tufts University Shool of Mediine, Boston, Massahusetts. This work was supported by National Institutes of Health grant AI (M.E.A.P.). Submitted for publiation: February 22, 1991; aepted June 24, Reprint requests: Dr. Pereira, Tufts University Shool of Mediine, 750 Washington Street, Box 041, Boston, MA moeba as a ause of disease in humans, fators responsible for tissue disruption and invasion remain largely unknown. We report here that Aanthamoeba speies expresses a membrane-assoiated neuraminidase ativity in both developmental forms of the parasite (sialidase, EC ). Beause mirobial neuraminidase has been impliated as a virulene fator in many human pathogens, suh as virus, 8 bateria, 9 " 11 and protozoa, the Aanthamoeba enzyme ould be a fator that determines the outome of Aanthamoeba infetion. Materials and Methods Materials Materials inluded 2'-(4-methylumbelliferyl)-a-D- N-aetylneuramini aid (4-MU-NANA), soybean trypsin inhibitor, phenylmethylsulfonyl fluoride (PMSF), Triton X-100, ethylenediaminetetraaeti aid (EDTA), ethyleneglyol-bis (/3-amino-ethyl ether) N,N'-tetraaeti aid (EGTA), and leupeptin. All were obtained from Sigma Chemial Co. (St. Louis, MO). Sodium dodeyl sulfate (SDS) was obtained from Bio-Rad Laboratories (Rihmond, CA). Proteose peptone and yeast extrat were obtained from Difo, Detroit, M\. Peanut agglutinin was purified as desribed previously. 14 Parasites Parasites were a gift of Dr. Govinda S. Visvesvara, Centers for Disease Control, Atlanta, Georgia. Trophozoites and ysts of A. astellanii strains ATCC and have been desribed previously 1516 and were used in all experiments. Tropho- 3061

2 3062 INVESTIGATIVE OPHTHALMOLOGY & VISUAL SCIENCE / November 1991 Vol. 32 zoites of several Aanthamoeba group II organisms isolated from the oraeal biopsy or sraping of Aanthamoeba keratitis patients also were studied, inluding strain RM, probably A. astellanii; strain MEEI:0184:1, probably A. astellanii, strain CDC: 0786:1, probably A astellanii; strain MEEI:0785:l, probably A. polyphaga; and strain CDCO 187:1, probably A. polyphaga. All strains isolated from linial speimens aused ytopathi effet in monkey kidney ell ultures and destroyed the ell monolayer within 7-10 days. Parasites were grown axenially in a proteose peptone-yeast extrat-gluose (PYG) medium, ph 6.5 at 30 C, in 15 X 125 mm borosiliate glass srew-ap tubes as desribed previously. 17 Trophozoites were harvested during the exponential phase of growth, usually at 1-2 X 10 6 ml" 1. Cysts from axenially grown amoeba were produed by inoulation of washed trophozoites from a 6-day-old ulture into an enystment medium 18 onsisting of NaCl 7 g/1" 1, CaCl g/r 1, MgCl g/t 1, Na 2 HPO g/r 1, KH 2 PO g/1" 1, FeSO g/r 1, ph 6.8 at 30 C and harvested 2 days later. Enumeration and morphologi harateristis of parasites were determined under phase ontrast mirosopy using an improved Neubauer hemoytometer. Preparation of Aanthamoeba Lysates Parasites were harvested by hilling the tubes on ie at 4 C for 15 min, pelleting at 500 X g for 10 min, and washing three times in phosphate-buffered saline (PBS, 20 mm sodium phosphate, ph 7.2, ontaining 150 mm sodium hloride). The trophozoites were lysed by soniation or addition of 1% Triton X-100 in PBS, ontaining 2 mm PMSF and inubated for 1 hr at 4 C. Beause it is diffiult to extrat proteins of mature ysts with nonioni detergents, ysts were harvested by entrifugation, washed and disrupted by soniation at 4 C with 5 to 10 pulses of 30 se eah at an output of 5 with a Branson 450 sonifier. Determination of Neuraminidase Ativity Neuraminidase ativity was assayed in two ways. The first method, peanut letin hemagglutination assay, is based on the ability of peanut letin to agglutinate desialylated human erythroytes as desribed. 14 Briefly, 50 /A of 10% washed, fresh, or glutaraldehydefixed human O+ erythroyte was inubated 2 hr at 30 C with 50 /A of live intat or lysed parasites or ulture onditioned medium, in a total volume of 200 /il 0.2 M Na aetate buffer, ph 5. At the end of the inubation time, the reation was stopped by entrifuging the samples for 30 se in a miroentrifuge, washing the erythroytes one in PBS-BSA (2 mg ml" 1 ), resuspending them in 250 n\ PBS-BSA, followed by titration against peanut agglutinin in mirotiter plates. Speifi neuraminidase ativities were expressed as the reiproal of the peanut agglutination titer hr" 1. The seond method, fluorometri assay, is based on the fluoresene produed by the speifi hydrolysis of 4-MU-NANA. 19 Final onentration of 4-MU-NANA was mm in the inubation mixture. The reation was stopped by addition of 1.5 ml of 0.5 M glyine NaOH ph 10.5, and the amount of released 4-MU was measured in a Perkin Elmer LS-5 fluoresene spetrophotometer with an exitation wavelength of 365 nm and an emission wavelength of 450 nm. An enzyme unit was defined as the amount of enzyme neessary to release 1 /imol of siali aid per minute under assay onditions. Heated inativated samples were used as ontrol. The influene of ph on the neuraminidase ativity was determined with both peanut letin hemagglutination and fluorometri assays in 0.2 M sodium aetate buffer (ph 3-5.5) and in 0.2 M sodium itrate buffer (ph 5-8) for 30 min at 30 C. The influene of temperature also was assayed with the two methods in 0.2 M sodium aetate buffer ph 5 for 30 min at 4,25, 30,37 and 56 C. The requirement of divalent ations was assessed by the influene of EDTA and EGTA at different onentrations, 2, 5, 10, 20 mm for 30 min at 30 C in 0.2 M sodium aetate buffer ph 5. Parasite Frationation Harvested trophozoites were washed three times for 10 min eah in PBS, resuspended (5 X 10 6 ells ml" 1 ) in antiprotease oktail (2 mm PMSF, 1% soybean trypsin inhibitor, 1% leupeptin, in PBS) and lysed by soniation as desribed. Lysates were entrifuged at 200 X g for 5 min at 4 C to remove unbroken ells and nulei, and membranes were pelleted at 100,000 X g for 1 hr at 4 C. The ytosoli fration was saved, and pelleted membrane proteins were extrated in antiprotease oktail ontaining 1% Triton X-100 for 1 hr on ie. Detergent-insoluble material was removed by entrifugation at 100,000 X g for 1 hr at 4 C in a Bekman L5-5OB ultraentrifuge. The pellet of Triton-X 100 insoluble fration, orresponding to the ytoskeleton was inubated for 1 hr on ie with 1% SDS in antiprotease oktail, ultraentrifuged as desribed, and the supernatant of this fration was mixed with Triton X-100 (1% [volume/volume] final onentration) to lower the final onentration of SDS to 0.1%. Release of Neuraminidase Ativity The release of neuraminidase in the Aanthamoeba-onditioned ulture medium was evaluated as follows. Cultures of A. astellanii in PYG medium

3 No. 12 NEURAMINIDASE ACTIVITY IN ACANTHAMOEDA SPECIES TROPHOZOITES A CYSTS / Pellegrin er ol 3063 were performed using an inoulum of 5.6 X 10 4 trophozoites ml" 1. At different time intervals, onditioned medium was olleted by entrifugation, supernatants filtered through a 0.22 fim nitroellulose filter, and assayed for neuraminidase ativity. Heat inativated samples and aliquots of mok inoulated PYG medium inubated at 30 C for different time intervals were used as ontrol. Protein Determination "a) o Q. CO 3000 i Protein onentration was determined using the dye-binding method of Bradford. 20 Results The neuraminidase ativity of A. astellanii and of Aanthamoeba group II strains of the linial isolates initially was evaluated using a fluorometri assay based on the ability of neuraminidase to release siali aid from 4-MU-NANA. All strains tested expressed signifiant enzyme ativity, but only and strains were used in all experiments desribed. The speifi ativities of ATCC and trophozoites were 215 ± 12 mu/mg of protein and 219 ± 32 mu/mg, respetively. The NA was onserved in both developmental forms of the parasite beause ysts and trophozoites showed omparable enzyme ativity (Table 1). The speifi ativity of the Aanthamoeba group II strains isolated from patients with keratitis was somewhat higher than in the nonoular strains, ranging from 196 to 315 mu/mg of protein. Among the linial isolates, three strains are lassified as "probably A. astellanii" and two as "probably A. polyphaga," suggesting that the enzyme may be onserved in other members of the genus. Time ourse analysis showed that the rate of siali aid release inreased rapidly, peaking at 15 min, and reahed a steady state level (Fig. 1). The optimum temperature for all strains was 30 C Table 1. Neuraminidase-speifi ativity of trophozoites and ysts of Aanthamoeba spp determined by 4-MU-NANA assay Strain designation A. astellanii, A. astellanii, ATCC A. astellanii, RM A. astellanii, MEEI:0184:1 A. astellanii, CDC:0786:l A. polyphaga, MEEI:O785:1 A. polyphaga, CDC:0187:1 Trophozoites (mu/mg) 215 ± 12(6) 219 ±32 (6) 315 ± 19(2) 248 ± 15(2) 303 ± 14(2) 196 ± 22 (2) 304 ± 27 (2) Cysts (mu/mg) 262 ± 50 (4) 222 ± 44 (4) Values are mean ± SD; values in parentheses are the number of determinations., not determined Time (min) Fig. 1. Time ourse of total lysate of/4, astellanii (open irle) and ATCC (losed irle) using 4-MU-NANA as substrate. Ativity is expressed in nanomolars of NANA released per milligram of protein of lysate. Assay was performed in 0.2 M sodium aetate buffer ph 5 at 30 C. Similar kinetis was obtained with the RM and CDC:0786:l strains of A. astellanii. (Fig. 2), a temperature that also is the optimum for the growth of parasites in vitro; 80% of the enzyme ativity was inhibited by heating the sample at 56 C, and the ativity was destroyed by boiling for 15 min. The influene of ph was studied in the range from 3 to 8. The maximal enzyme ativity was found to be at ph 5.0 (Fig. 3), with a sharp derease in ativity observed at ph values greater than 6.5 and less than 4. o> uu n. - i 1 I Temperature ( C) 9 5 Fig. 2. Influene of temperature on neuraminidase ativity of A. astellanii trophozoites (light olumn) and ATCC (dark olumn) using 4-MU-NANA as substrate. Trophozoite lysates were inubated with 4-MU-NANA at the indiated temperatures for 15 min in 0.2 M sodium aetate buffer ph 5.

4 3064 INVESTIGATIVE OPHTHALMOLOGY & VISUAL SCIENCE / November 1991 Vol i <D E 20 H h40 «"3 u PH Fig. 3. Influene of ph on neuraminidase ativity of A. astellanii trophozoites (lysate) using the fluorometri assay. Results are expressed in perent of the highest ativity. Assay was performed in 0.2 M sodium aetate buffer (ph 3-5.5) and in 0.2 M sodium itrate buffer (ph 5-8) for 30 min at 30 C. Similar ph urve was obtained with the RM and CDC:0786:l strains of A. astellanii. r-'-o Time (hours) Fig. 4. Release of the neuraminidase ativity in ulture medium. The ativity (open irle) is expressed in nanomolars of siali aid released per milligram of protein ontained in the medium. Assay was performed in 0.2 M sodium aetate buffer ph 5 for 30 min at 30 C. Culture medium supernatants were obtained at 24, 48, 72, 96, and 120 hr during the exponential growth phase. The number of ells (losed irle) in the ulture is expressed at the same times. Requirement for divalent ations was tested by assessing the effet of EDTA and EGTA in the range of from 1 to 20 mm, and the results indiated that neither EDTA nor EGTA affet enzyme ativity at the onentrations tested. Furthermore, addition of exogenous divalent ations (ie, Ca++, Mg++, Mn++, Zn++) in the range of 1-10 mm did not alter enzyme ativity (data not shown). Cell frationation experiments showed that the enzyme was mainly membrane assoiated beause 65% of the ativity was reovered in the pelleted membrane fration. On the ontrary, enzyme ativity was not deteted in the ytosoli or ytoskeleton frations. The neuraminidase was released into the ulture medium at the start of the logarithmi phase of growth (Fig. 4). This was demonstrated by removing samples of medium at different time intervals during parasite growth, entrifuging them for 10 min at 1,000 X g, filtering the supernatant through a ^m Millipore filter, and assaying the neuraminidase ativity of the filtrate. Interestingly, after 48 hr of ulture, the released neuraminidase ativity reahed a plateau and did not inrease proportionally with the number of parasites, although the number of ells inreased fivefold (Fig. 4). All experiments desribed thus far were performed using a syntheti substrate, 4-MU-NANA. As prelude to testing whether neuraminidase is involved in the pathogenesis of Aanthambeba infetion, we sought to determine if human erythroytes ould serve as a substrate for the parasite enzyme, using a peanut letin hemagglutination assay. 14 Peanut letin normally does not agglutinate mature red blood ells unless the ells are treated with neuraminidase to expose terminal letin binding sites (DGal 01->3 DGalNA). As shown in Figure 5, both live and lysed parasites release free siali aid from human erythroytes in a time-dependent fashion, and the kinetis of ativity losely resembles the one observed with the 4-MU- NANA method. Other series of experiments showed that ysts and trophozoites were equally effetive on a per partile basis (not shown). Optimum peanut agglutinin (PNA) titers were obtained after inubation of the mixtures at 30 C for 15 min at ph 5.0. o o M CO 3 (0 E0) \I G I / r i i i I I Time (min) Fig. 5. Time ourse of the neuraminidase ativity of live trophozoites (open irle) and total lysate (losed irle) of A. astellanii 76/2252 (3 X 10 6 ells ml" 1 ) using peanut hemagglutination. Assay was performed in 0.2 M sodium aetate buffer ph 5 at 30 C.

5 No. 12 NEURAMINIDASE ACTIVITY IN ACANTHAMOEDA SPECIES TROPHOZOITES A CYSTS / Pellegrin er ol 3065 Disussion The results presented here show that trophozoites and ysts of Aanthamoeba speies express a neuraminidase ativity that is ative against the syntheti substrate 4-MU-NANA and human erythroytes. A. astellanii ATCC and and Aanthamoeba group II strains isolated from ornea of patients with keratitis expresses a omparable amount of enzyme ativity. The enzyme is membrane-assoiated and is released into the growth medium at the start of the logarithmi phase of growth. The release of Aanthamoeba neuraminidase into the ulture medium during late logarithmi phase is not proportional to the number of parasites in ulture. It is not lear why the neuraminidase is not ontinuously released in the ulture medium, but it may be assoiated with ell density inhibition, suggesting its partiipation in early steps of the olonization proess. The presene of enzyme ativity in the ulture medium is not attributable to ell death beause in suh ases, one would expet the released enzyme ativity to inrease during the stationary phase of growth. Additionally, there was no evidene of trophozoite death in the growth onditions employed as evaluated by mirosopi observation of the parasite ultures. The biohemial properties of Aanthamoeba neuraminidase resembles other protozoan neuraminidases. Like the Aanthamoeba enzyme, the Naegleria fowleri neuraminidase is released into the ulture media, exhibits a ph optimum at 4.5-5, and is ion independent. 12 T. ruzi neuraminidase also is released into the ulture supernatant, but a signifiant amount appears to be membrane-assoiated. 21 The T. ruzi neuraminidase has an optimum ativity at ph and does not require divalent ations for full ativity. 22 In addition, the T. ruzi and Aanthamoeba neuraminidase share antigeni determinants beause a monolonal antibody against the former seem to ross-reat and inhibit the ativity of the Aanthamoeba enzyme (Pellegrin J-L J, et al, in preparation). Aanthamoeba is able to olonize the upper respiratory trat muosa in normal subjets, to ause keratitis in ontat lens wearers, and to ause disseminated infetions, notably granulomatous amoebi enephalitis, in immunosuppressed patients. 1 Beause neuraminidases frequently are produed by miroorganisms that have lose ontat with animals as pathogens or symbionts, they also ould be relevant in the pathogenesis of Aanthamoeba infetions. Aanthamoeba neuraminidase might enhane the olonization of siali aid-rih orneal and upper respiratory trat epitheliums, as does the neuraminidase of Influenza virus, 8 Corynebaterium diphtheriae, 9 Streptoous pneumoniae, 10 and Pseudomonas aeruginosa. 11 This enhanement is thought to our beause the neuraminidase dereases the visosity and adhesiveness of the respiratory muus, thereby failitating the mobility of the parasite. A similar role ould be postulated for the neuraminidase in the establishment of Aanthamoeba keratitis. Indeed, the innermost layer of the preoular tear film of the human eye is rih in muin. 23 The onentration of muin inreases from the external to the internal layer. 23 Like the respiratory trat muus, the oular muin film has been postulated to protet the oular surfae from infetion by trapping miroorganisms before they reah the apial ell membrane. 24 The environmental onditions of the tear film allow the ativity of Aanthamoeba neuraminidase. For example, the temperature of the tear film and the anterior ornea ranges from 35 C to 30 C. The ph of the tear film varies from 6.5 to 7.6, but an shift to more aid values with prolonged losure of the eyes, inflammatory onditions of the ornea or onjuntiva, and the presene of ontat lens. 23 Although the role of Aanthamoeba neuraminidase in the pathogenesis of keratitis is not lear, the fat that diret ontat is not neessary for ell damage to our 17 suggests that the release of parasite produts partiipates in the mehanism of tissue injury. Beause Aanthamoeba is able to desialylate human ells in vitro and a signifiant amount of the enzyme is released into the medium, the possibility exists that the neuraminidase ould partiipate in the degradation of sialylated glyoonjugates on oular muin or orneal epithelial surfae and modulate host ell-parasite interation. In this respet, evidene impliates an Aanthamoeba mannose-speifi letin in the reognition and phagoytosis of mammalian red blood ells. 25 The presene of suh mannose residues at the human orneal stromal matrix has been suggested by Conanavalin A binding, 26 and its expression seems enhaned in wounded orneas. 27 It generally is aepted that the ability of a miroorganism to produe disseminated infetion is related to its apaity to damage olonized tissues. Neuraminidase may be impliated with several other amoeba omponents and enzymes, suh as phospholipase A, sphingomyelinase, elastase, ollagenase, and a ytolyti and granule-assoiated ytotoxi ativity, in the destrution, penetration of muosal surfaes, and dissemination of Aanthamoeba Finally, neuraminidase-indued leavage of siali aid from glyoproteins and gangliosides of erebral tissue ould ontribute to the morbidity of granulomatous amoebi enephalitis. Suh an effet of neuraminidase was suggested in primary amoebi meningoenephalitis aused by Naegleria, also a free living amoeba. 12 In onlusion, we report for the first time the pres-

6 3066 INVESTIGATIVE OPHTHALMOLOGY b VISUAL SCIENCE / November 1991 Vol. 32 ene of a neuraminidase ativity in trophozoites and ysts of Aanthamoeba speies. The enzyme is membrane assoiated and is released in the ulture medium during the exponential phase of growth. Like the neuraminidase of other pathogeni miroorganisms, Aanthamoeba neuraminidase ould be relevant in olonizing the siali aid-rih orneal epithelium and in damaging erebral tissue in granulomatous amoebi enephalitis. Key words: neuraminidase, Aanthamoeba speies, siali aid, sialidase, keratitis, sialoglyoonjugate Aknowledgments The authors thank Dr. Gerald T. Keush and Dr. Bernard Leng for their advie and interest. Referenes 1. Ma P, Visvesvara GS, Martinez AJ, Theodore FH, Dagget P-M, and Sawyer TH: Naegleria and Aanthamoeba infetions: Review. Rev Infet Dis 12:490, Wang SS and Feldmann HA: Isolation of Hartmanella speies from human throats. N Engl J Med 277:1174, Visvesvara GS, Jones DB, Robinson NM: Isolation, identifiation and biologial haraterization of Aanthamoeba polyphaga from a human eye. Am J Trop Med Hyg 24:784, Preston TM and King CA: Ameboid loomotion of Aanthamoeba astellanii with speial referene to ell-substratum interations. J Gen Mirobiol 130:2317, Gonzalez MM, Gould E, Dikinson G, Martinez AJ, Visvesvara GS, Cleary TJ, and Hensley JT: Aquired immunodefiieny syndrome assoiated with Aanthamoeba infetion and other opportunisti organisms. Arh Pathol Lab Med 110:749, Wiley CA, Safrin RE, Davis CE, Lampert PW, Braude AI, Martinez AJ, and Visvesvara GS: Aanthamoeba meningoenephalitis in a patient with AIDS. J Infet Dis 155:130, Visvesvara GS and Stehr-Green JK: Epidemiology of free living ameba infetions. J Protozool 37:25S, Kilbourne ED: The influenza virus and influenza. New York, Aademi Press, Rosenberg A and Shengrund C-L: Sialidases. In Biologial Roles of Siali Aids, Rosenberg A and Shengrund C-L, editors. New York, Plenum Publishing Corp., 1976, pp Sanlon KL, Diven WF, and Glew RH: Purifiation and properties of Streptoous pneumoniae neuraminidase. Enzyme 41:143, Leprate R and Mihel-Briand Y: Extraellular neuraminidase prodution by a strain of Pseudomonas aeruginosa isolated from ystifibrosis.ann Mirobiol (Paris) 131:209, Eisen D and Franson RC: Aid-ative neuraminidases in growth media from ultures of pathogeni Naegleria fowleri and in soniates of rabbit alveolar marophages. Biohim Biophys Ata 924:369, Prioli RP, Mejia JS, and Pereira MEA: Monolonal antibodies against Trypanosoma ruzi neuraminidase reveal enzyme polymorphism, reognize a subset of trypomastigotes, and enhane infetion in vitro. J Immunol 144:4384, Pereira MEA: A rapid and sensitive assay for neuraminidase using peanut letin hemagglutination: Appliation to Vibrio holerae and Trypanosoma ruzi. J Immunol Methods 63:25, Castellani A: An ameba found in ulture of a yeast: Preliminary note. J Trop Med Hyg 33:160, De Jonkheere JF: Growth harateristis, ytopathi effet in ell ulture, and virulene in mie of 36 type strains belonging to 19 different Aanthamoeba spp. Appl Environ Mirobiol 39:681, Visvesvara GS and Balamuth W: Comparative studies on related free-living and pathogeni amebae with speial referene to Aanthamoeba. J Protozool 22:245, Jensen T, Barnes WG, and Meyers D: Axeni ultivation of large population of Aanthamoeba astellanii (JBM). J Parasitol 56:904, Potier M, Mameli L, Belisle M, Dallaire L, and Melanon SB: Fluorometri assay of neuraminidase with sodium (4-methylumbelliferyl-a-D-N aetylneuraminate) substrate. Anal Biohem 94:287, Bradford M: A rapid and sensitive method for the quantifiation of mirogram quantities of protein utilizing the priniple of protein-dye binding. Anal Biohem 72:248, Rosenberg I, Prioli RP, Ortega-Barria E, Pereira MEA: Stagespeifi, phospholipase C mediated release of Trypanosoma ruzi neuraminidase. Mol Biohem Parasitol 32:303, Pereira MEA: A developmentally regulated neuraminidase ativity in Tripanosoma ruzi. Siene 219:1444, Reords RE: The tearfilm.in Biomedial Foundations of Ophthalmology, Vol. 2, Tasman W and Jaeger EA, editors, Philadelphia, J.B. Lippinott, 1989, pp Wells PA and Hazlett LD: Complex arbohydrates at the oular surfae of the mouse: An ultrastrutural and ytohemial analysis. Exp Eye Res 39:19, Brown RC, Bass H, and Coombs JP: Carbohydrate binding proteins involved in phagoytosis by Aanthamoeba. Nature 254:434, Panjwani N, Moulton P, Alroy J, and Baum J: Loalization of letin binding sites in human, at, and rabbit orneas. Invest Ophthalmol Vis Si 27:1280, Spurr-Mihaud SJ, Barza M, and Gipson IK: An organ ulture system for study of adherene of Pseudomonas aeruginosa to normal and wounded orneas. Invest Ophtalmol Vis Si 29:379, Ferrante A and Bates EJ: Elastase in the pathogeni free-living Amoebae Naegleria and Aanthamoeba spp. Infet Immun 56:3320, He YG, Niederkorn JY, MCulley JP, Stewart GL, Meyer DR, Silvany R, and Dougherty J: In vivo and in vitro ollagenolyti ativity of Aanthamoeba astellanii. Invest Ophtalmol Vis Si 31:2235, 1990.