Acid Phosphatase Activity in Coxiella burnetii - Infection and Immunity

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The level of phosphatase activity detected in several isolates of. C. burnetii ... These data suggest that the acid phosphatase of the parasite may be a major ...
Vol. 61, No. 10

INFECrION AND IMMUNITY, Oct. 1993, p. 4232-4239

0019-9567/93/104232-08$02.00/0 Copyright X 1993, American Society for Microbiology

Acid Phosphatase Activity in Coxiella burnetii: a Possible Virulence Factor OSWALD G. BACA,12* MIRIAM J. ROMAN,' ROBERT H. GLEW,3 ROBERT F. CHRISTNER,l JOHN E. BUHLER,2 AND ADAM S. ARAGON2 Departments of Biology,'* Microbiology, 2 and Biochemistry,3 University of New Mexico, Albuquerque, New Me-xico 87131 Received 13 May 1993/Returned for modification 25 June 1993/Accepted 7 July 1993

High-speed supernatant fluids derived from sonicated CoxieUla burnetii contained considerable acid phosphatase activity when assayed by using 4-methylumbelliferylphosphate; they also contained a factor that blocked superoxide anion production by human neutrophils stimulated with formyl-Met-Leu-Phe. The pH optimum of the enzyme was approximately 5.0. The level of phosphatase activity detected in several isolates of C. burnetii implicated in acute (Nine Mile) and chronic (S Q217, PRS Q177, K Q154) Q fever was 25 to 60 times greater than that reported in other microorganisms, including Leishmania and LegioneUla spp. The enzyme was found in rickettsiae grown in different hosts (L929 cells and embryonated eggs) and, in the case of L929 cells, for both short periods (less than a month) and the long term (years). Cytochemical techniques coupled with electron microscopy localized the phosphatase activity to the periplasmic gap in the parasite. Ion-exchange chromatography revealed a major species of the enzyme and showed that the enzyme of the parasite was distinct from that of the host cell (L929 fibroblasts); its apparent molecular weight was 74,000. Phosphatase inhibitors (i.e., molybdate heteropolyanions) had differential effects on the phosphatases of the parasite and host cell. C. burnetii supernatant fluid inhibited superoxide anion production by formyl-Met-Leu-Phestimulated human neutrophils; molybdate inhibitors reversed the inhibition. Treatment of C. burnetii-infected L929 cells with one of the molybdate compounds (complex B') significantly reduced the level of infection and did not affect the viability or growth of the host cell. These data suggest that the acid phosphatase of the parasite may be a major virulence determinant, allowing the agent to avoid being killed during uptake by phagocytes and subsequently in the phagolysosome. The agent of Q fever in humans, Coxiella burnetii, resides and proliferates within acidic phagolysosomes of host cells, including phagocytes (2). Although this obligate intracellular rickettsial parasite contains superoxide dismutase and catalase (1), which probably protect it from host cell-generated superoxide anion and hydrogen peroxide, respectively, it is likely that other virulence determinants play a significant role in allowing the agent to thrive within phagolysosomes. Recently we reported that intact C. bumetii inhibits the respiratory burst of phagocytosing human neutrophils (3). In light of reports (13, 21, 24) that Legionella micdadei and Leishmania promastigotes possess acid phosphatases (ACPs) that inhibit the respiratory (oxidative) burst of neutrophils, we investigated the possibility that C. burnetii also possesses such a virulence factor, which would account for its capacity to inhibit the oxidative burst of the neutrophil. Both of these facultative parasitic agents also proliferate in phagolysosomes of macrophages. Their ACPs apparently modulate the respiratory burst of neutrophils by reducing the amount of the second messengers, inositol 1,4,5-triphosphate and sn-1,2-diacylglycerol, produced following receptor-mediated stimulation (12, 25). In this report we show that C. bumetii, does, indeed, possess significant ACP activity and that it is probably responsible for inhibiting the metabolic burst of human

MATERIALS AND METHODS C. burnetii propagation and purification. Several C. burnetii isolates implicated in acute and chronic Q fever (see Table 1) were cultivated in L929 fibroblasts or in embryonated eggs and subsequently purified from host cell components. L929 fibroblasts were infected with C. burnetii by established procedures (6, 7, 22). Normal and infected cells were maintained in suspension culture at 35°C. Rickettsiae were purified from infected L929 cells (continuously infected and maintained in culture for the periods indicated without freezing), and concentrations were determined as described by Baca et al. (7). Infected cells suspended in 2 mM magnesium acetate were disrupted in a Ten Broek homogenizer, and the released rickettsiae were purified from host components by differential centrifugation: alternating low-speed (150 x g for 10 min) and high-speed (10,000 x g for 30 min) centrifugations to sediment host debris at low speed and rickettsiae at high speed. The purified rickettsiae were enumerated by the methods of Silberman and Fiset (28) as modified by Williams et al. (33). C. burnetii organisms, grown in pathogen-free embryonated eggs, were purified from infected yolk sacs after homogenization, differential centrifugation, and banding in 30 to 60% (wtlwt) linear sucrose gradients (29). The preparations were monitored for microbial contaminants with the aid of thioglycolate broth and blood agar. Electron microscopy and ACP cytochemistry. Infected L929 cells and purified C. burnetii organisms were subjected to electron microscopy for localization of ACP activity within the parasite by the Gomori cytochemical technique (2, 15). The enzyme substrates used were 1.25% (wt/vol) P-glycerophosphate or p-nitrophenyl phosphate (disodium

neutrophils. (This work was presented, in part, at the 1992 General Meeting of the American Society for Microbiology, New Orleans, La. [abstr. no. B571.) *

Corresponding author. 4232

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salt) (Sigma Chemical Co., St. Louis, Mo.); 0.2% (wt/vol) lead (PbNO3) served as the capture agent. Cells and rickettsiae were fixed for 30 min in 0.1 M cacodylate buffer (pH 7.4) containing 2.5% (vol/vol) glutaraldehyde. After fixation, the cells were washed in three changes of ice-cold cacodylate buffer and then incubated for 2 h at 37°C in a shaking water bath with constant agitation (100 rpm) in reaction medium buffered to pH 5.0 with Tris-maleate. Control samples lacked the substrate in the reaction medium. The cells and rickettsiae were postfixed for 1 h in 1% (wttvol) OS04 buffered in cacodylate, embedded, sectioned, stained (with uranyl acetate and lead citrate), and examined by transmission electron microscopy (2). Standard ACP assay. ACP activity was determined fluorometrically with 4-methylumbelliferylphosphate (MUP) serving as the substrate (14). The standard assay was carried out for 15 min at 37°C in a 0.1-ml reaction mixture containing 0.2 M sodium acetate buffer (pH 5.5) and 5 mM MUP. One unit of enzyme activity is defined as the amount of enzyme required to convert 1 nmol of substrate to product per hour. Protein was determined by the Bradford (8) method with bovine serum albumin as the standard. Preparation of C. burnetii supernatant fluid and ion-exchange chromatography. Purified rickettsiae suspended in N-2hydroxyethylpiperazine-N'-2-ethanesulfonic acid (HEPES) buffer (0.01 M [pH 7.4] with 0.8% [wtlvol] NaCl) were subjected to ultrasonication (30 to 60 s at ice bath temperature) followed by centrifugation at 10,000 x g for 1 h at 4°C. The supernatant fluid was subjected to additional centrifugation at 100,000 x g for 1 h at 4°C. The resulting high-speed supernatant fluid was chromatographed on a QAE-Sephadex column and subjected to high-performance liquid chromatography (stationary-phase Superdex G-75 column). At each step, aliquots were assayed for ACP activity and used immediately in experimentation or stored at -70°C. In the initial stages of this investigation the sonicates were also subjected to three cycles of freezing and thawing. We subsequently found that freezing and thawing did not enhance ACP yields and therefore discontinued the practice. It was also found that freezing and thawing did not affect the results obtained (e.g., chromatography profiles and enzyme activities). Except when noted in this report, sonicated C. bumetii organisms were not also subjected to freezing and thawing. Preparation of human neutrophils and superoxide anion assay. Neutrophil suspensions were prepared from human peripheral blood by dextran sedimentation followed by density gradient centrifugation on Ficoll-Hypaque gradients and isotonic NH4C1 lysis (20, 24). The generation of superoxide anions by formyl-Met-Leu-Phe-stimulated neutrophils was measured as superoxide dismutase-inhibitable cytochrome c reduction by using a continuous assay method (20). Treatment of infected L929 cells with a heteromolybdate compound (complex B') and determination of degree of infection. The heteromolybdate compound complex B' was dissolved in sterile distilled water (10-mg/ml stock), and infected L929 cells (infected ca. 3,200 days earlier with phase I Nine Mile C. burnetii) were exposed to either 20 or 40 jig of the compound per ml for up to 2 weeks. The percentages of infected cells were determined as previously described by us (35, 36) by direct microscopic examination of Gimenezstained cells. A minimum of 300 cells were examined in each

prepared slide to determine the percentage of the population that was infected (one or more rickettsiae per cell). Duplicate flasks containing 10 ml of infected cells (starting cell concentration, 2.5 x 105/ml) were used for each concentra-

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TABLE 1. ACP activity in different isolates of C. burnetii associated with acute or chronic disease, cultivated in either L929 cells or embryonated eggs Isolate

Phase

Host

Nine Mile Nine Mile Nine Mile S Q217 S Q217 S Q217 PRS Q177 K Q154

Originally I I I Originally I I I I I

L929 (952 days)b L929 (28 days) Egg embryoc L929 (952 days) L929 (28 days) Egg embryo Egg embryo Egg embryo

Sp act (U/mg of protein)a 1,510 1,540 2,430 2,630 2,620 1,610 3,840 2,240

± 127

± ± ± ± ±

102 154 159 118 104 ± 477 ± 224

a Results represent the means + standard deviations of three independent with purified intact rickettsiae. experiments L929 cells infected for the number of days indicated. c The egg-grown C. burnetii organisms, obtained from

L. P. Mallavia, Washington State University, were in their third serial egg passage; they were in phase I. The Nine Mile and S Q217 isolates (both phase I) grown in L929 cells were previously cultivated in guinea pigs and subjected to three egg passages.

tion of the compound; duplicate control flasks received only the solvent (water). The cell populations were divided twice a week, and the concentration of molybdate was adjusted. Viability was determined by the dye exclusion technique

(35). RESULTS Detection of ACP C. burnetii isolates and optimum pH activity. Several isolates of C. burnetii, representative of the three major groups implicated in acute or chronic disease, exhibited significant levels of ACP activity when assayed fluorometrically with MUP serving as the substrate (Table 1). The rickettsiae were purified from host cells as described in Materials and Methods and included the isolates Nine Mile, which causes acute short-term disease, S Q217 (plasmidless) and K Q154 (QpRS plasmid) isolated from patients with chronic endocarditis, and PRS Q177 isolated from a goat cotyledon and from the same plasmid group (QpRS) as K Q154 (25). The assays were performed on intact organisms at 37°C in 0.1-ml reaction mixtures containing 0.2 M sodium acetate buffer (pH 5.5) and 5 mM MUP. Irrespective of the host from which the rickettsiae were isolated-L929 fibroblasts or embryonated eggs-the parasites exhibited significant levels of ACP activity. Also, rickettsiae derived from short-term (28 days)- and long-term (952 days)-infected L929 cells exhibited comparable levels of ACP activity (Table 1). Disrupted C. burnetii organisms (sonicated for 30 to 60 s at 0°C, in a Branson Sonifier with the microprobe) exhibited slightly higher levels of ACP activity than did the intact rickettsiae (specific activities, 1,430 + 157 for the intact organisms and 1,840 ± 147 for the sonicated organisms [results of four independent experiments]). The C. burnetii Nine Mile organisms were purified from L929 cells that had been infected for over 2,500 days. Sonication for additional periods (up to 4 min, which is sufficient to completely disrupt the rickettsiae [1]) did not result in increased ACP activity. These results suggested that the enzyme was localized at or near the parasite surface, i.e., in the periplasmic space. The ACP activity (on a per-milligram-of-protein basis) present in C. bumetii was greater than has been reported to date, to our knowledge, for any other microorganism; depending on the isolate, it was approximately 25 to 60 times that observed in Legionella micdadei (24).

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cells were obtained after homogenization with the Ten Broek homogenizer and centrifugation at 100,000 x g for 1 h, and they too were subjected to chromatography. The ACPs of L929 cells and parasites eluted at distinctly different NaCl concentrations (Fig. 3). The elution profile for C. bumetii shows at least one major peak (middle region) and indications of two minor peaks. Molecular mass of C. burnetii ACP. The apparent native molecular mass of the predominant C burnetii species was determined by high-performance liquid chromatography to be approximately 74 kDa (Fig. 4). When the host cell extract

180160>

140-

100-

Z~ 80C,)

O

60-

o

40-

e

20-

0

I 3

8 6 7 5 pH FIG. 1. Effect of hydrogen ion concentration on C. burnetii ACP activity. Niine Mile supernatant fluid (from 100,000 x g centrifugation) was pirepared and chromatographed through QAE-Sephadex (0 to 0.5 M NTaCi, 10 mM phosphate buffer [pH 6.4]) as described in Materials a Lnd Methods and in the legend to Fig. 3. The ACPs in the fractions otbtained were pooled, and the activity at various pHs was determined Iby using the following buffers: 0.2 M sodium acetate (pH 3 to 5.5), 0..2 M sodium cacodylate (pH 6), and 0.2 M Tris-HCI (pH 6.5 and 8). The Nine Mile organisms were isolated from L929 cells which had Ibeen infected for 3,052 days.

4

Optimu:m C. bumetii ACP actvlty was observed at pH 5.0 in 100,00(ex g supernatant fluids derived from sonicated Nine Mille C.buvaetio organisms (Fig. 1). Assays were

performec as the substrate

Presencoe of ACP in the C. burnetii periplasmic space. By using the (cytochemical technique of Gomori (2, 15), the ACP activity wras detected only in the periplasmic space of the Nine Mille and S Q217 isolates of C bumetii. Purified rickettsiae or parasites present in phagolysosomes of L929 cells were incubated with the phosphate substrate (13-glycerophosphalte orp-nitrophenylphosphate- lead nitrate was the capture a tgent). Following incubation, the samples were prepared Ifor electron microscopy (2) and thin sections were examined by transmission electron microscopy. The electron micr ographs (Fig. 2) clearly showed the presence Of electron-dlense reaction products (lead phosphate) in the periplasmilic space. With the glycerophosphate substrate approxim;ately 10% of rickettsiae gave a positive result; with p-nitropheatylphosphate, more than 50% did so. Ion-excibange chromatography clearly distinguished C. burnetii phosjphatase from that of the host cell. Purified Nine Mile C. bumetii organisms were disrupted by sonication while suspendecI in 0.01 M HEPES buffer (pH 7.4)-0.8% NaCl. The disrulpted organisms were centrifuged at 100,000 x g at 4°C for 1 h, and the supematant fluid was eluted through QAE-Sep]hadex with a linear gradient ranging from 0 to 0.5 M NaCl. Supernatant fluids from normal uninfected L929

was chromatographed on the same column, three distinct peaks of ACP activity were observed; they eluted in fractions corresponding to molecular masses of 2.5, 10.5, and 50 kDa. All three of these peaks eluted after the Coxiella ACP (data not shown). Since few enzymes are 10 kDa or smaller,

speculate that the relatively low apparent molecular masses of two of the three host cell ACPs may be attributed to their interaction with the column resin. Supernatant fluids from 100,000 x g centrifugation of the Nine Mile rickettsiae and L929 cells were analyzed in a stationary-phase Superdex

we

G-75 column eluted with phosphate-buffered saline at 120

lb/in2. Eluted fractions were assayed for phosphatase activity. Molecular mass standards were also chromatographed on the column to establish the molecular mass of the ACP.

Effect of a heteromolybdate complex on C. burnetii and host

cell phosphatase activity. It was demonstrated previously that a number of heteromolybdate compounds inhibit ACP activity (23). The potential inhibitory effects of various chemicals on the ACP activity associated with intact Nine Mile C. bumnetii and supernatant fluids from 10,000 x g centrifugation of infected and uninfected L929 cells were investigated (Table 2). The compounds tested

included sodium tartrate,

sodium fluoride, and two heteromolybdates synthesized

by Michael Pope, Georgetown University. The two heteromolybdates studied are designated as complex B'

{[C(NH2)3]2(CH3)2AsMo4O15H} and complex E2 [(NH4)6 As2Mo18062xH2O]. Complex B' significantly reduced the

ACP activity of intact C. bumetii (over 50% inhibition) and only slightly inhibited the ACP activity of uninfected L929 cells. Although complex E2 seemed to have a slightly stronger inhibitory effect on the C. burnetii ACP (intact organisms) than on that of the uninfected host cell, the difference is probably not significant. The effect of the molybdate compounds on the C. burnetii ACP activity present in 100,000 supernatant fluids was even more pronounced. For example, 25 and 50 ,uM concentrations of B' inhibited the C. burnetii ACP by 59 and 70%, respectively; 25 and 50 ,uM concentrations of E2 inhibited the rickettsial ACP by 72 and 80%, respectively. The differential effect of these two heteromolybdate compounds is an additional indication that the ACPs of parasite and host are distinct; it is also indirect evidence that the ACP associated with the parasite is C. bumnetii specific (i.e., produced by the parasite). Other potential phosphatase inhibitors (sodium fluoride and sodium tartrate) were examined; however, neither compound

FIG. 2. Detection of ACP in the periplasmic spaces of the S Q217 (A) and Nine Mile (B and C) isolates of C. burnetii present in phagolysosomes (panels A and B) of L929 fibroblasts or purified from L929 cells (panel C). The electron-dense reaction products (arrows) indicate the presence of ACP and were generated by usingp-nitrophenylphosphate (panel A) or 1-glycerophosphate (panels B and C) as the substrate. Organisms in panel D are control rickettsiae processed at the same time as those in panel C but without substrate. The L929 cells had been continuously infected for 1,058 days (A) or 2,562 days (B); the purified C. bumetii organisms (panels C and D) were from L929 cells that had been continuously infected for 2,893 days. Bars, 2 pum (panels A and D), 0.6 ,um (panel B), and 1.1 pum (panel C).

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ACID PHOSPHATASE ACTIVITY IN COXIELLA BURNETII

N_&WqL

_;

ir

4235

BACA ET AL.

4236

IN ECT. IMMUN.

900-

E 700= 600z

500> 400-

300-

C

200-

IL

n( ioo

TABLE 2. Differential effect of two molybdate compounds on the ACP activity of Nine Mile C. bumetii and uninfected L929 cellsa

Ro

800-

None Complex B' Complex E2

I KR6 1' 3

Sp act (U/mg of protein)b

Inhibitor

NM 0

5' 7

0

0

9 11 3 15 17

192 23 2527 29 31 33 35

FRACTION NUMBER

FIG. 3. Chromatography of C. bumnetii and L929 ACPs on a QAE-Sephadex column. Purified Nine Mile C. burnetii organisms (9 mg or 3.3 x 1011 rickettsiae) in 3 ml of HEPES buffer (0.01 M [pH 7.4] with 0.8% NaCl) were lysed by being subjected to sonication (Branson Sonifier; 30 s with the microprobe). L929 cells (7.3 x 108, in 10 ml of HEPES buffer-0.8% NaCl) were disrupted in a Ten Broek homogenizer maintained at 4°C. After centrifugation at 100,000 x g for 1 h at 4°C, the supernatant fluids from the parasites and L929 cells were independently chromatographed through a QAE-Sephadex column (30 mm by 10 mm [inner diameter]) with a NaCl linear gradient ranging from 0 to 0.5 M, which was initiated at fraction 3 and terminated at fraction 35. Fractions (0.5 ml) were collected and assayed for ACP activity. The assays were carried out at 37°C in 0.1 ml of reaction mixture containing 0.2 M sodium acetate buffer (pH 5.5) and 5 mM MUP as substrate. O, L929 ACP activity; *, C. bumetii ACP activity.

1,120 ± 232 1,090 + 113 676 ± 253

1,380 + 94 652 ± 80 533 ± 88

rickettsiae. The supernatant fluid contained significant ACP activity (1,488 U/mg of protein). Human neutrophils were prepared from peripheral blood as described in Materials and Methods. Superoxide anion production by formyl-Met-LeuPhe-stimulated neutrophils was measured by a continuous cytochrome c reduction method (20). In the presence of 10 p,l of the C. bumnetii supernatant preparation (which contained 4 U of phosphatase activity), the neutrophils (106 cells in 1.0 ml of buffer) did not generate significant levels of superoxide 0.10LO

0.08-

LU 0.06() z

m 0.040 m

Cl)

0.02-

60 v 0

-lE

C. bumetii

a The compounds were tested in the standard ACP assay with MUP as the substrate. The complex B' concentration was 100 p.M; the E2 concentration was 50 pM. b Results represent the means ± standard deviations of four independent experiments. c Uninfected L929 cells were suspended in HEPES buffer (0.01 M [pH 7.4] with 0.8% NaCi), homogenized with the aid of a Ten Broek homogenizer, and centrifuged at 10,000 x g for 1 h at 4'C. The supernatant fluid was assayed for ACP activity.

significantly affected the activity of the enzymes of the parasite and host cell. Inhibition of superoxide anion production by human neutrophils coincubated with C. burnetii supernatant fluid. Supernatant fluids were prepared from sonicated Nine Mile C burnetii organisms by subjecting the sonicates to centrifugation at 10,000 x g for 1 h to remove cell debris and intact

L929 supernatante

0

t1LLLI[

30' 60- 90 120 150 180 210 240 270

TIME (sec)

: 50-

st40-

0

w

co

I- 300.

VO Q-.20-

C

1

6

8

10 12 14 16 18 20 24 26

FRACTION NUMBER FIG. 4. Apparent molecular mass of C. burnetii ACP. The molecular mass of C. burnetii ACP was determined by high-performance liquid chromatography as described in the text. The ACP was from rickettsiae derived from L929 cells that had been infected for approximately 3,000 days.

FIG. 5. Apparent inhibition of superoxide anion production by human neutrophils coincubated with C. burnetii supernatant fluid. Sonicated (30 s in a Branson S75 Sonifier fitted with a microprobe) Nine Mile C bumetii organisms (3 mg of purified rickettsiae per ml) were centrifuged at 10,000 x g for 1 h. The resulting supernatant fluid was assayed for ACP activity (1,488 U/mg of protein). Human neutrophils were prepared from peripheral blood by dextran sedimentation followed by density gradient centrifugation on FicollHypaque gradients and isotonic NH4C1 lysis (20, 24). The generation of superoxide anions by formyl-Met-Leu-Phe-stimulated neutrophils was measured as superoxide dismutase-inhibitable cytochrome c (0.05 mM) reduction by using a continuous assay method (20). Experimental neutrophils (0) (106) in 1.0 ml of Krebs-RingerPhosphate solution (pH 7.4) were preincubated at 37°C (2 to 3 min) with 10 p,l (4 U of phosphatase activity) of the C burnetii supernatant fluid; control untreated (-) neutrophils (106) received 10 PI1 of buffer. Measurement of superoxide anion production began with the addition of 10-7 M formyl-Met-Leu-Phe. Absorbance readings were made at 30-s intervals during the 5-min incubation period. The untreated neutrophils produced significant amounts of superoxide anion (i.e., increased absorbance) during the course of the experiment, whereas the experimental neutrophils treated with C. bumetii supernatant fluid did not (i.e., they exhibited only the innate baseline absorbance, which was approximately 0.07).

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0.16

?. .

0.120.10-

Ul

.4 0.06-

0.02-

DAYS TREATED

0.002

FIG. 7. Treatment of C 30

60

120

150

180

210

240

270

TME (see)

FIG. 6. Suiperoxide anion production by human neutrophils exposed to C. bumnetii 100,000 x g supernatant fluid in the presence and absence c)f heteromolybdates. Nine Mile C. burnetii organisms were purified from L929 cells that had been continuously infected for ca. 3,000 dlays. Superoxide anion production by the formyl-MetLeu-Phe-stimiulated neutrophils was assayed before (control) (0) and after exp(Dsure to 100,000 x g supernatant fluid containing 5 U of ACP activi ty alone (@) or in combination with the heteromolybdates E2 (A) and B' (A). The innate baseline absorbance was approximatel)y 0.07.

anion during the course of the continuous assay when compared with control untreated neutrophils (Fig. 5). Similar results were obtained with 50 pl of the C. burnetii supernatant preparation. When C. bumetii supernatant fluid from centrifugation at 10,000 x g was boiled, its capacity to inhibit superoxide anion production by neutrophils (data not shown) was destroyed; this is evidence that the inhibitor is probably a protein (i.e., an enzyme such as ACP). Although the enzyme has not been purified to homogeneity, it was enriched sevenfold by subjecting the Nine Mile C. burnetii 10,000 x g supernatant fluid to 100,000 x g centrifugation for 60 min followed by chromatography on a hydroxylapatite column. These enrichment steps resulted in concomitantly enhanced capacity to inhibit superoxide anion production by human neutrophils (data not shown). That ACP in the supernatant fluid is responsible for the suppressed anion production is also strongly indicated by the following results, obtained with the heteromolybdate phosphatase inhibitors. Inhibitory effect of C. burnetii supernatant fluid on superoxide anion production by neutrophils blocked by heteromolybdate compounds. Inclusion of either heteromolybdate E2 or B' blocked the C. bumnetii supernatant fluid from inhibiting superoxide anion production by human neutrophils (Fig. 6). That these compounds have been shown to inhibit ACPs from other sources (23) implicates the C burnetii ACP as the inhibitory factor in the supernatant fluid. This conclusion is indirectly supported by the following results. Heteromolybdate complex B' reduced the level of C. burnetii infection. Treatment of C bumetii-infected L929 cell populations with the heteromolybdate complex B' for a period of 15 days resulted in markedly decreased levels of infection (Fig. 7). The experiment depicted shows that the percentage of infected cells decreased in a dose-dependent manner

bumnetii-infected L929 cells with heter-

omolybdate complex B'. L929 cells continuously infected for ap-

proximately 3,200 days with Nine Mile C. burnetii organisms were treated

with complex B' (20

SLg/ml [0] and

40

.ig/ml [V]) for 15 days;

control infected cells (0) received the solvent (water) only. Aliquots were

removed on the indicated days, and the cells were stained and

examined microscopically to determine the percentage of infected cells (see Materials and Methods). L929 cell viabilities were determined at each sampling and were .98%. Control, uninfected L929 cell populations also exhibited .98% viability (data not shown). Each point on the curves represents the average of two independently treated (or control) flasks. The infectivity counts obtained for each point did not vary by more than 10%. These results are representative of two independently conducted experiments.

down to the 20 to 30% range after 11 days of treatment; untreated control populations were approximately 80% infected. After 15 days of continuous treatment, about 40% of the L929 cells were infected; in contrast, approximately 90% of the control untreated cells were infected. Also notable is the observation that the treated populations continued to proliferate with similar viabilities (298%) as control untreated infected (and uninfected) cells; this indicates that the chemical was relatively nontoxic to the host cell.

DISCUSSION In this report evidence has been presented that C. bumetii innate ACP activity. The enzyme was detected in several isolates representative of strains implicated in acute or chronic disease and was localized in the periplasmic space. The level of activity found in the parasite far exceeds that reported for other organisms, including Legionella and Leishmania spp. (21, 24). Supematant fluid derived from the parasite significantly depressed the metabolic burst of human neutrophils stimulated by the formylated peptide formyl-Met-Leu-Phe; inclusion of heteromolybdate phosphatase inhibitors relieved the inhibition. That the heteromolybdate complex B' reduced the level of infection of host L929 cells indicates that it may be specifically targeting the Coxiella ACP while sparing the host cell ACP. This is consistent with the observation that the compound had a greater inhibitory effect on the parasite ACP than on that of the L929 fibroblasts. Collectively, these results strongly indicate that ACP is the factor responsible for the inhibitory effect of the rickettsial extract. Previously we conclusively demonstrated that C. bumetii grows within phagolysosomes of host cells (2). Because of the detection of typical lysosomal markers ACP and 5'nucleotidase activity within parasite-containing vacuoles, possesses

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Burton et al. (10, 11) had earlier concluded that C. burnetii (Nine Mile isolate) proliferated within phagolysosomes of host cells. They also reported that the rickettsiae generated ACP reaction product within the periplasmic space when grown in Vero cells but did not do so when grown in L929 cells. These authors speculated that the pleomorphic appearance of the rickettsiae within Vero cells indicated that they were undergoing degradation by host lysosomal enzymes and that the ACP in the parasite periplasm was of host origin, having penetrated into the parasites (no viability studies were performed). As shown in our studies, we have discovered ACP activity in several isolates of C. burnetii grown in two different host systems, L929 cells and chicken embryos (Table 1). That Burton et al. (11) did not detect ACP in the periplasm of C. burnetii organisms grown in L929 cells may be explained by their use of P-glycerophosphate as the substrate. As shown above, we found that use of ,B-glycerophosphate resulted in only about 1 in 10 rickettsiae positive for ACP in the periplasmic gap whereas use of p-nitrophenylphosphate revealed about 50% ACP-positive organisms. These results suggest that thep-nitrophenylphosphate penetrates into the periplasmic gap better than the -glycerophosphate does; alternatively, p-nitrophenylphosphate may be a significantly better substrate for ACP. The interpretation by Burton et al. that the ACP they detected in the C. bumetii periplasm (grown in Vero cells) is host derived is probably incorrect; in all probability the phosphatase is encoded in and produced by the parasite. The importance of the oxidative metabolic burst in the destruction of intracellular parasites by phagocytes has been demonstrated by several investigators (4, 5, 9, 19, 27). We recently reported that phagocytosis of opsonized or unopsonized C. bumetii organisms failed to trigger a significant production of superoxide anion by human neutrophils (3). By failing to elicit an adequate metabolic burst (as indicated by superoxide anion production) during phagocytosis by neutrophils, C burnetii resembles other intracellular pathogens such as Mycobactenium leprae (17), Toxoplasma gondii (34), Leishmania donovani (16), and Legionella micdadei (13, 18, 31), an organism that is phylogenetically related to C. burnetii (30, 32). Successful parasitization of phagocytes by C. bumetii may be due, in part, to the inability of the phagocyte to generate adequate concentrations of microbicidal oxygen metabolites during and after rickettsial entry. That the C. bumnetii ACP may play a role in shutting down the metabolic burst and concomitant superoxide anion production is indicated by these studies. One should bear in mind the likelihood that the C bumetii periplasmic ACP plays a role in dephosphorylating various compounds, e.g., host nucleotides, that may subsequently be rephosphorylated during or after transit through the cytoplasmic membrane of the parasite. We are now in the process of purifying and further characterizing the ACP. One of our first goals will be to investigate the substrate specificity of the purified enzyme. ACKNOWLEDGMENTS We thank Louis Mallavia for providing the purified egg-grown C. burnetii organisms used in this study. We are also indebted to Michael Pope, Department of Chemistry, Georgetown University, Washington, D.C., for his generous gift of the heteromolybdate compounds. We gratefully acknowledge the assistance provided by Andrzej Pastuzyn and colleagues of the Department of Biochemistry Protein Core Facility, UNM School of Medicine. This work was supported by U.S. Public Health Service grant R01-AI-32492 from the National Institute of Allergy and Infectious

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Diseases and by a grant (to R.H.G.) from the Research Allocations Committee of the UNM School of Medicine. REFERENCES 1. Akporiaye, E. T., and 0. G. Baca. 1983. Superoxide anion production and superoxide dismutase and catalase activities in Coxiella bumetii. J. Bacteriol. 154:520-523. 2. Akporiaye, E. T., J. D. Rowatt, A. A. Aragon, and 0. G. Baca. 1983. Lysosomal response of a murine macrophage-like cell line persistently infected with Coaxiella bumetii. Infect. Immun. 40:1155-1162. 3. Akporiaye, E. T., D. Stefanovich, V. Tsosie, and 0. Baca. 1990. Coxiella bumetii fails to stimulate human neutrophil superoxide anion production. Acta Virol. 34:64-70. 4. Allen, R. C. 1977. Evaluation of serum opsonic capacity by quantitating the initial chemiluminescent response from phagocytizing polymorphonuclear leukocytes. Infect. Immun. 15:828833. 5. Babior, B. M., R. S. Kipnes, and J. T. Curnutte. 1973. Biological defense mechanisms: the production by leukocytes of superoxide, a potential bactericidal agent. J. Clin. Invest. 52:741-744. 6. Baca, O., T. Scott, E. Akporiaye, R. DeBlassie, and H. Crssman. 1985. Cell cycle distribution patterns and generation times of L929 fibroblast cells persistently infected with Coxiella bumetii. Infect. Immun. 47:366-369. 7. Baca, 0. G., E. T. Akporiaye, A. S. Aragon, I. L. Martinez, M. V. Robles, and N. L. Warner. 1981. Fate of phase I and phase II Coxiella bumetii in several macrophage-like cell lines. Infect. Immun. 33:258-266. 8. Bradford, M. M. 1976. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal. Biochem. 72:248-254. 9. Buchmuller, Y., and J. Mauel. 1981. Studies on the mechanisms of macrophage activation: possible involvement of oxygen metabolites in killing of Leishmania enrietii by activated mouse macrophages. J. Reticuloendothel. Soc. 29:181-192. 10. Burton, P. R., N. Kordova, and D. Paretsky. 1971. Electron microscopic studies of the rickettsia Coxiella bumetii: entry, lysosomal response, and fate of rickettsial DNA in L-cells. Can. J. Microbiol. 17:143-158. 11. Burton, P. R., J. Stueckemann, R. M. Welsh, and D. Paretsky. 1978. Some ultrastructural effects of persistent infections by the rickettsia Coxiella bumetii in mouse L cells and green monkey kidney (Vero) cells. Infect. Immun. 21:556-566. 12. Das, S., A. K. Saha, A. T. Remaley, R. H. Glew, J. N. Dowling, M. Kajiyoshi, and M. Gottlieb. 1986. Hydrolysis of phosphoproteins and inositol phosphates by cell surface phosphatase of Leishmania donovani. Mol. Biochem. Parasitol. 20:143-153. 13. Dowling, J. N., A. K. Saha, and R. H. Glew. 1992. Virulence factors of the family Legionellaceae. Microbiol. Rev. 56:32-60. 14. Glew, R. H., M. S. Czuczman, W. F. Diven, R. L. Berens, M. T. Pope, and D. E. Katsoulis. 1982. Partial purification and characterization of particulate acid phosphatase of Leishmania donovani promastigotes. Comp. Biochem. Physiol. 72B:581-590. 15. Gomori, G. 1952. Microscopic histochemistry, principles and practice, p. 189-194. University of Chicago Press, Chicago. 16. Haidaris, C. G., and P. F. Bonventre. 1982. A role for oxygendependent mechanisms in killing of Leishmania donovani tissue forms by activated macrophages. J. Immunol. 129:850-855. 17. Holzer, T. J., K. E. Nelson, V. Schauf, R. G. Crispen, and B. R. Andersen. 1986. Mycobacterium leprae fails to stimulate phagocytic cell superoxide anion generation. Infect. Immun. 51:514520. 18. Horwitz, M. A., and S. C. Silverstein. 1981. Interaction of the legionnaires' disease bacterium (Legionella pneumophila) with human phagocytes. II. Antibody promotes binding of L. pneumophila to monocytes but does not inhibit intracellular multiplication. J. Exp. Med. 153:398-406. 19. Murray, H. W., and Z. A. Cohn. 1979. Macrophage oxygendependent antimicrobial activity. I. Susceptibility of Toaxoplasma gondii to oxygen intermediates. J. Exp. Med. 150:938949. 20. Newburger, P. E., M. E. Chovaniec, and H. J. Cohen. 1980.

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