Characterization and Sequencing of a Respiratory Burst-inhibitingAcid Phosphatase from Francisella tularensis*

(Received for publication, October 16, 1995, and in revised form, January 16, 1996)

Thomas J. Reilly‡§, Gerald S. Baron¶i, Francis E. Nano¶, and Mark S. Kuhlenschmidt‡**

From the ‡Department of Pathobiology, College of Veterinary Medicine, University of Illinois, Urbana, Illinois 61801 andthe ¶Department of Biochemistry and Microbiology, University of Victoria, Victoria, British Columbia, Canada V8W 3P6

Acid phosphatases (Acp) of intracellular pathogenshave recently been implicated as virulence factors thatenhance intracellular survival through suppression ofthe respiratory burst. We describe here the identifica-tion, purification, characterization, and sequencing of anovel burst-inhibiting acid phosphatase from the facul-tative intracellular bacterium, Francisella tularensis.Similar to other the burst-inhibiting Acps, F. tularensisAcp (AcpA) is tartrate-resistant and has broad substratespecificity. The AcpA enzyme is unique, however, in thatit is easily released from the bacterial cell in solubleform, is a basic enzyme, suppresses the respiratoryburst of not only fMet-Leu-Phe but also phorbol 12-my-ristate 13-acetate-stimulated neutrophils and does notfit into any of the three currently recognized classes ofacid phosphatase. We also report the complete nucleo-tide sequence of the gene acpA, encoding AcpA, and thededuced primary structure of its encoded polypeptide.Comparative sequence analyses of AcpA is discussed. Toour knowledge, this is the first report describing thecloning and sequencing of a burst-inhibiting acidphosphatase.

Acid phosphatases (EC 3.1.3.2) are a ubiquitous class ofenzymes that catalyze the hydrolysis of phosphomonoesters atan acidic pH. In addition to mobilization of phosphate, somemembers of this class of enzymes perform many essential bio-logical functions including regulation of metabolism, energyconversion, and signal transduction. These enzymes have beenidentified and characterized from many eukaryotic and pro-karyotic sources and comprise several distinct subgroups basedon substrate specificity, molecular weight, and sensitivity toknown inhibitors.In the past decade, a new emphasis has been placed on

understanding the role acid phosphatases may play in micro-bial pathogenesis. Comprehensive studies of acid phosphatasespurified from Leishmania donovani (1) and Legionella micda-

dei (2) suggest that members of a class of tartrate-resistant,nonspecific acid phosphatases (TRAPs)1 may play a crucial rolein the survival of intracellular pathogens within a host’s phag-ocytic cells. An exciting discovery in these studies was thatTRAPs purified from these organisms suppressed the respira-tory burst of activated human neutrophils (3, 4). Althoughinformation is now becoming available about some of the enzy-matic, biochemical, and biophysical properties of the burst-inhibiting TRAPs, unequivocal proof of the role of these en-zymes as virulence factors in vivo has yet to be obtained.Progress toward this goal is currently limited by the lack ofprotein or gene sequence information and the absence of iso-genic TRAP mutants.Francisella tularensis is the etiologic agent of the potentially

fatal human disease tularemia and is capable of survival andmultiplication within a host’s professional phagocytes as wellas nonphagocytic cells (5, 6). Although many studies have beenconducted into the host’s immune response to Francisella in-fection, until recently relatively little attention has been fo-cused on biochemical characterization of purified macromole-cules which may function as virulence factors in theseorganisms (7). In initial studies, we found a particular strain ofF. tularensis (ATCC 6223, B38) to be enriched in acid phospha-tase activity. The Acp specific activity in this strain was greaterthan previously reported for any other bacterial or protozoanorganism. It was also easily solubilized in the absence of de-tergents allowing relatively large amounts of enzyme to bepurified to apparent homogeneity. We describe here the iden-tification, purification, and characterization of some of theunique properties of this burst-inhibiting acid phosphatase(AcpA) as well as its complete primary structure derived fromcloning and nucleotide sequencing of the AcpA gene (acpA).

EXPERIMENTAL PROCEDURES

Bacterial Strains and Materials—F. tularensis strains (ATCC 6223and 29684) were purchased from American Type Culture Collection(Rockville, MD), and strain NDBR 101 LVS was obtained from TheNational Drug Company (Philadelphia, PA). Francisella novicida waspurchased from the ATCC (15482). Strains of Mycobacteria were pro-vided by Dr. John Urbance (University of Illinois, Urbana, IL). Bacte-riological media including Bacto Cystine Heart agar (CHA) and Iso-Vitalex were obtained from Baxter (McGraw Park, IL). All otherchemicals, unless stated otherwise, were purchased from Sigma andwere of the highest purity available. Chromatography resins were pur-

* This work was supported in part by Medical Research Council ofCanada Grant MT11668 (to F. E. N.). The costs of publication of thisarticle were defrayed in part by the payment of page charges. Thisarticle must therefore be hereby marked “advertisement” in accordancewith 18 U.S.C. Section 1734 solely to indicate this fact.The nucleotide sequence(s) reported in this paper has been submitted

to the GenBankTM/EMBL Data Bank with accession number(s)L39831.§ Supported in part by a United States Department of Agricultural

Sciences National Needs Graduate Fellowship Program Grant 87-GRAD-9-0088. This work is in partial fulfillment for the degree Doctorof Philosophy in the Dept. of Pathobiology, College of Veterinary Med-icine, University of Illinois.

i Supported by a fellowship from the Natural Sciences and Engineer-ing Research Council of Canada.** Recipient of a grant from the University of Illinois Research Board.

To whom correspondence should be addressed.

1 The abbreviations used are: TRAP, L-(1)-tartrate-resistant acidphosphatase; acpA, acid phosphatase encoding gene; BSA, bovine se-rum albumin; CHA, Cystine Heart agar; fMLP, N-formyl-methionyl-leucyl-phenylalanine; AcpA, Francisella tularensis acid phosphatase;HPLC, high pressure liquid chromatography; MES, 2-(N-morpholino)ethanesulfonic acid; MUP, 4-methylumbelliferylphosphate; PMA, phor-bol 12-myristate 13-acetate; PAGE, polyacrylamide gel electrophoresis;PLC, phospholipase C; FPLC, fast protein liquid chromatography; IP3,inositol 1,4,5-trisphosphate; PTPase, peptide-tyrosine phosphatase;PIP, phosphatidylinositol phosphate; CHAPS, 3-[(3-cholamidopro-pyl)dimethylammonio]-1-propanesulfonic acid.

THE JOURNAL OF BIOLOGICAL CHEMISTRY Vol. 271, No. 18, Issue of May 3, pp. 10973–10983, 1996© 1996 by The American Society for Biochemistry and Molecular Biology, Inc. Printed in U.S.A.

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chased from Pharmacia Biotech Inc. Protein electrophoresis reagentsand ampholytes were obtained from Bio-Rad Laboratories. SDS-PAGEmolecular weight standards were obtained from Integrated SeparationSystems (Hyde Park, MA) or NOVEX (San Francisco, CA). Heteropoly-molybdate complexes were gifts from Dr. Robert Glew (University ofNew Mexico School of Medicine, Albuquerque, NM).Culture Conditions—F. tularensis strains 6223, 29684, NDBR 101

LVS, and F. novicida were cultured on hemoglobin-enriched BactoCystine Heart agar for 1–5 days at 37 °C. The organisms were passagedonce after being received from ATCC; aliquots were then frozen at280 °C and used for inoculation of CHA for purification of the enzyme.Bacteria were harvested by scraping the cultures from the agar. Har-vested material was suspended in 100 ml of buffer A (50 mM sodiumacetate buffer, pH 6.0).Screening of F. tularensis Hydrolase Activities—Bacterial cultures

from CHA were resuspended to a protein concentration of 1 mg/ml,200-ml aliquots were added to api-ZYM® strips (bioMerieux Vitek, Inc.,Hazelwood, MO), the strips were incubated for 12 h at 37 °C and thenanalyzed for semiquantitation of F. tularensis hydrolase activities ac-cording to the manufacturer’s instructions.Enzyme Assays—Acp activity was measured fluorometrically using

an Aminco-Bowman spectrophotofluorometer. The 0.3-ml standard as-say mixture contained 0.2 M sodium acetate buffer, pH 6.0, 1.0 mM

4-methylumbelliferyl phosphate (MUP), and varying amounts of en-zyme. The mixtures were incubated at 37 °C for 15 min and 1.2 ml of 0.5M glycine, pH 10, was added to stop the reaction. Under these condi-tions, enzyme activity was linear with the amount of enzyme added.During kinetic experiments, enzyme activity was linear with time for atleast 60 min. Only initial rates (slopes within the first 15 min) wereused for calculation of enzyme activity and associated kinetic parame-ters. One unit of enzyme activity is defined as the amount of enzymerequired to convert 1 nmol of substrate to product per h. Assays todetermine the pH optimum were performed using either 0.2 M MES or0.2 M HEPES as the buffer, and the final substrate concentration was1.0 mM. Determination of the Michaelis-Menten constant for MUP andtyrosine phosphate was performed using 0.06 unit of AcpA and a widerange (Km/10 to 5 Km) of each substrate. Replicates of five were testedat each substrate concentration. Data were analyzed using a nonlinear,least squares regression computer program (8) graciously supplied byDr Stephen P. J. Brooks, Carleton University, Ottawa, Canada. Phos-pholipase C (PLC) activity was measured by monitoring the hydrolysisof p-nitrophenylphosphorylcholine as described previously (9).Substrate Specificity Assays—Substrate specificity was determined

by measuring the release of inorganic phosphate from phosphomo-noester substrates (including MUP) using the method of Lanzetta et al.(10). This assay was also used for the determination of the pH optimumof AcpA for phosphomonoesters other than MUP. Phosphatidylinositolphosphates were assayed in the presence of 1.0% Triton X-100.Peptide-tyrosine Phosphatase Activity of AcpA—A synthetic peptide

p60src (TEPQpYQPGE) containing a single phosphorylated tyrosinewas synthesized by the University of Illinois Genetic Engineering fa-cility according to a previously described method (11). Purity of thepeptide was assessed by reversed-phase HPLC on a Vydac 218TP54analytical column, and the product was found to be 98% pure. Massspectrometry analysis of the peptide gave the expected molecular ion,and the amino acid analysis was within 5% of the expected values in allcases. AcpA catalyzed dephosphorylation of the monophosphorylpeptideand determination of kinetic parameters were performed as describedabove.Purification of F. tularensis Acid Phosphatase (Acp)—All procedures

were conducted at 4 °C unless otherwise noted. The bacterial culture(16 g obtained by scraping bacteria growth from 100 CHA plates (150mm)) was suspended in buffer A and homogenized using a motor drivenPotter-Elvehjem homogenizer. An equal volume of an extraction bufferconsisting of buffer A containing 2 M NaCl, 0.5% sodium cholate, 0.2 mM

EDTA, 0.2 mM dithiothreitol, 75 mg/ml phenylmethylsulfonyl fluoride,and 5 mg/ml Pepstatin A was added to the homogenate, the mixture wasstirred for 12 h and centrifuged at 200,000 3 g for 1.5 h. The superna-tant, at a protein concentration of 5 mg/ml, was dialyzed for 12 h at4–6 °C against three changes (6 liters each) of buffer A. This dialyzedsupernatant, designated supernatant I, was again centrifuged at200,000 3 g to remove a precipitate which had formed during dialysis.This second supernatant, containing 97% of the starting activity, wasdesignated supernatant II. Supernatant II (210 ml) was applied to aS-Sepharose cation exchange column (3 3 18 cm) pre-equilibrated withbuffer A. The column was washed with 500 ml of buffer A and a 0–0.5M linear NaCl gradient (600 ml) in buffer A was applied to the columnat 0.5 ml/min. A single peak of phosphatase activity was eluted between

0.17 and 0.26 M NaCl. Active fractions were pooled and concentrated byultrafiltration. The concentrated sample was then applied and eluted(0.2 ml/min) from a Sephadex G-100 superfine column (1.5 3 95 cm)equilibrated in buffer A containing 0.3 M NaCl. The sample eluted as asingle peak, and fractions containing Acp activity were pooled andconcentrated as described above. The sample (1.2 ml) was then appliedin four separate 0.3-ml aliquots to a Superdex 75 HR 10/30 FPLCcolumn and eluted with buffer A containing 0.3 M NaCl at 0.5 ml/min.Fractions were collected, analyzed for Acp activity, and monitored forprotein purity by SDS-PAGE. Enzymatic activity in fractions otherthan those two containing the highest activities were contaminated andthus not pooled. The purification results are summarized in Table II.Radioiodination—Iodination of AcpA was performed using IODO-

GEN (Pierce). Ten mg of pooled Acp from the Superdex 75 column wasadded to an IODOGEN-coated tube containing 10 ml of 0.5 M Tris buffer,pH 7.5, and 0.5 mCi of 125I. The reaction was incubated at roomtemperature for 3 min, after which 200 ml of a 10 mg/ml solution of KIwas added to stop the reaction. Labeled enzyme was separated fromunincorporated 125I by desalting on a GF-5 Excellulose column (PierceChemical Co.) pretreated with 1.0 ml of a 10 mg/ml suspension of BSAand equilibrated in 0.5 M Tris, pH 7.5. Void volume fractions containingradioactivity were pooled and analyzed by SDS-PAGE andautoradiography.Preparation of Rabbit Anti-F. tularensis Acp (AcpA) Antisera—Puri-

fied AcpA (719 mg) was dialyzed against 0.9% NaCl, filter-sterilized,and emulsified in complete Freund’s adjuvant. The immunogen wasthen injected subcutaneously at multiple sites into a New ZealandWhite rabbit. Twenty six days after primary immunization, the im-mune response was boosted by a single subcutaneous injection with 200mg of purified AcpA emulsified in Ribi Adjuvant (Ribi Biologicals).Serum was collected by ear vein puncture 7 days following the secondinjection.Purification of Anti-Acp Antibodies—Monospecific anti-AcpA anti-

bodies (IgG) were purified from anti-AcpA antisera by repeated absorp-tion and centrifugation with nonrelevant antigen as described previ-ously (12). Nonrelevant antigen used was either pellet I obtainedfollowing removal of supernatant I during purification of AcpA as de-scribed above or an E. coli Y1090 freeze-thaw extract. Anti-AcpA IgGwas then purified from the adsorbed antiserum by protein A-Sepharoseaffinity chromatography.Polyacrylamide Gel Electrophoresis and Detection of Acid Phospha-

tase by Western Blot—Sodium dodecyl sulfate-PAGE was performed asdescribed by Laemmli (13). Polyacrylamide gels were 3% T stacking and7.5% T resolving. Molecular weight of acid phosphatase was estimatedusing NOVEX Mark 12 molecular weight standards and GelReader forMacintosh Version 2.0 software (University of Illinois National Centerfor Supercomputing Applications). Western blot detection of AcpA wasperformed as described previously (14). Purified rabbit anti-F. tularen-sis AcpA antibody was used as the primary antibody (1:12,000 dilution),and goat anti-rabbit IgG (H 1 L) was conjugated to alkaline phospha-tase as the secondary antibody (1:1000 dilution).Isoelectric Focusing—Purified acid phosphatase (7.5 3 104 units) was

applied to an LKB 8100 Ampholine apparatus in a 5–25% (w/w) linearsucrose gradient containing 4% (w/v) ampholytes (pH 3–10). Cathodeand anode buffer were 1.0 M NaOH and 1.0 M H3PO4, respectively.Focusing was performed at 3 watts for 72 h at 15 °C.Mass Spectrometry of F. tularensis Acid Phosphatase—The purified

acid phosphatase was subjected to matrix-assisted laser desorptiontime of flight mass spectrometry using a VG TofSpec mass spectrome-ter. Approximately 5 pmol of AcpA was embedded in a matrix of sina-pinic acid and irradiated at 337 nm. The instrument is equipped with a33 nm nitrogen laser with a 5-ns maximum pulse width, a 50-mJminimum output, and a 150 3 250 micron spot size. Data acquisitionand processing were performed by a VG OPUS data system and aVAXstation 4000 computer.Isolation of Neutrophils—Neutrophil-enriched cell fractions were iso-

lated from freshly collected normal porcine blood (50 ml) as describedpreviously (15). The neutrophil fraction was resuspended in HEPES/NaCl buffer (200 mMHEPES, 0.9% NaCl, pH 7.3) to 13 107 cells/ml andstored on ice until use (within 2 h of isolation) in respiratory burstassays.Measurement of Respiratory Burst in Neutrophils—Respiratory burst

activity of isolated porcine neutrophils was measured in the presenceand absence of AcpA by following the production of superoxide usingmodifications of a previously described method (16). Briefly, the super-oxide dismutase-inhibitable reduction of ferricytochrome c at 550 nmwas continuously measured at 37 °C using either a Beckman DU-50spectrophotometer or an Aminco dual-beam recording spectrophotom-

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eter (DW 2000). The standard assay (0.4 ml) was performed in HEPES/NaCl buffer containing 25 mM HEPES, 150 mM NaCl, 0.90 mM CaCl2,and 0.50 mM MgCl2, pH 6.8 instead of the modified Dulbecco’s phos-phate-buffered saline medium. O2

2 production was initiated by eitherthe addition of 1 ml of PMA (1 mg/ml in dimethyl sulfoxide) or 5 ml offMLP (100 mM in dimethyl sulfoxide). The purified acid phosphatasesample used in these studies was also analyzed for superoxide dis-mutase activity (17), catalase activity (18), and as a direct scavenger ofsuperoxide using a xanthine oxidase assay (19).Cyanogen Bromide Cleavage of AcpA—Cyanogen bromide cleavage of

AcpA was done as described previously (20). Briefly, 50 mg of AcpA wasdigested in the dark at 20 °C for 20 h. The resultant peptide sample wasanalyzed by gel electrophoresis using 10–20% polyacrylamide gradientgels. Separated peptides were electroblotted to PVDF using standardconditions. The membrane was then partially destained, air-dried andsubmitted to the University of Illinois Molecular Genetics Facility forsequence analyses.N-terminal Amino Acid Sequence—The N-terminal amino acid se-

quence of AcpA was performed using automated Edman degradationand a model 470A Applied Biosystems gas phase Sequencer equippedwith a 120A phenylthiohydantoin-amino acid analyzer by the GeneticEngineering Facility of the University of Illinois Biotechnology Center.Oligonucleotide Synthesis and Gene Cloning—A nondegenerate oli-

gonucleotide (59-ACI GAT GTI AAT AAT III AAA CCI AAT GAT TATGG-39) was prepared (Applied Biosystems 319 DNA Synthesizer) byreverse translation of the N-terminal peptide sequence. The codonusage in the valAB locus of F. novicida (21) was used as a guide indesigning the oligonucleotide. The oligonucleotide was 39-end-labeled(ECL 39-oligolabeling system, Amersham) as per manufacturer’s in-structions and used to screen a F. tularensis ATCC 29684 genomiclibrary of partial Sau3AI fragments cloned into the BamHI site of thephagemid vector pTZ18U (Bio-Rad). One of the hybridizing clones con-tained a 1.3-kilobase DNA insert. Partial sequencing of this insertrevealed one open reading frame (ORF) with a deduced amino acidsequence identical with the N-terminal amino acid sequence and to thesequence of an internal CNBr-generated peptide. This insert was usedas a probe to identify a 3.1-kilobase HindIII fragment of F. novicidaDNA that was cloned into pUC18 (22). BLASTP and BLASTX (23) wereused to search for amino acid sequence similarities among the databases available on-line throughout the National Center for Biotechnol-ogy Information. Pairwise alignments were done using FASTA (24) andmodified by inspection. A 1798-base pair region was sequenced on bothstrands using a commercial T7 DNA polymerase (Sequenase, U. S.Biochemical Corp.) or Taq DNA polymerase (TaqTrack, Promega) usingboth universal and custom-designed primers. The gene encoding AcpAwas designated acpA and was assigned the GenBank accession numberL39831.We chose to sequence the F. novicida acpA gene to facilitate future

genetic experiments which can most easily be done in F. novicida.Although 16 S RNA and DNA relatedness (25) studies clearly identify F.novicida as a F. tularensis strain (26), biohazard rules place strictureson the transfer of genes between F. novicida and F. tularensis.Protein Determination—Protein concentrations were determined us-

ing bicinchoninic acid (Micro BCA Protein Assay Reagent, Pierce) asdescribed previously (27). Human albumin/g-globulin protein standard(Sigma) was used as a standard.

RESULTS

Detection of Acid Phosphatase Activity in F. tularensis—Acidphosphatase specific activities varied markedly between spe-cies of Francisella and among strains of F. tularensis. F. tula-rensis strain 6223 displayed the highest specific activity. It wasgenerally in excess of 18,000 units/mg (13,000 to 30,000) andrepresents, to our knowledge, the highest specific activity everreported for a bacterial or protozoan acid phosphatase. In com-parison to other Acp-enriched intracellular pathogens (Table I),F. tularensis strain 6223 Acp specific activity is greater than 10times that of L. micdadei (28), more than 4 times that ofCoxiella burnetii strain PRS Q177 strain (29), and about twicethat of the protozoan parasite, L. donovani (3). Acp specificactivity in strain NDBR 101 was 550 to 3089 units/mg, whereasstrain 29684 Acp specific activity was only 100 units/mg. TheAcp specific activity of F. novicida was approximately 1700units/mg.The rather wide variation in acid phosphatase specific activ-

ity among members of the Francisella genus may correlatewith the passage history of individual strains. During experi-ments aimed at optimizing expression of Acp, we observed alarge decrease in Acp specific activity upon repeated passage ofstrain 6223 on CHA (data not shown). A loss of almost 90%(8–10-fold reduction) of the starting Acp specific activity wasseen following 9 passages. The reduction was most likely notdue to the accumulation of reversible inhibitors since washingthe cells in physiological saline followed by extraction of Acpfailed to increase Acp specific activity, and mixing of extractsfrom passaged cultures with purified AcpA did not result in theinhibition of the activity of the purified enzyme. Furthermore,detection of AcpA by Western blot analysis indicated a markedreduction in anti-AcpA reactive material following 9 passagesas compared to that found in initial cultures (data not shown).Therefore, single passage F. tularensis (6223) was selected asthe source for enzyme purification.AcpA Purification—In initial attempts to solubilize the en-

zyme, we found at least 70% of the phosphatase activity couldbe extracted with 1 M NaCl alone; including sodium cholate inthe extraction buffer resulted in complete solubilization of theenzyme. Furthermore, essentially no difference in total AcpAactivity was observed in the extracted material compared to theactivity exhibited by intact bacteria (Table II). All of the enzy-matic activity detected in intact bacteria was solubilized by thecholate NaCl extraction buffer and remained in the superna-tant following extensive dialysis and two centrifugations(200,000 3 g, 1.5 h). The soluble AcpA was completely retainedduring loading at pH 6 on cation exchange resins S-Sepharoseand Mono S and eluted as a single peak of activity between 0.17

TABLE IISummary of the purification of AcpA

Acid phosphatase was measured using MUP as substrate as de-scribed under “Experimental Procedures.” 1 unit 5 1 nmol of MUPhydrolyzed/h.

Purificationstep Total activity Total

protein Specific activity Purification Yield

units 3 107 mg units 3 104/mg -fold %

Whole cells 4.06 3036 1.34 1 (100)Super I 4.41 1062 4.15 3 109Super II 3.94 801 4.92 4 97S-Sepharose 3.83 88.6 43.23 32 94SephadexG-100

3.36 6.1 551 411 83

Superdex 75 1.05 1.1 955 713 26

TABLE IComparison of acid phosphatase activities among various

microorganismsCrude extracts of the indicated organisms were prepared adjusted to

a protein concentration of 1 mg/ml and acid phosphatase specific activ-ity was determined using MUP as described under “Experimental Pro-cedures.”

Strain Acp specific activity

Francisella tularensis (ATCC 29684) 100Emeria vermiformis 135a

Mycobacteria chelonae 204Escherichia coli Y1090 372Mycobacterium thume 388Salmonella typhimurium SB137 532Legionella micdadei 640a

Escherichia coli K300 791Francisella novicida 1729Francisella tularensis LVS NDBR 101 3087Mycobacterium sengal 3628Coxiella burnetii PRS Q177 3840a

Leishmania donovani 8360a

Francisella tularensis B-38 (ATCC 6223) 18138a Values obtained from the literature (see text).

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M and 0.26 M NaCl (Fig. 1A). AcpA eluted in the breakthroughvolume, however, during attempted anion exchange chroma-tography on either Q-Sepharose or Mono Q at pH 7.3.The material recovered from cation exchange chromatogra-

phy was enriched 32-fold in acid phosphatase activity andcontained 94% of the starting activity. Gel filtration chroma-tography through Sephadex G-100 superfine (Fig. 1B) resultedin an additional 13-fold increase in specific activity with 83%recovery of the applied activity. Final purification of the en-

zyme was achieved by gel filtration FPLC (Fig. 1C). This stepresulted in a further 1.7-fold increase in specific activity with26% of the sample recovered in a single protein peak coincidentwith AcpA activity. The apparently low recovery from theFPLC column is explained by the conservative pooling of AcpAactive fractions as described under “Experimental Procedures.”The actual recovery was approximately 75%, but only the twofractions containing the highest AcpA activity were pooled forfurther analyses. Overall, AcpA was purified 713-fold over thatin intact bacteria (Table II). The purification behavior of AcpAfrom strains 6223, NDBR 101, and 29684 and the results ofcomparative molecular weight (Fig. 2A) and immunoreactivitywith rabbit anti-Ft (6223) AcpA IgG (Fig. 2B) suggested theenzyme is very similar in all strains of F. tularensis. Also, theenzyme activity chromatographed as a single entity throughout

FIG. 1. Purification steps of F. tula-rensis acid phosphatase. For A–C,AcpA activity (●) and protein concentra-tion (E). A, S-Sepharose cation exchangechromatography of Supernatant II con-taining AcpA using a 0 to 0.5 M NaCllinear gradient (O) as described under“Experimental Procedures.” Twenty-one6.0-ml fractions (38–58) found to containAcpA activity eluted between 0.17 and0.26 M NaCl. B, Sephadex G-100 Super-fine chromatography of pooled and con-centrated AcpA from S-Sepharose (5.3 ml,6.7 mg/ml protein). Application and elu-tion of AcpA to this gel filtration resin wasperformed as described under “Experi-mental Procedures.” C, Superdex 75 HR10/30 FPLC chromatography of a 0.3-mlaliquot of pooled Acp activity from Seph-adex G-100. D, SDS-PAGE separation ofsamples from the purification procedure.From left to right: lane 1, Novex Mark 12molecular weight standards; lane 2, 30 mgof whole F. tularensis; lane 3, 30 mg ofsupernatant I; lane 4, 30 mg of superna-tant II; lane 5, 30 mg of S-Sepharose pool(fractions 38–58); lane 6, 30 mg of Seph-adex G-100 pool (fractions 47–59); lane 7,8 mg of AcpA from Superdex 75 FPLC.

FIG. 2. SDS-PAGE and Western blot analyses of acid phospha-tase from three strains of F. tularensis. A, Novex standards, asdescribed for Fig. 1 (lane 1), 30 mg of extracted proteins from F. tula-rensis strains NDBR 101, 29684, and 6223 (lanes 2–4), and 8 mg ofpurified acid phosphatase from these same strains (lanes 5–7) weresubjected to SDS-PAGE and stained with Coomassie Blue R-250. B,Western blot analysis of blotted acid phosphatases from F. tularensisstrains NDBR 101, 29684, and 6223 (lanes 1–3) using rabbit anti AcpA(6223) IgG.

FIG. 3. Evaluation of AcpA purity by radioiodination of pooledfractions from Superdex 75 chromatography. Ten mg of pooledAcpA from Superdex 75 gel filtration chromatography was iodinated asdescribed under “Experimental Procedures” and subjected to SDS-PAGE and autoradiography. The position of molecular weight markersare shown on the far left of the autoradiograph. The 5 lanes to the rightof the markers (lane 1) contain 2, 4, 6, 8, and 10 ml, respectively, of the1.5-ml void volume from the desalting column. Molecular weight stand-ards are: b-galactosidase (116,000), phosphorylase b (95,000), BSA(68,000), glutamic dehydrogenase (55,000), carbonic anhydrase(29,000), and lysozyme (14,000).

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all purification steps suggesting that multiple acid phosphata-ses may not exist in F. tularensis in contrast to the resultsreported for some other facultative intracellular organisms(1, 2).AcpA Purity and Molecular Weight—The purity of AcpA was

assessed in several experiments. 1) SDS-PAGE of samples ob-tained throughout the purification procedure demonstrate thepresence of an ;57-kDa protein which was continuously en-riched as the purification proceeded and electrophoresed as asingle Coomassie Blue or silver (data not shown)-stained bandfollowing recovery from the final FPLC gel filtration step (Fig.1D). 2) In an effort to visualize minor protein contaminants orthose which may be refractory to staining, the purified AcpAfraction (Superdex 75 fraction) was radioiodinated, subjected toSDS-PAGE, and the 125I-labeled proteins were visualized byautoradiography. A single major band was seen on autoradio-graphs as increasing amounts of the radiolabeled AcpA fractionwere applied to the SDS-PAGE gel (Fig. 3). This band, com-prising 98% of the total signal as measured by quantitativedensitometry, had a molecular weight of approximately57,000. 3) N-terminal amino acid sequence analysis throughthe first 20 amino acids revealed the presence of a singlethreonine residue at the N terminus of the sequence(TDVNNSKPNDYGTLVKIEQK).The molecular mass of the purified enzyme was determined

by gel filtration chromatography, SDS-PAGE, and matrix-as-sisted laser desorption time of flight MS. Superdex 75 FPLC gelfiltration chromatography gave a partition coefficient for AcpAof 0.09 (Fig. 4A). This value was compared to the regression

FIG. 4. Estimation of the molecularweight of AcpA. A, regression line (●) ofthe log molecular weight of the gel filtra-tion standards versus their respectivepartition coefficients: BSA (67,000 Kav 50.035), ovalbumin (43,000 Kav 5 0.165),chymotrypsin (25,000 Kav 5 0.324), andRNase A (13,700 Kav 5 0.501). Elutionposition and partition coefficient of AcpAare indicated by arrow. B, regression lineof log molecular weight standards (Fig. 1)versus electrophoretic mobility. Mobilityand estimated molecular weight of AcpAare indicated by the arrow. C, matrix-assisted laser desorption time of flightprofile of purified AcpA. M1 5 m/z55759.4. Matrix, sinapinic acid; laserwavelength, 337 nm.

FIG. 5. Determination of pH optimum. A–D, purified AcpA wasincubated with 1 mM indicated substrate in either 0.2 M MES (●, pKa6.10) or 0.2 M HEPES (E, pKa 7.48) at varying pH values. Acp activitywas determined by the Lanzetta assay for inorganic phosphate asdescribed under “Experimental Procedures.” Data are plotted as per-cent of optimal activity for each substrate. A, 59-AMP; B, Glc-6-PO4; C,MUP; D, tyrosine phosphate.

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line generated from the four molecular weight standards, andthe Kav corresponded to an apparent molecular weight of56,000. A similar value, 57,000, was obtained with SDS-PAGE(Fig. 4B) using both reducing and nonreducing conditions (datanot shown). Finally, mass spectrometry of AcpA indicated asingly charged species at 55,759 atomic mass units with a massaccuracy of 0.1% (Fig. 4C).AcpA pH Optimum and Isoelectric Point—The purified AcpA

behaved as an acid phosphatase (acid pH optimum) in allbuffers tested (Fig. 5). Although the activity was slightly less inMES and HEPES than in acetate buffer, the optimal pH was6.0 and activity was markedly diminished at 2 pH units to

either side of this optimum. The pH optimum was independentof phosphomonoester substrates assayed including adenosinemonophosphate (Fig. 5A), glucose 6-phosphate (Fig. 5B), tyro-sine phosphate (Fig. 5D), and phosphorylated tyrosine residue

FIG. 6. Purified AcpA (7.5 3 104 units) was mixed into a 5–25%w/w sucrose gradient containing 4% w/v Ampholytes pH 3–10and focused in a LKB 8100 isoelectric focusing column at 3 wattsfor 72 h at 15 °C. After focusing, the gradient was fractionated into1.0-ml fractions from which Acp activity (●) and pH (E) values weredetermined.

FIG. 7. Estimation of the Km and Vmax for AcpA with threedifferent substrates. Each substrate was incubated with purifiedAcpA at final concentrations from 0.04 to 1.6 mM in 0.2 M sodiumacetate buffer, pH 6.0. The reactions were incubated for 15 min at37 °C; quantitation of phosphatase activity was performed using theassay for inorganic phosphate as described under “Experimental Pro-cedures.” Each point represents the average of 5 separate samples foreach concentration indicated. A and B show substrate saturation andLineweaver-Burk plot of AcpA incubated with MUP (●) and tyrosinephosphate (E). C and D show similar plots when the phosphorylatedsubstrate p60src was used as substrate. Inset of C is the pH optimum ofAcpA’s PTPase activity.

TABLE IIISubstrate specificity of F. tularensis acid phosphatase

All substrates were tested at a final concentration of 1 mM exceptphosvitin and yeast mannan (10 mg/ml). Inositol phosphates and phos-phatidylinositol phosphates were used at concentrations of 40 mM in 1%Triton X-100. All measurements were made in quadruplicate and in atleast two separate experiments.

Substrate Relative activity

% MUP

MUP 100O-Phospho-DL-tyrosine 102Phenylphosphate 98Inositol 1-phosphate 93AMP 93ATP 89p-Nitrophenyl phosphate 89Mannose 6-phosphate 85Phosphoenolpyruvate 82Phospho-L-serine 79Fructose 1,6-bisphosphate 76Glucose 6-phosphate 76b-NADP 63Fructose 6-phosphate 60Pyridoxal phosphate 58Ribose 5-phosphate 56O-Phospho-DL-threonine 51Inositol 4-phosphate 28Inositol 1,4,5-trisphosphate 15Inositol cyclic phosphate 3PIP ,1PIP2 ,1Phytic acid ,1Yeast mannan ,1Phosvitin ,1Cysteamine phosphate NDa

a ND, none detected.

TABLE IVEffects of various compounds on F. tularensis acid phosphatase

Acid phosphatase activity was tested in the presence and absence ofeach of the indicated inhibitors as described under “Experimental Pro-cedures.” Each inhibitor, solubilized in dH2O, was tested for effect onpH of the assay before use as an inhibitor.

Inhibitor I50% Max concentration tested

mM

Mercury chloride 0.5 5 mM

Mb complex E2 5 10 mM

Ferrous sulfate 22 150 mM

Arsenic acid 36 250 mM

Hydroxymercuriphenylsulfonate 40 160 mM

Zinc chloride 50 100 mM

Cupric sulfate 56 56 mM

Ammonium molybdate 90 100 mM

Sodium vanadate 162 162 mM

Silver nitrate 200 1 mM

Sodium phosphate 1,800 15 mM

Sodium dithionate 14,900 25 mM

Dithiothreitol 25,000 25 mM

CHAPS NIa 0.1%Triton X-100, 114 NI 1%Okadaic acid NI 1 mM

EDTA, EGTA NI 20 mM

Sodium fluoride NI 20 mM

L-(1)-Sodium tartrate NI 50 mM

Threonine NI 10 mM

Serine NI 10 mM

Glycerol NI 1.4 M

a NI, no inhibition.

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of p60src (Fig. 7C, inset). When the purified AcpA was subjectedto isoelectric focusing (pH 3–12), a single peak of activity wasfound at pH 9.2 (Fig. 6). The basic pI of this enzyme is consist-ent with its fractionation behavior during ion exchange chro-matography (Fig. 1A).Substrate Specificity—AcpA has a broad in vitro substrate

specificity (Table III). Sixteen of the 26 phosphomonoesterstested were hydrolyzed at greater than 50% the rate of MUP.The most active physiological substrates included tyrosinephosphate, AMP, ATP, and mannose 6-phosphate. Of the ino-sitol phosphates tested, the monophosphates were preferredsubstrates. Inositol 1-monophosphate was hydrolyzed at nearthe same rate as MUP while inositol 4-phosphate was hydro-lyzed at only 28% the rate of MUP. Inositol 1,4,5-trisphosphate(IP3) was also recognized as a substrate, although it was hy-drolyzed at only 15% the rate of MUP. Inositol cyclic phosphatewas the most slowly hydrolyzed substrate and may be a conse-

quence of its cyclic nature. In contrast to some of the inositolphosphates which were good substrates for AcpA, phosphati-dylinositol phosphate derivatives, PIP and PIP2, were poorsubstrates. In general, these studies showed that smallphosphomonoesters were more easily hydrolyzed than larger ormultiphosphorylated compounds. For example, yeast mannan,phosvitin, and phytic acid were not recognized as substrates byAcpA. The acidic pH optimum of AcpA, and, more importantly,its inability to hydrolyze the thiophosphate substrate, cysteam-ine phosphate, which is an alkaline phosphatase-specific sub-strate, is consistent with the designation of AcpA as an acidphosphatase.Determination of Kinetic Parameters and Peptide-tyrosine

Phosphatase (PTPase) Activity of AcpA—The Km of AcpA forMUP and tyrosine phosphate was estimated to be 0.25 mM and0.27 mM, respectively (Fig. 7) at pH 6.0. In addition to tyrosinephosphatase activity, AcpA displayed readily measurable PTP-ase activity. The Km of the monophosphorylated peptide p60src

(determined by the release of inorganic phosphate) was 0.34mM. The Vmax values were 9.6 3 106, 8.0 3 106, and 6.7 3 106

nmol of Pi released per h per mg of enzyme for MUP, tyrosinephosphate, and p60src, respectively (Fig. 7, B and D).Effect of Inhibitors—To further characterize and classify this

new AcpA, we measured the effects of acid phosphatase inhib-itors. As shown in Table IV, the enzyme is not inhibited byL-(1)-sodium tartrate, sodium fluoride, okadaic acid, divalentcation chelators (EDTA, EGTA), or detergents (CHAPS, TritonX-100, Triton X-114). However, the enzyme was sensitive to theearly transition metal oxyanions such as molybdate and vana-date. As is true of the acid phosphatases described for otherintracellular pathogens (2, 28), this enzyme was sensitive tothe heteropolymolybdate complex E2. Monofunctional sulfhy-dryl group reagents such as mercury and silver inhibited theenzyme by 50% at 0.5 mM and 290 mM, respectively. Hy-droxymercuriphenylsulfonate, a potent inhibitor of bovine liveracid phosphatase (30) inhibited AcpA by 50% at a concentrationof 50 mM. Zinc was also found to be an inhibitor of the enzyme;50 mM ZnCl2 inhibited AcpA activity by 50%. Inorganic phos-phate was found to be a competitive inhibitor with a Ki ofapproximately 50 mM (data not shown). Glycerol, serine, andthreonine had no inhibitory effect.AcpA-mediated Inhibition of the Respiratory Burst in Neu-

trophils—In preliminary experiments, we found that a highspeed supernatant from a crude F. tularensis extract contain-ing an intense, heat-labile acid phosphatase activity was capa-ble of dose-dependent inhibition of fMLP-activated porcineneutrophils. When this supernatant was subjected to gel filtra-tion chromatography, the AcpA and respiratory burst inhibi-tory activities eluted coincidentally. To determine if AcpA wasresponsible for burst inhibition, porcine neutrophils weretreated with the purified enzyme prior to activation with eitherfMLP or PMA. Under these conditions, AcpA caused a dose-de-pendent inhibition of the respiratory burst when added toeither fMLP- or PMA-activated porcine neutrophils (Figs. 8, Aand B). The inhibition was also seen when AcpA was addedfollowing PMA or fMLP addition but required larger amountsof enzyme, monitoring superoxide formation for longer times,and was seen only after a lag of 2–3 min following addition ofAcpA except when the highest amounts of AcpA were used(data not shown). A greater inhibitory effect was obtained bypreincubation of the neutrophils with AcpA prior to activation(Fig. 8C). Maximum burst inhibition was seen following prein-cubation for 15 min at 37 °C. Heat-inactivated AcpA had noeffect on the rate of superoxide formation in activated neutro-phils (Fig. 8A). We also did not detect catalase or superoxidedismutase activities in AcpA (data not shown), and AcpA had

FIG. 8. AcpA-mediated inhibition of the respiratory burst inporcine neutrophils. Isolated porcine neutrophils (1 3 107 cells/ml)were incubated with purified AcpA prior to addition of either PMA orfMLP. Superoxide anion production was determined by continuousspectrophotometric measurements of the reduction of ferricytochrome cat 550 nm. A, each point (v) represents the mean of the rate of cyto-chrome c reduction 6 S.D. from 5 separate experiments by porcineneutrophils after a 15-min preincubation with AcpA and activated withPMA; f, heat-inactivated AcpA (100 °C, 15 min). B, comparison of theamount of superoxide anion production as measured by reduction offerricytochrome c by fMLP-stimulated porcine neutrophils after a 15-min exposure to 1000 units of AcpA (OO) or without prior exposure toAcpA (— ——). C, effect of increasing preincubation times of porcineneutrophils with purified AcpA (8000 units) on production of superoxideanion in PMA-activated neutrophils.

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no inhibitory effect on the rate of xanthine oxidase-catalyzedgeneration of superoxide (data not shown). Thus, it is unlikelythis enzyme affects the respiratory burst indirectly throughelectron scavenging.Nucleotide Sequencing and Deduced Primary Structure of

acpA—To further characterize the structure and function ofAcpA, we cloned and sequenced the AcpA structural gene(acpA). A nondegenerate oligonucleotide was prepared andused to screen a F. tularensis ATCC 29684 and subsequently aF. novicida genomic library (see “Experimental Procedures”).The complete acpA nucleotide sequence and derived primarystructure is shown in Fig. 9. The first 21-amino acid sequenceof the open reading frame, prior to the N-terminal Thr residueof AcpA, contains many of the functional elements of a stand-ard Gram-negative signal peptide (31). The next 20-amino aciddeduced sequence is identical with the N-terminal sequence(TDVNNSKPNDYGTLVKIEQK) determined by Edman degra-dation of the purified AcpA. Furthermore, the deduced se-quence (MYPNAKNPEGE) at position 422–454 was identicalwith the peptide sequence determined by Edman degradationof a CNBr fragment of AcpA. The molecular weight of thesignal peptide cleaved AcpA predicted from the nucleotide se-quence (55,593) is in close agreement with the molecularweight of AcpA (55,759) determined by mass spectrometry.These data strongly indicate the nucleotide sequence presentedin Fig. 9 contains the complete open reading frame of the acpAgene.

Comparative sequence analyses (Blast, National Center forBiotechnology Information) indicate acpA has no overall se-quence similarity to other known acid phosphatases, but it ispartially similar to bacterial phosphatidylcholine phospho-lipases (PLC-N and PLC-H) identified in Pseudomonas aerugi-nosa (32, 33). The amino acid sequence of PLC-N is 40% ho-mologous to PLC-H (33). The majority of this homology lieswithin the amino two-thirds of the proteins’ sequence while theremaining one-third shows very little homology. AcpA showsan overall sequence identity of 16% to either PLC-N or PLC-H.For comparison, the sequence alignment of AcpA to PLC-N isshown in Fig. 10. Considering both identical and conservedamino acid residues, AcpA shows an overall sequence similar-ity to PLC-N of 51%. In preliminary experiments, phospho-lipase C activity was detected in AcpA using the syntheticsubstrate p-nitrophenylphosphorylcholine assayed at pH 7.3but not at pH 6.0, the pH optimum for phosphomonoesteraseactivity. The phospholipase C specific activity of AcpA (610nmol of p-nitrophenylphosphorylcholine hydrolyzed/h/mg), al-though comparable to that of a commercial Clostridium phos-pholipase (1040 nmol/h/mg (Sigma, Type XIV)), was approxi-mately 3–4 orders of magnitude lower than its phosphomon-oesterase specific activity assayed at pH 7.3 (1.5 3 106 nmol ofMUP/h/mg) and pH 6.0 (9.5 3 106 nmol/h/mg). There was nodetectable phosphomonoesterase activity, using MUP as a sub-strate, in the Clostridium PLC preparation.

FIG. 9. Nucleotide sequence of acpA gene and deduced primary structure of AcpA polypeptide. Gene cloning and nucleotidesequencing was performed as described under “Experimental Procedures.” AcpA gene sequences plus 59 and 39 noncoding regions are shownnumbered from 1, the start of the 59 noncoding region. The acpA gene open reading frame begins at nucleotide 203 and runs through nucleotide1773 before encountering a stop codon (*). The acpA orf is preceded by a putative ribosome binding site (SD) 5 bp upstream from the start codon.Putative 210 and 235 promoter regions are underlined. The single underlined segment which follows in the open reading frame is the start of theAcpA N-terminal peptide which is identical with that obtained by Edman degradation of the purified enzyme. The double underlined segment 39to the AcpA N-terminal sequence is the deduced amino acid sequence identical with a CNBr peptide sequence prepared from AcpA.

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DISCUSSION

Members of the genus Francisella are facultative intracellu-lar pathogens and were found to harbor varying amounts ofacid phosphatase activity in crude extracts. One strain in par-ticular, ATCC 6223, produced the highest levels of acid phos-phatase thus far reported for a protozoan or bacterial pathogenand was chosen for purification of the enzyme. The Acps fromall Francisella strains were examined and found to be indistin-guishable in purification, molecular weight, and reaction withrabbit anti-AcpA polyclonal antibody. These data suggest F.tularensis, in contrast to L. donovani and L. micdadei whichcontain multiple Acp types (1, 34), produce a single Acppolypeptide that is similar, if not identical, in all members ofthe genus. F. tularensis (strain 6223) is remarkable in that it ishighly enriched in a respiratory burst-inhibiting acid phospha-tase. Using a specific activity for the purified enzyme of 1 3 107

units/mg and a molecular mass of 56,000 Da, we estimate thereare approximately 50,000 AcpA molecules produced per viablebacterial cell when cultured on hemoglobin-enriched CHA.This number was, however, dependent on the strain and pas-sage history.The physical and chemical properties of AcpA indicate this

enzyme is unique not only among burst-inhibiting acid phos-phatases but also among acid phosphatases in general. AcpA,in contrast to burst-inhibiting Acps (1, 2, 29), is easily releasedfrom the bacterial cell in soluble form, is a basic enzyme, andsuppresses the respiratory burst of not only fMLP but alsoPMA-stimulated neutrophils. AcpA is also much more sensitiveto inhibition by molybdate compounds than other burst-inhib-iting Acps. As shown in Table IV, these compounds inhibit 50%

of the activity of AcpA at concentrations that are 100 and 1000times lower than the I50 values for either Leishmania or Legio-nella acid phosphatases (1, 2).The recognized classes of acid phosphatases include high and

low molecular weight acid phosphatases, some protein phos-phatases specific for phosphoserine or phosphothreonine andpurple acid phosphatases (35). The purple acid phosphatasesare readily distinguished from other acid phosphatases by theirpurple color in solution, which is due to the presence of abinuclear iron center or iron-zinc center (36). AcpA is not pur-ple in solution, and preliminary x-ray diffraction and protonaccelerator studies of AcpA crystals did not indicate the pres-ence of any metal cofactors.2 Results from our inhibitor studiesalso suggest the enzyme is not a serine/threonine-specific pro-tein phosphatase. This class of protein phosphatases, consist-ing of groups 1, 2A, 2B, and 2C, is either acutely sensitive tookadaic acid or has an absolute requirement for divalent cat-ions (37, 38). AcpA is resistant to okadaic acid and retains fullactivity in 20 mM EDTA.AcpA also does not fit into either the high or low molecular

weight class of acid phosphatases. High molecular weight acidphosphatases differ in several respects from their low molecu-lar weight counterparts. A comparison of the class-distinctiveproperties of the high and low molecular weight Acps to thoseof AcpA is shown in Table V. According to its molecular weight,AcpA should be classified as a high molecular weight Acp.However, it has broad substrate specificity and is resistant totartrate and fluoride, which are common inhibitors of highmolecular weight acid phosphatases.Although AcpA was shown to have PTPase activity but it did

not possess an unambiguous phosphate binding loop signaturesequence, (H/V)C(X)5R(S/T)(G/A/P), present in Yop51 and morethan 40 other PTPases (39). We did find a possible phosphatebinding loop (C(X5)KSG) in AcpA (Fig. 10, residues 237–245) inwhich the critical arginine residue found in all PTPs is replacedby a lysine, and this may explain why AcpA still retains PTP-ase activity. P-loop motifs found conserved in GTP- and ATP-binding proteins also have the general sequence G(X)4GK(T/S)in which a lysine residue is conserved in all cases (40). It istempting to speculate that AcpA has a diverged cysteine activesite, phosphate binding loop in which an arginine has beenconservatively replaced by a lysine. The lack of a consensusPTPase P-loop, however, precludes its classification as aPTPase.Inhibition of AcpA activity by monofunctional sulfhydryl in-

hibitors including mercuric ions, silver, and hydroxymercuri-phenylsulfonate suggests this enzyme may possess a cysteineactive site and may therefore be classified as a “low molecularweight” acid phosphatase despite its high molecular weight.This is not without precedent since a cysteine active site, lowmolecular weight TRAP that has high molecular mass (35kDa), has been described (41).Interestingly, comparative nucleotide sequence analyses re-

vealed partial homology to known phosphatidylcholine phos-pholipases (PLC) of P. aeruginosa but failed to reveal homologyto any known acid phosphatase and did not detect the presenceof any known acid phosphatase, protein-tyrosine phosphatase,or phospholipase signature motifs. In preliminary experiments,we were able to detect phospholipase C activity in the purifiedAcpA when assayed using a synthetic substrate, p-nitrophen-ylphosphorylcholine, at pH 7.0 but not at pH 6.0, the pH opti-mum for phosphomonoesterase activity. The phospholipase Cspecific activity of AcpA, although comparable to that of acommercial Clostridium phospholipase, was 3000 times lower

2 E. Garman, personal communication.

FIG. 10. Amino acid alignment between AcpA and PLC-N. Dou-ble stars indicate identity and single stars indicate aligned amino acidswith similar contributions to secondary structure.

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than its phosphomonoesterase specific activity. The markedlyhigher rate of hydrolysis of monophosphate esters at acidic andneutral pH compared to phosphodiester substrates, includingthe p-nitrophenylphosphorylcholine phospholipase C sub-strate, supports the designation of AcpA as an acid phospha-tase in spite of its partial sequence similarity to P. aeruginosaPLC. Unequivocal demonstration of PLC activity of AcpA mustawait further studies using natural substrates.The mechanism(s) by which any acid phosphatase sup-

presses the respiratory burst has also not been determined. Aproposed mechanism for Leishmania and Legionella Acp medi-ated inhibition of the fMLP-stimulated respiratory burst isAcp-catalyzed depletion of PIP2 and IP3 (4). In this mechanism,it is not clear, however, whether depletion of PIP2 and IP3 poolsoccurs by direct hydrolysis of these intermediates or whetherAcp is somehow interfering with plasma membrane signaltransduction mechanisms. It has yet to be shown that anyburst-inhibiting Acp gains entry or accessibility to PIP2 or IP3pools within the neutrophil or macrophage. In the case forAcpA, it seems unlikely that depletion of PIP2 and IP3 poolsaccounts for all the observed inhibition since PIP2 and IP3 arerelatively poor substrates for AcpA, and AcpA also inhibitsPMA-stimulated porcine neutrophils which is an PIP2/IP3 in-dependent superoxide anion production pathway (42). Further-more, it is unlikely that AcpA gains access to the neutrophilcytoplasm. In preliminary experiments using radioiodinated,catalytically active AcpA, we found no evidence for uptake ofexogenously added AcpA into neutrophils over a 2-h time pe-riod even though burst inhibition occurred within the first 15min. Thus, it seems more likely that AcpA inhibits the respi-ratory burst by hydrolysis of neutrophil surface-exposed sub-strates that are involved in signal transduction pathways nec-essary for burst activation or maintenance.The broad substrate specificity of AcpA including its tyrosine

phosphatase (PTPase) and phospholipase C activities may pro-vide clues to possible mechanisms of respiratory burst inhibi-tion. Dephosphorylation of multiple targets including phos-phatidylcholine, protein tyrosine phosphates, secondarymessengers, or other low molecular weight substrates criticalto phagocyte activation such as ribose 5-phosphate, NADPH, orATP may explain why this particular acid phosphatase inhibitsthe respiratory burst of both fMLP or PMA-stimulatedneutrophils.Whether AcpA’s burst-inhibiting activity is relevant to the

pathogenicity of F. tularensis or secondary to even more impor-tant microbial physiologic processes remains to be determined.There is no unequivocal proof that any of the burst-inhibitingAcps function as virulence factors in vivo. In our opinion, iden-tification of these enzymes as virulence factors must awaitconstruction and use of isogenic Acp-negative mutant strains inboth in vitro and in vivo infectivity experiments. Until now,

there has been no nucleotide sequence information reported forany burst-inhibiting Acp. The results of cloning and sequencingof the AcpA gene reported here should help in the design ofexperiments aimed at elucidating the physiological function ofAcpA and to directly test its role, if any, in F. tularensisvirulence.

Acknowledgments—We would like to thank Dr. Jim C. Williams ofthe Food and Drug Administration for our first samples of F. tularensis.We are also grateful to Dr. Graeme Laver (The Australian NationalUniversity) for growing AcpA crystals and Dr. Elspeth Garman (Uni-versity of Oxford) for her preliminary AcpA x-ray diffraction and protonacceleration studies. We would also like to acknowledge the Universityof Illinois Biotechnology and Mass Spectrometry Laboratories for theirefforts in obtaining the N-terminal sequences and the matrix-assistedlaser desorption mass spectrometry determined molecular mass ofAcpA. We also thank Dr. Saul Roseman, The Johns Hopkins University,for his many helpful suggestions in the preparation of this manuscript.

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TABLE VComparison of AcpA to low and high molecular weight Acp classes

The class-distinctive physicochemical properties of known high and low molecular mass acid phosphatases are compared to those of AcpA

Property Low molecular mass High molecular mass AcpA

Molecular mass 14–18 kDa 40–60 kDaa 56 kDaSubunit composition Monomeric Multimeric MonomericSubstrate specificity Narrow Broad BroadTartrate inhibition No Yes NoFluoride inhibition No Yes NoHg21/Ag21 inhibition Yes No YesActive site Cys Yes No ? (Yes)b

RHG motif No Yes NoPTPase activity Yes No YesPLC activity ? ? Yes

a Subunit molecular mass.b Preliminary data based on inhibition studies (see text). For review see Ref. 35.

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Thomas J. Reilly, Gerald S. Baron, Francis E. Nano and Mark S. KuhlenschmidtFrancisella tularensisfrom

Characterization and Sequencing of a Respiratory Burst-inhibiting Acid Phosphatase

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