(Received for publication, October 16, 1995; and in revised form, January 16, 1996)
From the
Acid phosphatases (Acp) of intracellular pathogens have recently been implicated as virulence factors that enhance intracellular survival through suppression of the respiratory burst. We describe here the identification, purification, characterization, and sequencing of a novel burst-inhibiting acid phosphatase from the facultative intracellular bacterium, Francisella tularensis. Similar to other the burst-inhibiting Acps, F. tularensis Acp (AcpA) is tartrate-resistant and has broad substrate specificity. The AcpA enzyme is unique, however, in that it is easily released from the bacterial cell in soluble form, is a basic enzyme, suppresses the respiratory burst of not only fMet-Leu-Phe but also phorbol 12-myristate 13-acetate-stimulated neutrophils and does not fit into any of the three currently recognized classes of acid phosphatase. We also report the complete nucleotide sequence of the gene acpA, encoding AcpA, and the deduced primary structure of its encoded polypeptide. Comparative sequence analyses of AcpA is discussed. To our knowledge, this is the first report describing the cloning and sequencing of a burst-inhibiting acid phosphatase.
Acid phosphatases (EC 3.1.3.2) are a ubiquitous class of enzymes that catalyze the hydrolysis of phosphomonoesters at an acidic pH. In addition to mobilization of phosphate, some members of this class of enzymes perform many essential biological functions including regulation of metabolism, energy conversion, and signal transduction. These enzymes have been identified and characterized from many eukaryotic and prokaryotic sources and comprise several distinct subgroups based on substrate specificity, molecular weight, and sensitivity to known inhibitors.
In the past decade, a new emphasis
has been placed on understanding the role acid phosphatases may play in
microbial pathogenesis. Comprehensive studies of acid phosphatases
purified from Leishmania donovani(1) and Legionella micdadei(2) suggest that members of a
class of tartrate-resistant, nonspecific acid phosphatases (TRAPs) ()may play a crucial role in the survival of intracellular
pathogens within a host's phagocytic cells. An exciting discovery
in these studies was that TRAPs purified from these organisms
suppressed the respiratory burst of activated human
neutrophils(3, 4) . Although information is now
becoming available about some of the enzymatic, biochemical, and
biophysical properties of the burst-inhibiting TRAPs, unequivocal proof
of the role of these enzymes as virulence factors in vivo has
yet to be obtained. Progress toward this goal is currently limited by
the lack of protein or gene sequence information and the absence of
isogenic TRAP mutants.
Francisella tularensis is the etiologic agent of the potentially fatal human disease tularemia and is capable of survival and multiplication within a host's professional phagocytes as well as nonphagocytic cells(5, 6) . Although many studies have been conducted into the host's immune response to Francisella infection, until recently relatively little attention has been focused on biochemical characterization of purified macromolecules which may function as virulence factors in these organisms(7) . In initial studies, we found a particular strain of F. tularensis (ATCC 6223, B38) to be enriched in acid phosphatase activity. The Acp specific activity in this strain was greater than previously reported for any other bacterial or protozoan organism. It was also easily solubilized in the absence of detergents allowing relatively large amounts of enzyme to be purified to apparent homogeneity. We describe here the identification, purification, and characterization of some of the unique properties of this burst-inhibiting acid phosphatase (AcpA) as well as its complete primary structure derived from cloning and nucleotide sequencing of the AcpA gene (acpA).
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 strictures on the transfer of genes between F. novicida and F. tularensis.
The rather wide variation in acid phosphatase specific activity among members of the Francisella genus may correlate with the passage history of individual strains. During experiments aimed at optimizing expression of Acp, we observed a large decrease in Acp specific activity upon repeated passage of strain 6223 on CHA (data not shown). A loss of almost 90% (8-10-fold reduction) of the starting Acp specific activity was seen following 9 passages. The reduction was most likely not due to the accumulation of reversible inhibitors since washing the cells in physiological saline followed by extraction of Acp failed to increase Acp specific activity, and mixing of extracts from passaged cultures with purified AcpA did not result in the inhibition of the activity of the purified enzyme. Furthermore, detection of AcpA by Western blot analysis indicated a marked reduction in anti-AcpA reactive material following 9 passages as compared to that found in initial cultures (data not shown). Therefore, single passage F. tularensis(6223) was selected as the source for enzyme purification.
Figure 1:
Purification steps of F. tularensis acid phosphatase. For A-C, AcpA activity ()
and protein concentration (
). A, S-Sepharose cation
exchange chromatography of Supernatant II containing AcpA using a 0 to
0.5 M NaCl linear gradient (-) as described under
``Experimental Procedures.'' Twenty-one 6.0-ml fractions
(38-58) found to contain AcpA activity eluted between 0.17 and
0.26 M NaCl. B, Sephadex G-100 Superfine
chromatography of pooled and concentrated AcpA from S-Sepharose (5.3
ml, 6.7 mg/ml protein). Application and elution of AcpA to this gel
filtration resin was performed as described under ``Experimental
Procedures.'' C, Superdex 75 HR 10/30 FPLC chromatography
of a 0.3-ml aliquot of pooled Acp activity from Sephadex G-100. D, SDS-PAGE separation of samples from the purification
procedure. From left to right: lane 1, Novex
Mark 12 molecular weight standards; lane 2, 30 µg of whole F. tularensis; lane 3, 30 µg of supernatant I; lane 4, 30 µg of supernatant II; lane 5, 30
µg of S-Sepharose pool (fractions 38-58); lane 6, 30
µg of Sephadex G-100 pool (fractions 47-59); lane 7,
8 µg of AcpA from Superdex 75 FPLC.
The material recovered from cation exchange chromatography was enriched 32-fold in acid phosphatase activity and contained 94% of the starting activity. Gel filtration chromatography through Sephadex G-100 superfine (Fig. 1B) resulted in an additional 13-fold increase in specific activity with 83% recovery of the applied activity. Final purification of the enzyme was achieved by gel filtration FPLC (Fig. 1C). This step resulted in a further 1.7-fold increase in specific activity with 26% of the sample recovered in a single protein peak coincident with AcpA activity. The apparently low recovery from the FPLC column is explained by the conservative pooling of AcpA active fractions as described under ``Experimental Procedures.'' The actual recovery was approximately 75%, but only the two fractions containing the highest AcpA activity were pooled for further analyses. Overall, AcpA was purified 713-fold over that in intact bacteria (Table 2). The purification behavior of AcpA from strains 6223, NDBR 101, and 29684 and the results of comparative molecular weight (Fig. 2A) and immunoreactivity with rabbit anti-Ft(6223) AcpA IgG (Fig. 2B) suggested the enzyme is very similar in all strains of F. tularensis. Also, the enzyme activity chromatographed as a single entity throughout all purification steps suggesting that multiple acid phosphatases may not exist in F. tularensis in contrast to the results reported for some other facultative intracellular organisms(1, 2) .
Figure 2: SDS-PAGE and Western blot analyses of acid phosphatase from three strains of F. tularensis. A, Novex standards, as described for Fig. 1(lane 1), 30 µg of extracted proteins from F. tularensis strains NDBR 101, 29684, and 6223 (lanes 2-4), and 8 µg of purified acid phosphatase from these same strains (lanes 5-7) were subjected to SDS-PAGE and stained with Coomassie Blue R-250. B, Western blot analysis of blotted acid phosphatases from F. tularensis strains NDBR 101, 29684, and 6223 (lanes 1-3) using rabbit anti AcpA(6223) IgG.
Figure 3:
Evaluation of AcpA purity by
radioiodination of pooled fractions from Superdex 75 chromatography.
Ten µg of pooled AcpA from Superdex 75 gel filtration
chromatography was iodinated as described under ``Experimental
Procedures'' and subjected to SDS-PAGE and autoradiography. The
position of molecular weight markers are shown on the far left of the autoradiograph. The 5 lanes to the right of the
markers (lane 1) contain 2, 4, 6, 8, and 10 µl,
respectively, of the 1.5-ml void volume from the desalting column.
Molecular weight standards are: -galactosidase (116,000),
phosphorylase b (95,000), BSA (68,000), glutamic dehydrogenase
(55,000), carbonic anhydrase (29,000), and lysozyme
(14,000).
The molecular mass of the purified enzyme
was determined by gel filtration chromatography, SDS-PAGE, and
matrix-assisted laser desorption time of flight MS. Superdex 75 FPLC
gel filtration chromatography gave a partition coefficient for AcpA of
0.09 (Fig. 4A). This value was compared to the
regression line generated from the four molecular weight standards, and
the K corresponded to an apparent molecular
weight of 56,000. A similar value, 57,000, was obtained with SDS-PAGE (Fig. 4B) using both reducing and nonreducing
conditions (data not shown). Finally, mass spectrometry of AcpA
indicated a singly charged species at 55,759 atomic mass units with a
mass accuracy of 0.1% (Fig. 4C).
Figure 4:
Estimation of the molecular weight of
AcpA. A, regression line () of the log molecular weight
of the gel filtration standards versus their respective
partition coefficients: BSA (67,000 K
=
0.035), ovalbumin (43,000 K
= 0.165),
chymotrypsin (25,000 K
= 0.324), and RNase
A (13,700 K
= 0.501). Elution position and
partition coefficient of AcpA are indicated by arrow. B,
regression line of log molecular weight standards (Fig. 1) versus electrophoretic mobility. Mobility and estimated
molecular weight of AcpA are indicated by the arrow. C,
matrix-assisted laser desorption time of flight profile of purified
AcpA. M
= m/z 55759.4. Matrix,
sinapinic acid; laser wavelength, 337 nm.
Figure 5:
Determination of pH optimum. A-D, purified AcpA was incubated with 1 mM indicated substrate in either 0.2 M MES (,
pK
6.10) or 0.2 M HEPES (
,
pK
7.48) at varying pH values. Acp
activity was determined by the Lanzetta assay for inorganic phosphate
as described under ``Experimental Procedures.'' Data are
plotted as percent of optimal activity for each substrate. A,
5`-AMP; B, Glc-6-PO
; C, MUP; D,
tyrosine phosphate.
Figure 7:
Estimation of the K and V
for AcpA with three different
substrates. Each substrate was incubated with purified AcpA at final
concentrations from 0.04 to 1.6 mM in 0.2 M sodium
acetate buffer, pH 6.0. The reactions were incubated for 15 min at 37
°C; quantitation of phosphatase activity was performed using the
assay for inorganic phosphate as described under ``Experimental
Procedures.'' Each point represents the average of 5 separate
samples for each concentration indicated. A and B show substrate saturation and Lineweaver-Burk plot of AcpA
incubated with MUP (
) and tyrosine phosphate (
). C and D show similar plots when the phosphorylated
substrate p60
was used as substrate. Inset of C is the pH optimum of AcpA's PTPase
activity.
Figure 6:
Purified AcpA (7.5 10
units) was mixed into a 5-25% w/w sucrose gradient
containing 4% w/v Ampholytes pH 3-10 and focused in a LKB 8100
isoelectric focusing column at 3 watts for 72 h at 15 °C. After
focusing, the gradient was fractionated into 1.0-ml fractions from
which Acp activity (
) and pH (
) values were
determined.
Figure 8:
AcpA-mediated inhibition of the
respiratory burst in porcine neutrophils. Isolated porcine neutrophils
(1 10
cells/ml) were incubated with purified AcpA
prior to addition of either PMA or fMLP. Superoxide anion production
was determined by continuous spectrophotometric measurements of the
reduction of ferricytochrome c at 550 nm. A, each
point (
) represents the mean of the rate of cytochrome c reduction ± S.D. from 5 separate experiments by porcine
neutrophils after a 15-min preincubation with AcpA and activated with
PMA;
, heat-inactivated AcpA (100 °C, 15 min). B,
comparison of the amount of superoxide anion production as measured by
reduction of ferricytochrome c by fMLP-stimulated porcine
neutrophils after a 15-min exposure to 1000 units of AcpA
(-) or without prior exposure to AcpA (- -
-). C, effect of increasing preincubation times of
porcine neutrophils with purified AcpA (8000 units) on production of
superoxide anion in PMA-activated
neutrophils.
Figure 9: Nucleotide sequence of acpA gene and deduced primary structure of AcpA polypeptide. Gene cloning and nucleotide sequencing was performed as described under ``Experimental Procedures.'' AcpA gene sequences plus 5` and 3` noncoding regions are shown numbered from 1, the start of the 5` noncoding region. The acpA gene open reading frame begins at nucleotide 203 and runs through nucleotide 1773 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 -10 and -35 promoter regions are underlined. The single underlined segment which follows in the open reading frame is the start of the AcpA N-terminal peptide which is identical with that obtained by Edman degradation of the purified enzyme. The double underlined segment 3` to the AcpA N-terminal sequence is the deduced amino acid sequence identical with a CNBr peptide sequence prepared from AcpA.
Comparative sequence analyses (Blast,
National Center for Biotechnology Information) indicate acpA has no overall sequence similarity to other known acid
phosphatases, but it is partially similar to bacterial
phosphatidylcholine phospholipases (PLC-N and PLC-H) identified in Pseudomonas aeruginosa(32, 33) . The amino
acid sequence of PLC-N is 40% homologous to PLC-H(33) . The
majority of this homology lies within the amino two-thirds of the
proteins' sequence while the remaining one-third shows very
little homology. AcpA shows an overall sequence identity of 16% to
either PLC-N or PLC-H. For comparison, the sequence alignment of AcpA
to PLC-N is shown in Fig. 10. Considering both identical and
conserved amino acid residues, AcpA shows an overall sequence
similarity to PLC-N of 51%. In preliminary experiments, phospholipase C
activity was detected in AcpA using the synthetic substrate p-nitrophenylphosphorylcholine assayed at pH 7.3 but not at pH
6.0, the pH optimum for phosphomonoesterase activity. The phospholipase
C specific activity of AcpA (610 nmol of p-nitrophenylphosphorylcholine hydrolyzed/h/mg), although
comparable to that of a commercial Clostridium phospholipase
(1040 nmol/h/mg (Sigma, Type XIV)), was approximately 3-4 orders
of magnitude lower than its phosphomonoesterase specific activity
assayed at pH 7.3 (1.5 10
nmol of MUP/h/mg) and pH
6.0 (9.5
10
nmol/h/mg). There was no detectable
phosphomonoesterase activity, using MUP as a substrate, in the Clostridium PLC preparation.
Figure 10: Amino acid alignment between AcpA and PLC-N. Double stars indicate identity and single stars indicate aligned amino acids with similar contributions to secondary structure.
Members of the genus Francisella are facultative
intracellular pathogens and were found to harbor varying amounts of
acid phosphatase activity in crude extracts. One strain in particular,
ATCC 6223, produced the highest levels of acid phosphatase thus far
reported for a protozoan or bacterial pathogen and was chosen for
purification of the enzyme. The Acps from all Francisella strains were examined and found to be indistinguishable in
purification, molecular weight, and reaction with rabbit anti-AcpA
polyclonal antibody. These data suggest F. tularensis, in
contrast to L. donovani and L. micdadei which contain
multiple Acp types(1, 34) , produce a single Acp
polypeptide that is similar, if not identical, in all members of the
genus. F. tularensis (strain 6223) is remarkable in that it is
highly enriched in a respiratory burst-inhibiting acid phosphatase.
Using a specific activity for the purified enzyme of 1 10
units/mg and a molecular mass of 56,000 Da, we estimate there are
approximately 50,000 AcpA molecules produced per viable bacterial cell
when cultured on hemoglobin-enriched CHA. This number was, however,
dependent on the strain and passage history.
The physical and
chemical properties of AcpA indicate this enzyme is unique not only
among burst-inhibiting acid phosphatases but also among acid
phosphatases in general. AcpA, in contrast to burst-inhibiting
Acps(1, 2, 29) , is easily released from the
bacterial cell in soluble form, is a basic enzyme, and suppresses the
respiratory burst of not only fMLP but also PMA-stimulated neutrophils.
AcpA is also much more sensitive to inhibition by molybdate compounds
than other burst-inhibiting Acps. As shown in Table 4, these
compounds inhibit 50% of the activity of AcpA at concentrations that
are 100 and 1000 times lower than the I values for either Leishmania or Legionella acid
phosphatases(1, 2) .
The recognized classes of acid
phosphatases include high and low molecular weight acid phosphatases,
some protein phosphatases specific for phosphoserine or
phosphothreonine and purple acid phosphatases (35) . The purple
acid phosphatases are readily distinguished from other acid
phosphatases by their purple color in solution, which is due to the
presence of a binuclear iron center or iron-zinc center(36) .
AcpA is not purple in solution, and preliminary x-ray diffraction and
proton accelerator studies of AcpA crystals did not indicate the
presence of any metal cofactors. ()Results from our
inhibitor studies also suggest the enzyme is not a
serine/threonine-specific protein phosphatase. This class of protein
phosphatases, consisting of groups 1, 2A, 2B, and 2C, is either acutely
sensitive to okadaic acid or has an absolute requirement for divalent
cations(37, 38) . AcpA is resistant to okadaic acid
and retains full activity in 20 mM EDTA.
AcpA also does not fit into either the high or low molecular weight class of acid phosphatases. High molecular weight acid phosphatases differ in several respects from their low molecular weight counterparts. A comparison of the class-distinctive properties of the high and low molecular weight Acps to those of AcpA is shown in Table 5. According to its molecular weight, AcpA should be classified as a high molecular weight Acp. However, it has broad substrate specificity and is resistant to tartrate and fluoride, which are common inhibitors of high molecular weight acid phosphatases.
Although AcpA was shown to have PTPase
activity but it did not possess an unambiguous phosphate binding loop
signature sequence, (H/V)C(X)R(S/T)(G/A/P),
present in Yop51 and more than 40 other PTPases(39) . We did
find a possible phosphate binding loop (C(X
)KSG)
in AcpA (Fig. 10, residues 237-245) in which the critical
arginine residue found in all PTPs is replaced by a lysine, and this
may explain why AcpA still retains PTPase activity. P-loop motifs found
conserved in GTP- and ATP-binding proteins also have the general
sequence G(X)
GK(T/S) in which a lysine residue is
conserved in all cases(40) . It is tempting to speculate that
AcpA has a diverged cysteine active site, phosphate binding loop in
which an arginine has been conservatively replaced by a lysine. The
lack of a consensus PTPase P-loop, however, precludes its
classification as a PTPase.
Inhibition of AcpA activity by monofunctional sulfhydryl inhibitors including mercuric ions, silver, and hydroxymercuriphenylsulfonate suggests this enzyme may possess a cysteine active site and may therefore be classified as a ``low molecular weight'' acid phosphatase despite its high molecular weight. This is not without precedent since a cysteine active site, low molecular weight TRAP that has high molecular mass (35 kDa), has been described(41) .
Interestingly, comparative nucleotide sequence analyses revealed partial homology to known phosphatidylcholine phospholipases (PLC) of P. aeruginosa but failed to reveal homology to any known acid phosphatase and did not detect the presence of any known acid phosphatase, protein-tyrosine phosphatase, or phospholipase signature motifs. In preliminary experiments, we were able to detect phospholipase C activity in the purified AcpA when assayed using a synthetic substrate, p-nitrophenylphosphorylcholine, at pH 7.0 but not at pH 6.0, the pH optimum for phosphomonoesterase activity. The phospholipase C specific activity of AcpA, although comparable to that of a commercial Clostridium phospholipase, was 3000 times lower than its phosphomonoesterase specific activity. The markedly higher rate of hydrolysis of monophosphate esters at acidic and neutral pH compared to phosphodiester substrates, including the p-nitrophenylphosphorylcholine phospholipase C substrate, supports the designation of AcpA as an acid phosphatase in spite of its partial sequence similarity to P. aeruginosa PLC. Unequivocal demonstration of PLC activity of AcpA must await further studies using natural substrates.
The mechanism(s) by which any acid phosphatase
suppresses the respiratory burst has also not been determined. A
proposed mechanism for Leishmania and Legionella Acp
mediated inhibition of the fMLP-stimulated respiratory burst is
Acp-catalyzed depletion of PIP and
IP
(4) . In this mechanism, it is not clear,
however, whether depletion of PIP
and IP
pools
occurs by direct hydrolysis of these intermediates or whether Acp is
somehow interfering with plasma membrane signal transduction
mechanisms. It has yet to be shown that any burst-inhibiting Acp gains
entry or accessibility to PIP
or IP
pools
within the neutrophil or macrophage. In the case for AcpA, it seems
unlikely that depletion of PIP
and IP
pools
accounts for all the observed inhibition since PIP
and
IP
are relatively poor substrates for AcpA, and AcpA also
inhibits PMA-stimulated porcine neutrophils which is an
PIP
/IP
independent superoxide anion production
pathway(42) . Furthermore, it is unlikely that AcpA gains
access to the neutrophil cytoplasm. In preliminary experiments using
radioiodinated, catalytically active AcpA, we found no evidence for
uptake of exogenously added AcpA into neutrophils over a 2-h time
period even though burst inhibition occurred within the first 15 min.
Thus, it seems more likely that AcpA inhibits the respiratory burst by
hydrolysis of neutrophil surface-exposed substrates that are involved
in signal transduction pathways necessary for burst activation or
maintenance.
The broad substrate specificity of AcpA including its tyrosine phosphatase (PTPase) and phospholipase C activities may provide clues to possible mechanisms of respiratory burst inhibition. Dephosphorylation of multiple targets including phosphatidylcholine, protein tyrosine phosphates, secondary messengers, or other low molecular weight substrates critical to phagocyte activation such as ribose 5-phosphate, NADPH, or ATP may explain why this particular acid phosphatase inhibits the respiratory burst of both fMLP or PMA-stimulated neutrophils.
Whether AcpA's burst-inhibiting activity is relevant to the pathogenicity of F. tularensis or secondary to even more important microbial physiologic processes remains to be determined. There is no unequivocal proof that any of the burst-inhibiting Acps function as virulence factors in vivo. In our opinion, identification of these enzymes as virulence factors must await construction and use of isogenic Acp-negative mutant strains in both in vitro and in vivo infectivity experiments. Until now, there has been no nucleotide sequence information reported for any burst-inhibiting Acp. The results of cloning and sequencing of the AcpA gene reported here should help in the design of experiments aimed at elucidating the physiological function of AcpA and to directly test its role, if any, in F. tularensis virulence.
The nucleotide sequence(s) reported in this paper has been submitted to the GenBank(TM)/EMBL Data Bank with accession number(s) L39831[GenBank].