Inhibition of AcpA phosphatase activity with ascorbate attenuates Francisella tularensis intramacrophage survival

DOI: 10.1074/jbc.M109.039511

Source and publish data:

Journal of Biological Chemistry p. 5171 - 5177 (2010)

Update date:2022-08-11

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Authors:

McRae, Steven

Pagliai, Fernando A.

Mohapatra, Nrusingh P.

Gener, Alejandro

Mahmou, Asma Sayed Abdelgeliel

Gunn, John S.

Lorca, Graciela L.

Gonzalez, Claudio F.

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Article abstract of DOI:10.1074/jbc.M109.039511

Acid phosphatase activity in the highly infectious intracellular pathogen Francisella tularensis is directly related with the ability of these bacteria to survive inside host cells. Pharmacological inactivation of acid phosphatases could potentially help

Full text of DOI:10.1074/jbc.M109.039511

THE JOURNAL OF BIOLOGICAL CHEMISTRY VOL. 285, NO. 8, pp. 5171–5177, February 19, 2010

Inhibition of AcpA Phosphatase Activity with Ascorbate

□

Attenuates Francisella tularensis Intramacrophage Survival

Received for publication, June 30, 2009, and in revised form, December 11, 2009 Published, JBC Papers in Press, December 22, 2009, DOI 10.1074/jbc.M109.039511

Steven McRae^‡1, Fernando A. Pagliai^‡, Nrusingh P. Mohapatra^§, Alejandro Gener^‡1,

Asma Sayed Abdelgeliel Mahmou^‡, John S. Gunn^§, Graciela L. Lorca^‡2, and Claudio F. Gonzalez^‡2,3

From the ^‡Department of Microbiology and Cell Science, Institute of Food and Agricultural Sciences, University of Florida,

Gainesville, Florida 32610-3610 and the ^§Center for Microbial Interface Biology, Department of Molecular Virology, Immunology

and Medical Genetics, and Department of Internal Medicine, Division of Infectious Diseases, The Ohio State University,

Columbus, Ohio 43210

Acid phosphatase activity in the highly infectious intracellu-

lar pathogen Francisella tularensis is directly related with the

ability of these bacteria to survive inside host cells. Pharmaco-

logical inactivation of acid phosphatases could potentially help

in the treatment of tularemia or even be utilized to neutralize

the infection. In the present work, we report inhibitory com-

pounds for three of the four major acid phosphatases produced

by F. tularensis SCHU4: AcpA, AcpB, and AcpC. The inhibitors

were identified using a catalytic screen from a library of chemi-

cals approved for use in humans. The best results were obtained

against AcpA. The two compounds identified, ascorbate (K_i

380 ؎ 160 ␮M) and 2-phosphoascorbate (K_i

3.2 ؎ 0.85 ␮M)

inhibit AcpA in a noncompetitive, nonreversible fashion. A

potential ascorbylation site in the proximity of the catalytic

pocket of AcpA was identified using site-directed mutagenesis.

The effects of the inhibitors identified in vitro were evaluated

using bioassays determining the ability of F. tularensis to survive

inside infected cells. The presence of ascorbate or 2-phos-

phoascorbate impaired the intramacrophage survival of F. tula-

rensis in an AcpA-dependent manner as it was probed using

knockout strains. The evidence presented herein indicated that

ascorbate could be a good alternative to be used clinically to

improve treatments against tularemia.

host cell endosomes by hydrolyzing the phosphoester (P-O)

bonds of phosphorylated molecules. Acid phosphatases have

been identified in Coxiella (5), Bordetella (6), and Legionella (7,

8), and their activity has been directly linked with virulence in

some of these organisms. Several of these enzymes have been

purified, their biochemical characteristics described, and their

structure solved. Despite evidence suggesting that these phos-

phatases suppress the respiratory burst of human neutrophils

(7, 9, 10), their natural substrates, biological role, and molecular

events involved during infection are still under study.

A combination of several factors makes it difficult to identify

the intracellular substrate that these enzymes utilize to aid the

bacteria during the infection process. For example, phosphate

is the most abundant functional group present in the intracel-

lular pool of cellular metabolites (11). In addition, most of the

phosphatases previously studied have very low sequence

homology and little substrate specificity, being able to release

phosphate from a vast array of phosphorylated compounds in

vitro (12). This biochemistry makes it challenging to rationally

elaborate specific inhibitory compounds to be used in vivo. We

should note, in addition to these biochemical problems, the

further complexity of working with highly infectious intracel-

lular microorganisms.

Taking advantage of two important characteristics of these

enzymes, (i) their relaxed substrate specificity and (ii) their abil-

ity to work in a broad range of pH, we set up a rapid, simple, and

reliable screen for F. tularensis phosphatase inhibitors. These

characteristics allowed the use of p-nitrophenyl phosphate

(pNPP)⁴as a model substrate for our screening. Using this tech-

nique we performed an unbiased enzymatic screening looking

for chemical compounds that inhibit the F. tularensis phospha-

tase activity in vitro. Envisioning a potential utilization in

humans, we restricted our search to arrays of drugs approved by

the Federal Drug Administration (FDA). Our hypothesis was

that several natural/synthetic chemical scaffolds present in

these libraries can interact with the purified acid phosphatases,

blocking their specific activities. In the present work, we

describe the biochemical study of the inhibitory compounds

selected for three of the enzymes studied. We offer a molecular

Francisella tularensis, the causative agent of human tulare-

mia, is a highly infectious bacterium that could represent a

serious bio-threat if genetically modified to resist antibiotic

treatment (1–3). Four acid phosphatases are encoded by F.

tularensis: AcpA, AcpB, AcpC, and Hap. A recent work indi-

cated that a quadruple knockout mutant strain showed attenu-

ated virulence and diminished macrophage vacuolar escape (4),

thus the pharmacological inhibition of acid phosphatase activ-

ity is an excellent target to prevent the proliferation of these

bacteria inside infected cells.

Acid phosphatases are produced by many intracellular path-

ogenic organisms. These enzymes are able to function inside

□S

The on-line version of this article (available at http://www.jbc.org) contains

supplemental Figs. S1 and S2.

¹Undergraduate students of the MCB4905 Program, University of Florida.

²Both authors contributed equally to this work.

⁴The abbreviations used are: pNPP, p-nitrophenyl phosphate; CHA, on cys-

teine heart agar; TSB, tryptic soy broth; MES, 4-morpholineethanesulfonic

acid; MOPS, 4-morpholinepropanesulfonic acid; CHES, 2-(cyclohexylamino)-

ethanesulfonic acid; LVS, live vaccine strain.

³To whom correspondence should be addressed: Genetic Institute, Rm. 306,

University of Florida, Gainesville, FL 32610-3610. Tel.: 352-273-8088; Fax:

352-273-8284; E-mail: cfgonzalez@ufl.edu.

FEBRUARY 19, 2010•VOLUME 285•NUMBER 8

JOURNAL OF BIOLOGICAL CHEMISTRY 5171

AcpA Inactivation by Ascorbate Affects F. tularensis Survival

interpretation of the mechanism for the best inhibitory com- AGA for Arg and ATA for Ile). The cells were grown in LB with

pound obtained for AcpA. The effects observed in vitro were 1 ^Msorbitol and 2.5 mM betaine at 23 °C to an A₆₀₀of ϳ0.6, and

validated using bioassays. We found that the ability of F. tula- expression was induced with 0.4 m^Misopropyl 1-thio-␤-D-ga-

rensis to proliferate inside macrophages was severely affected lactopyranoside. After addition of isopropyl 1-thio-␤-D-galac-

when treated with the compound selected in our screen.

topyranoside, the cells were incubated with shaking at (17 °C)

overnight as described (15). The cells were harvested, resus-

pended in binding buffer (500 m^MNaCl, 5% glycerol, 20 mM

EXPERIMENTAL PROCEDURES

Bacterial Strains—Francisella novicida U112 (JSG1819) and Tris-HCl, pH 8.2, 15 mM imidazole), flash-frozen in liquid N₂,

F. novicida ⌬acpA JSG2660 (4) were routinely cultured at 37 °C and stored at Ϫ70 °C. The thawed cells were disrupted by pas-

on cysteine heart agar (CHA) (Hi-Media Laboratories, India) or sage through a French press, and the lysate was clarified by

in modified tryptic soy broth (TSB) (Difco Laboratories, centrifugation (30 min at 17,000 rpm) and passed through a

Detroit, MI) containing 35 ␮g/ml ferric pyrophosphate and metal chelate affinity-column charged with Ni^2ϩ. After the col-

0.1% cysteine hydrochloride. CHOC II plates (Difco Laborato- umn was washed, the protein was eluted from the column in

ries) were used for CFU enumeration. To obtain minimal inhib- elution buffer (binding buffer with 250 m^Mimidazole). The

itory concentrations, F. novicida wild-type and mutant strains purified proteins were dialyzed against 20 m^MTris-HCl, pH 7.9,

from 24-h CHA plates containing antibiotics were inoculated 500 m^MNaCl, 1 mM dithiothreitol, and concentrated using a

into modified TSB, as well as Chamberlain’s medium, and the Vivaspin 20 concentrator (Sartorius Biolab Products, Ger-

optical densities of the cultures were determined at 2-h inter- many). Protein concentration was estimated using the Bio-Rad

vals. All manipulations with F. novicida were performed in a protein assay kit (Bio-Rad).

class II biological safety laboratory. Escherichia coli DH5␣ cells

Enzymatic Assays—Phosphatase activity was determined by

were used to carry and propagate all vectors. When required for continuous reading at 412 nm for a period of 40 min at 37 °C

E. coli growth, Luria-Bertani medium (Difco Laboratories), was using pNPP as substrate. For AcpA the standard 150-␮l assay

supplemented with ampicillin (100 ␮g/ml) as needed. All anti- mixture contained 25 m^M(MES), pH 6.2, 1 mM pNPP, 1 ␮g/ml

biotics and chemicals were purchased from Sigma-Aldrich.

AcpA (unless stated otherwise). Under these conditions,

DNA Manipulations and Gene Cloning—Standard methods enzyme activity was linear with time for at least 60 min. The

were used for site-directed mutagenesis, chromosomal DNA amount of p-nitrophenol released was quantified at A₄₁₂using

Ϫ1

isolation, restriction enzyme digestion, agarose gel electro- the extinction coefficient ⑀ ϭ 16,300 M cm as described

phoresis, ligation, and transformation (13). Plasmids were iso- previously (16). Enzymatic activity was also determined using

lated using spin miniprep kits (Qiagen), and PCR products were 2-phosphoascorbate as a substrate using malachite green

purified using QIAquick purification kits (Qiagen). Muta- ammonium molybdate detection reagent (17).

genesis was performed using the QuikChange site-directed

The pH optimum was determined using overlapping buffers

mutagenesis kit (Stratagene). For protein expression and puri- at 25 m^M: MES (pH 6.0–6.6), MOPS (pH 6.6–7.0), HEPES (pH

fication, the selected gene was amplified from F. tularensis tula- 6.8–8.0), Tris (pH 8.2–8.8), CHES (pH 8.6–9.6), Maleate (pH

rensis SCHU4 chromosomal DNA (kindly provided by Dr. 5.6–6.4), citrate (pH 5.6–6.4), and acetate (pH 6.0–6.6). The

Wehrly, National Institutes of Health) by PCR. The primers effects of cations on the hydrolysis of pNPP were determined

used were: for AcpA (FTT0221): acpAFw, 5Ј-atgaagctcaata- using 1 m^Meach of Fe^2ϩ, Cd^2ϩ, Co^2ϩ, Mn^2ϩ, Ca^2ϩ, Fe^3ϩ

aaattactttagg-3Ј and acpARv, 5Ј-ttagtttaatttatccactactaatcctg- Mg^2ϩ, Ni^2ϩ, Cu^2ϩ, and Zn^2ϩin the form of chloride salts. Sat-

3Ј; for AcpB (FTT0156): acpBFw, 5Ј-atgacgcaacaacaagttatt- uration kinetics was assessed over a range of substrate concen-

agc-3Ј and acpBRv, 5Ј-ctaaaatcttgagttctcac-3Ј; for AcpC trations (0.05–15 m^M). Kinetic parameters (K_mand V_max) were

(FTT0620): acpCFw, 5Ј-atgagacaaataatattaatctttgtg-3Ј and determined by nonlinear curve fitting using Origin 8 software

acpCRv, 5Ј-ttattgccagctgccataca-3Ј; and Hap (FTT1064): (Northampton, MA).

hapFw, 5Ј-atgagaaaaatattcactatcg-3Ј and hapRv, 5Ј-ctatgttggt-

Inhibitor Screening Assays—The inhibitor screening assays

gtagaaactt-3Ј. The extra sequences 5Ј-ttgtatttccagggc-3Ј and were performed in 96-well format with 1152 small molecules

5Ј-caagcttcgtcatca-3Ј were added to the 5Ј-end of the forward found in the Prestwick chemical library (Prestwick Chemical,

and reverse primers, respectively, for the ligation-independent France) at a final concentration of 1.3 ␮g/ml. The dosage

cloning using a BD-infusion^TMCF Dry-Down PCR cloning kit dependence of potential inhibitors was tested in different buff-

(BD Biosciences). The plasmid p15TV-L (GenBank^TMacces- ers at concentrations ranging from 15 m^Mto 50 nM. The equa-

sion number EF456736) obtained from the Structural Genom- tion used for calculation of the inhibition constant for compet-

ics Consortium (Toronto) was used as a vector for all clones itive inhibition was K_mЈ ϭ (K_m*(1 ϩ [I]/K_i), and V_maxЈ ϭ

(13). This construct provides for an N-terminal hexahistidine

V_max/(1 ϩ [I]/K_i) for noncompetitive inhibitions. All measure-

tag separated from the protein by a tobacco etch virus protease ments were made in triplicate and in at least two separate

recognition site (ENLYFQ2GS). To achieve better protein experiments.

recovery the p15TV-L vector was further engineered using

Construction of the LVS acpA Mutant—The acpA (FTL_158)

inverse PCR by adding the sequence MPLGKNVKKK upstream gene deletion in live vaccine strain (LVS) was constructed as

of the His tag (14) to improve the solubility of the protein.

described with other genes (18). In brief, a 1100-bp upstream

Protein Purification—The His-tagged fusion proteins were fragment was amplified from LVS chromosomal DNA (forward

overexpressed in E. coli BL21-Star(DE3) cells (Stratagene) har- primer, 5Ј-acgcgtcgacGGA GTT AGT GAT TTA GTT GCA

boring an extra plasmid encoding three rare tRNAs (AGG and ATA GGT GTT GC-3Ј; reverse primer, 5Ј-cgcggatccGCT TCA

5172 JOURNAL OF BIOLOGICAL CHEMISTRY

VOLUME 285•NUMBER 8•FEBRUARY 19, 2010

AcpA Inactivation by Ascorbate Affects F. tularensis Survival

TABLE 1

TAT GAT ACC TTT AGT TGT TAG ATT CAA AGG AAA

Kinetic parameters of purified F. tularensis acid phosphatases

TAT TAA TAA C-3Ј), digested with SalI and BamHI and

inserted into SalI/BamHI-digested pJC84 plasmid. The result-

ing plasmid was digested with BamHI and XmaI and ligated

into this was a 1100-bp downstream fragment of acpA (forward

primer, 5Ј-cgcggatccAAA TAT TTA CTC GGT AAG TTG

CTT TAA TCT AGT ATT TTC GC-3Ј; reverse primer, 5Ј-tcc-

ccccgggGCT AAA GAT AAG GGC ATA AAG ACT ATC AAA

GAG AG-3Ј). The final construct named pJC84-AcpA was

transformed into LVS strain by electroporation and was pro-

V_max

K_m

k_cat

_cat/K_m

␮mol min^Ϫ1mg

^Ϫ1s

Ϫ1

AcpA

AcpB

AcpC

Hap

0.25 Ϯ 0.05

11.84 Ϯ 0.87

2.03 Ϯ 0.08

0.30 Ϯ 0.00

0.78 Ϯ 0.18

3.11 Ϯ 0.28

0.88 Ϯ 0.13

0.68 Ϯ 0.01

0.24

4.48

0.97

0.23

3.0 ϫ 10²

1.44 ϫ 10³

1.10 ϫ 10³

0.34 ϫ 10³

^aEstimated by nonlinear regression analysis using MicroCal Origin 8.

^bk_catwas determined based on the monomeric molecular weight of each protein.

cessed as described earlier (18). The LVS-acpA mutant was ent pH values (range 5–7.5), combined with salts and reducing

confirmed by PCR and Southern blot analysis. compounds. Unexpectedly, it was possible to recover active

In Vitro Minimal Inhibitory Concentration Assay—The min- protein only after 18–24 h dialysis against 25 m^MHEPES, pH

imal inhibitory concentration assay was performed in 96-well 7.5, containing 500 m^MNaCl and 1 mM dithiothreitol. The pro-

format. ^L-Ascorbic acid and 2-phospho-L-ascorbic acid (Sigma) cedure was systematically repeated, and the results were con-

were prepared fresh and diluted in recommended diluents to a sistent and reproducible. All enzymes were preserved in dialysis

final concentration of 500 m^M. Overnight cultures grown in buffer with 5% glycerol at Ϫ80 °C. Thawed aliquots demon-

TSB with 0.1% cysteine hydrochloride were diluted to 2 ϫ 10⁷strated good activity after a year of preservation in these

CFU/ml in TSB. Diluted cultures (50 ␮l) were placed in 96-well conditions.

microtiter dishes and mixed with various amounts of inhibitors

Biochemical Properties of the Francisella Acid Phosphatases,

(50 ␮l, 2ϫ concentration) diluted in TSB. Cells and peptides Establishment of Enzymatic Screen Conditions—Although the

were incubated at 37 °C overnight in a humid environment. biochemical characteristics of F. tularensis AcpA were previ-

Wells were evaluated for growth visually to assess bacterial via- ously described by Reilly et al. (10, 19), we carried out a set of

bility. Controls contained TSB in place of inhibitors.

preliminary assays to establish the conditions that would allow

Intramacrophage Survival Assay—Intramacrophage survival the screening of a large number of small molecules in a high

assay was performed in J774.1 and phorbol 12-myristate 13-ac- throughput manner. In all cases, the enzyme activity was de-

etate (10 n^M/ml) induced THP-1 macrophages. In brief, 2 ϫ 10⁵pendent on the chemical composition of the buffer utilized. We

macrophages were seeded in 24-well tissue culture plates. determine the best buffers for each enzyme: MES or MOPS, pH

Monolayers were infected with F. novicida, LVS, and their acpA 6.2, for AcpA, MOPS, pH 6.4, for AcpC, and HEPES, pH 7.0, for

mutants at multiplicity of infection of 50:1 in the presence or AcpB and Hap. Buffers were used at a 25 m^Mconcentration. No

absence of the AcpA inhibitors (10, 30, or 100 m^M). After 2 h of differences in activity were detected in the presence or absence

infection monolayers were washed and treated with fresh of the His₆tag in the purified protein (data not shown). AcpB,

media containing gentamicin (50 ␮g/ml) for 30 min to remove AcpC, and Hap activities were enhanced by the presence of

extracellular bacteria. Cells were washed and replenished with Mg^2ϩor Co^2ϩ(ϳ2- to 2.5-fold). The presence of Fe^2ϩstimu-

fresh media containing gentamicin 10 ␮g/ml and the AcpA lated AcpC (40% increase) and Hap (20% increase), but the

inhibitors. The cells were lysed at different time points with activity of AcpA and AcpB was diminished by 60% to 80%. Nev-

0.05% SDS, diluted in phosphate-buffered saline, and plated on ertheless, the cations with a positive effect were not necessary

CHOC-II plates to enumerate the CFU.

to visualize the enzyme activity using colorimetric methods or

Macrophages viability was evaluated by trypan blue dye to perform classic saturation assays. Therefore, no cofactors

exclusion assay, and their ability to produce interleukin 1␤ were added to perform the saturation assays with AcpA, AcpB,

in the presence of ascorbate and 2-phosphoascorbate was and AcpC. On the contrary, Hap required 0.1 m^Mof Mg^2ϩin

assessed by using a DuoSet ELISA kit after induction with 10 ng the reaction mixture to use pNPP efficiently and reach full sat-

of purified Salmonella lipopolysaccharide. Results are shown in uration. All the enzymes displayed a characteristic Michaelis-

supplemental Fig. S2 .

Menten saturation curve. The kinetic parameters of the puri-

fied enzymes are summarized in Table 1. All proteins showed

similarly low catalytic efficiency. AcpB displayed the lowest

RESULTS

Protein Purification—The four main acid phosphatases of F. affinity (3.11 m^M) toward the model substrate, whereas the

tularensis were successfully cloned, overexpressed, and puri- other three phosphatases have K_mvalues in a narrow compara-

fied. AcpB produced the best yield; 1 mg/liter of culture media ble range (0.68–0.88 m^M).

was centrifuged, whereas AcpA, AcpC, and Hap were obtained

Screening methodology was optimized to follow enzyme

at low yields (50–100 ␮g/liter of culture centrifuged). After activity in a continuous fashion for 30–40 min. The best con-

purification the enzymes were individually dialyzed against ditions to measure AcpA activity was obtained with 1 ␮g/ml

saline Tris-HCl buffer, pH 7.0, prior to enzymatic assays. Tris- protein in 25 m^MMES, pH 6.2, at 37 °C. Similar activity was

HCl buffer, pH 8.00, was the best choice to purify AcpB as a low obtained in MOPS and acetate buffer as described before (10).

amount of protein was recovered when using HEPES buffer. AcpB was used at 0.5 ␮g/ml and AcpC at 1 ␮g/ml. The substrate

Additionally, AcpB lost activity quickly when dialyzed against (pNPP) was used at the same concentration (1 m^M) for all pro-

Tris-HCl buffer, pH 7.0. To overcome this problem we set up a teins. This concentration is slightly above the K_mof AcpA and

systematic dialysis protocol using a battery of buffers at differ- AcpC (0.78 Ϯ 0.18 and 0.88 Ϯ 0.13 m^M, respectively). Keeping

FEBRUARY 19, 2010•VOLUME 285•NUMBER 8

JOURNAL OF BIOLOGICAL CHEMISTRY 5173

AcpA Inactivation by Ascorbate Affects F. tularensis Survival

TABLE 2

Biochemical characteristics of F. tularensis acid phosphatases

inhibitors

Enzyme

Small molecule

K_i^a

Interaction

␮M

AcpA

Ascorbic acid

380.0 Ϯ 160

NC^b

(ϩ)-5,6-O-Isopropylidene-L-ascorbic 3.27 Ϯ 0.85

acid

2-Phospho-^L-ascorbic acid

2-Sulfate-^L-ascorbic

0.32 Ϯ 0.16

ND^c

6-Palmitate-L-ascorbic

AcpB Thonzonium bromide

Procainamide hydrochloride

AcpC Atracurium besylate

Myricetin

0.59 Ϯ 0.23

14.8 Ϯ 3.4

342.0 Ϯ 50

154.0 Ϯ 89

190.0 Ϯ 30

C^d

Kaempferol

^aCalculated from at least three independent determinations using different concen-

tration of inhibitors.

^bNC, no competitive interaction.

^cND, inhibition was not detected.

^dC, competitive interaction.

FIGURE 1. Effect of small molecules on AcpA phosphatase activity.

Enzyme activity was measured using 1 mM pNPP as substrate in 25 mM MES,

pH 6.2, buffer. A control assay without inhibitor was always included in the

plates as positive internal standard. The development of yellow color was

the substrate concentration respect to K_min a range of 1.1–1.3

was considered optimal to maximize the sensitivity of the

method. AcpB, on the contrary, showed low affinity toward

pNPP. In this particular case we kept the substrate concentra-

tion lower than the K_mdue to the high velocity of the enzyme

(Table 1). A lower substrate concentration with respect to K_m

minimized the noise generated by the product released after

hydrolysis, improving the potential to identify enzyme inhibi-

tors. Due to low Hap recovery, low hydrolysis rate, and purity

degree, this enzyme could not be screened.

quantified by continuous reading at 412 nm, and the p-nitrophenol released

Ϫ1

by the action of the enzyme was calculated using ⑀ ϭ 17.7 mM cm^Ϫ1

Screening was performed in 96-well format in a final volume of 300 ␮l with 1

␮g/ml of reaction mixture of purified enzyme. The enzyme activity was

assayed against 1152 small molecules dispensed in the Prestwick Chemical

Library. The effects of ascorbate on AcpA activity, indicated by the arrowhead,

inhibited Ͼ40% the release of p-nitrophenol and was selected for further

studies.

Using the ascorbate scaffold as a guide, the effect of four

other compounds with different functional groups on the

High Throughput Screening for AcpA, AcpB, and AcpC ascorbate skeleton was assayed. The inhibition constants

Inhibitors—We performed an unbiased screen of AcpA, AcpB, obtained are shown in Table 2. We found that 2-phospho-^L-

and AcpC against a library of 1152 small molecules by following ascorbic acid and 5,6-isopropylidene-^L-ascorbic acid were

the time course of pNPP hydrolysis. All compounds that stronger inhibitors than ^L-ascorbic acid, whereas 2-sulfate-L-

decreased the enzyme velocity by Ͼ40% were selected and indi- ascorbic acid and 6-palmitate-^L-ascorbic acid had no effect on

vidually analyzed. Once the best hits for each enzyme were AcpA activity. The highest inhibition was achieved with

identified, the inhibition constants from saturation kinetics 2-phosphoascorbate (apparent K_iof 0.32 Ϯ 0.16 ␮M). Satura-

were estimated. The results obtained are summarized in Table tion kinetics determined that 2-phosphoascorbate as well as

2. A typical chart with the results of the screening assay 5,6-isopropylidene ascorbate K_iof 3.27 Ϯ 0.85 ␮M) are non-

obtained for AcpA is depicted in Fig. 1. When available, com- competitive inhibitors of AcpA (Fig. 2). Because the former

pounds with related chemical scaffold were assessed, and their compound has a phosphate group, we used malachite green

kinetic parameters were determined.

(17) to determine if 2-phosphoascorbate could serve as an enzy-

Two inhibitors, thonzonium bromide and procainamide, matic substrate for AcpA. After incubating a reaction mix with

were identified in the primary screen for AcpB. Further char- AcpA (1–5 ␮g/ml) for more than 4 h using 2-phospho-L-ascor-

acterization of their kinetic parameters showed they are com- bic acid as a substrate, no pyrophosphate was released (data not

petitive inhibitors with K_iin the low micromolar range (0.59 Ϯ shown). These results demonstrated that 2-phosphoascorbate

0.23 ␮M and 14.8 Ϯ 3.4 ␮M, respectively, Table 2). Similarly, two is not an enzymatic substrate for AcpA.

competitive inhibitors were identified for AcpC, atracurium

Analysis of AcpA Histidine Ascorbylation—The modification

besylate and myricetin, with K_iof 342 Ϯ 50 ␮M and 154 Ϯ 89 of the kinetic parameters by ascorbate, 2-phospho-^L-ascorbate,

␮M, respectively (Table 2). Kaempherol, a flavonoid similar to and 5,6-isopropylidene ascorbate on AcpA activity indicated

myricetin, also showed competitive inhibition with similar K_ithat the inhibition is noncompetitive and irreversible.

(Table 2).

Although the crystal structure of AcpA is available (20), it was

The results obtained in the primary screen of AcpA showed not possible to identify the specific sites of ligand-protein inter-

that ascorbate decreased the enzyme V₀by 53% (Fig. 2). The action by in silico modeling due to the kind of inhibition iden-

derived kinetic parameters showed that ascorbate behaves as a tified. However, it is well known that histidine, lysine, and argi-

noncompetitive inhibitor with apparent K_iof 380 Ϯ 160 ␮M. nine residues are the main targets of the Maillard reaction and

Once AcpA reacted with ascorbate, the activity was not recov- that these residues can be modified by glycation or ascorbyla-

ered even after exhaustive dialysis. This loss of activity may tion (21). A previous analysis of the protein structure indicates

indicate that the enzyme modification was irreversible and that the catalytic site has four histidine residues, His-106, His-

occurs by covalent modification.

287, His-288, and His-350 (20). These observations, together

5174 JOURNAL OF BIOLOGICAL CHEMISTRY

VOLUME 285•NUMBER 8•FEBRUARY 19, 2010

AcpA Inactivation by Ascorbate Affects F. tularensis Survival

FIGURE 2. AcpA enzyme kinetics in presence of inhibitors. Assays were performed in triplicate in 96-well plates using the model substrate pNPP in 25 mM

MESbuffer, pH6.2, with1␮g/mlpurifiedenzyme. Enzymeactivitywasmonitoredcontinuously, andtheinitialvelocity(V₀)wascalculatedfromthelinearrange.

The kinetic parameters were estimated by nonlinear regression analysis using MicroCal Origin 8. Symbols: The control assay (no inhibitor added) is indicated by

f in both panels. The symbols used to identify the concentrations of ascorbate are: A, 0.05 mM (ꢀ), 0.25 mM (F), 0.5 mM (Œ), and 0.75 mM (E). 2-Phosphorascor-

bate results are depicted in B and the concentrations used were 0.05 ␮M (ꢀ), 0.25 ␮M (F), 0.5 ␮M (Œ), and 0.75 ␮M (E). The values used in each figure are the

average of three independent determinations. The chemical scaffolds of the inhibitors used in each case are shown as sodium salts.

with the higher inhibition effects of 2-phosphoascorbate on novicida was tested at 10, 30, and 100 m^Mthroughout the assay

AcpA activity, suggested potential ascorbylation sites. Conse- against each of the strains described. We found that the highest

quently, we conducted site-directed mutagenesis on the four concentration was toxic to the macrophages as evidenced by vital

His residues of the active site (His-106 3 Ala, His-287 3 Ala, staining with Trypan blue, whereas the addition of either 30 m^M

His-288 3 Ala, and His-350 3 Ala), and the mutant proteins ascorbate or 2-phosphoascorbate to macrophages caused no del-

were successfully purified. Replacement of His with Ala at posi- eterious effect on viability and interleukin production (Fig. S2 ). At

tions 106, 288, and 350 rendered inactive enzymes (data not 10 m^Ma 2 log decrease in the intramacrophage survival of F. novi-

shown). In the His-287 3 Ala mutant the enzyme activity cidawasobservedwhilethestrongesteffectwasobtainedat30m^M

decreased ϳ10-fold related to the wild type (0.025 Ϯ 0.006 (Fig. 3A). Similar results were observed for F. tularensis LVS (Fig.

Ϫ1

␮mol min mg^Ϫ1), however 2-phosphoascorbate no longer 3C). The specificity of the inhibition was assayed by testing the

inhibited the enzyme activity (supplemental Fig. S1 ).

effect of the inhibitors on the survival of the respective isogenic F.

Intramacrophage Survival Assays—F. tularensis acid phos- tularensis novicida ⌬acpA (Fig. 3B) and F. tularensis LVS ⌬acpA

phatase A contributes to 90% of the total phosphatase activity (Fig. 3D) under the same conditions. Although the ⌬acpA mutant

displayed by this bacterium. Its biological role is still obscure, survives less than the wild-type strain within macrophages, no

but this enzyme has been indicated as a major F. tularensis effect was observed for any of the inhibitors (Fig. 3, B and D). This

virulence factor (4, 22). Since we successfully identified a com- resultindicatesthatthechemicals specificallytargetedAcpA. Sim-

pound that specifically inhibits AcpA activity, a set of bioassays ilar results were obtained using either JT774.1 or THP-1 macro-

were performed to validate the biological relevance of the bio- phage cell lines.

chemical results herein described. The assays were carried out

CONCLUSIONS

using two different wild-type strains, F. tularensis novicida

U112 and F tularensis LVS, as well as with their respective

⌬acpA mutant strains.

Using a systematic enzyme screening approach we were able

to select chemical compounds with the ability to inhibit three of

To determine if the AcpA inhibitors affected the growth/ the four primary F. tularensis acid phosphatases. Exhaustive

survival of Francisella, growth inhibition assays were per- analysis using enzymatic inhibition kinetics indicated that

formed in liquid culture in presence of the chemical com- AcpB and AcpC were competitively inhibited. On the other

pounds selected. None of the AcpA inhibitors affected the hand, ascorbate and ascorbate derivatives inhibited AcpA in a

viability of the strains when used at concentrations lower than nonreversible, noncompetitive fashion. Several lines of evi-

40 m^M. The minimal inhibitory concentrations for the com- dence suggest that AcpA activity is critical for F. tularensis sur-

pounds assayed (ascorbate and 2-phosphoascorbate) were sim- vival inside infected cells and that purified AcpA could inhibit

ilar and higher than 100 m^M.

the respiratory burst of porcine neutrophils (10) High expres-

Because AcpA has been related to the ability of F. tularensis to sion of the AcpA gene was detected in pathogenic F. tularensis

survive inside the host phagocytic cells (4), we evaluated the effect when it was compared with nonvirulent strains (23), and ⌬acpA

of the inhibitors using macrophage cell lines. The effect of ascor- mutants were less virulent than the isogenic wild-type strain (4,

bate and 2-phosphoascorbate on the intracellular growth of F. 22, 24). Because of its importance in the pathogenesis of this

FEBRUARY 19, 2010•VOLUME 285•NUMBER 8

JOURNAL OF BIOLOGICAL CHEMISTRY 5175

AcpA Inactivation by Ascorbate Affects F. tularensis Survival

tein surface. The results herein

obtained with Ala replacement

assays are in agreement with the

importance previously predicted for

the histidine patch (20). The

replacement of any His residues

(106, 287, 288, and 350) drastically

affected the catalytic abilities of this

protein. Notwithstanding, the low

residual activity displayed by the

His-287 3 Ala mutant was useful to

identify these residues as putative

ascorbylation targets, because the

activity of this mutant protein was

not inhibited by the presence of

2-phosphoascorbate.

Chemical reactions between

ascorbate, or the products of oxida-

tive ascorbate degradation with pro-

teins, can occur in vitro under phys-

iological conditions (25). Some

evidence suggests that these reac-

tions also occur in the same way in

vivo (26). The question arises as to

whether or not the biological func-

tion of AcpA can be hampered by

this modification. Can ascorbate be

used to inhibit AcpA activity and

directly affect the ability of F. tula-

rensis to survive inside macro-

phages? Our results indicate that F.

tularensis does not replicate and is

killed inside macrophages in the

presence of ascorbate.

FIGURE 3. F. tularensis intramacrophage survival assays performed in the presence of AcpA-specific

inhibitors. The assay was carried out using J774.1 murine macrophage cell line infected with F. tularensis

novicida (A) and F. tularensis novicida ⌬acpA (B). The second assays was carried out with phorbol 12-myristate

13-acetate-induced THP-1 macrophage cell line infected with F. tularensis LVS (C) and F. tularensis LVS ⌬acpA

(D). Mutantstrainswereusedtodemonstratethespecificactionoftheinhibitorsselected. Survivalisexpressed

inCFU/mlcountedinagarplatesaftermacrophageswerelysedby0.05%SDStreatment. Inhibitorswereadded

to the cell culture at 30 mM final concentration and kept throughout the experiment until the cells were

collected. Symbols: Control assay (no inhibitor added) (f); ascorbate 10 mM (ꢀ); 2-phosphoascorbate 10 mM

(ꢀ); ascorbate 30 mM (F); and 2-phosphoascorbate (‚) 30 mM. The assays were performed in triplicate.

The clinical importance of ascor-

organism, we focused our work on evaluating the mechanisms bate has been extensively studied and analyzed (27–29). How-

of AcpA inhibition and validating the biochemical results in ever, a biochemical mechanism by which ascorbate inhibits

intramacrophage survival. Ascorbate, as well as sugars, react enzymatic activities with a clear in vivo impact has not been

nonenzymatically with proteins by forming covalent adducts described. The discussion of the clinical importance of vitamin

with amino acids. This biochemical process is known as the C is well known and out of the scope of this report. Dr. Robert F.

Maillard reaction (21). The amino acids lysine, arginine, and Klenner has published hundreds of clinical cases of viral and

histidine were previously identified as the preferred targets of bacterial infections treated successfully with massive intrave-

protein ascorbylation (21). Any of these residues present in nous doses of vitamin C (30). He described toxin neutralization,

AcpA can be equally ascorbylated, and consequently the mech- antibiotic effects, and stimulation of the immune system. Per-

anism affecting activity is difficult to elucidate. However, when haps the results herein discussed can offer some insight at the

2-phosphoascorbate was tested, the K_ivalues achieved were molecular level to help further understanding of the positive

three orders of magnitude lower than those obtained with effects of ascorbate in clinical cases described more than 50

ascorbate. Although this interaction is noncompetitive, this years ago.

observation directed our research to analyze the residues in

Acknowledgments—We thank Dr. Max Teplitski for his valuable

comments, Beverly Driver for her technical assistance, and Ricardo

Valladares for the proofreading of the manuscript.

close proximity of the catalytic pocket. Previous analysis of the

structure of AcpA revealed a patch of four His residues on

the solvent side of the enzyme, contiguous to the active residue

(Ser-175). It was proposed that these histidines work as the

general base to activate a water molecule during catalysis (20).

Our hypothesis was that the electrostatic interaction between

the positively charged His patch and the negatively charged

phosphate group of 2-phosphoascorbate can enhance the inter-

action of this compound with this precise location on the pro-

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