Running title: Alkaline phosphatase inhibition and Francisella survival 1 Francisella DnaK Inhibits

24 Pages·2012·1.03 MB·English

JBC Papers in Press. Published on August 24, 2012 as Manuscript M112.404400
The latest version is at http://www.jbc.org/cgi/doi/10.1074/jbc.M112.404400
*Running title: Alkaline phosphatase inhibition and Francisella survival

Francisella DnaK Inhibits Tissue Nonspecific Alkaline Phosphatase**
Bernard P. Arulanandam1†, Senthilnath Lakshmana chetty1†, Jieh-Juen Yu1, Sean Leonard1, Karl
Klose1, Janakiram Seshu1, Andrew Cap2, James J. Valdes3, and James P. Chambers1
1From the Department of Biology, The University of Texas at San Antonio, San Antonio, TX 78249
2US Army Institute of Surgical Research, Ft. Sam Houston, TX 78234
3US Army Edgewood Chemical Biological Command, Aberdeen Proving Ground, MD 21010
*Running title: Alkaline phosphatase inhibition and Francisella survival
†Authors contributed equally
To whom correspondence should be addressed: James P. Chambers, Department of Biology, The
University of Texas at San Antonio, One UTSA Circle, San Antonio, TX, USA, Tel.: (210)-458-
5663; FAX: [phone]; E-mail: [email protected]
D
Keywords: Francisella, alkaline phosphatase, virulence, host response___________________________ o
w
n
lo
Background: Pulmonary Francisella infection analysis indicated the inhibitory factor to be ad
e
d
resulted in reduction of plasma alkaline the heat shock protein DnaK. Incubation fro
phosphatase activity. with increasing amounts of anti-DnaK m
h
antibody reduced the inhibitory effect in a ttp
Results: Francisella heat shock protein DnaK dose-dependent manner. Furthermore, DnaK ://w
w
binds to alkaline phosphatase reducing contains an adenosine triphosphate binding w
enzymatic activity. domain at its N-terminus, and addition of .jbc
.o
adenosine triphosphate enhances dissociation rg
Conclusion: A Francisella protein component b/
of DnaK with its target protein, e.g., alkaline y
responsible for alkaline phosphatase inhibition gu
phosphatase. Addition of adenosine e
was identified. triphosphate resulted in decreased DnaK co- st o
n
A
uSdseigefendn ibfsieyc. a a n bcea:c te Wriael pparethseongte na tnoo evveal dme ethceh ahnoisstm’s ipFmhramonsupcnihsoaeptllarase-ecm ipeidataisa tteeddw ella lkaawlsii nthe repdhuoctsaipolhknaa tlainoseef pril 3, 201
inhibition further supporting the binding of 9
SUMMARY Francisella DnaK to alkaline phosphatase.
Release of DnaK via secretion and/or
Following pulmonary infection with bacterial cell lysis into the extracellular
Francisella tularensis, we observed an milieu and inhibition of plasma alkaline
unexpected but significant reduction of phosphatase could promote an orchestrated,
alkaline phosphatase, an enzyme normally inflammatory response advantageous to
up-regulated following inflammation. Francisella.
However, no reduction was observed in mice
infected with a closely related Gram negative Francisella tularensis is a facultative
pneumonic organism (Klebsiella pneumonia) intracellular, Gram negative bacterium that
suggesting the inhibition may be Francisella causes the zoonotic disease pulmonary tularemia
specific. In similar fashion to in vivo (1,2). Several F. tularensis species and
observations, addition of Francisella lysate to subspecies are recognized including (i) F.
exogenous alkaline phosphatase (tissue tularensis subsp. tularensis (type A), (ii) F.
nonspecific isozyme) is inhibitory. Partial tularensis subsp. holarctica (type B), (iii) F.
purification and subsequent proteomic tularensis subsp. mediasiatica, and (iv) F.
1

Copyright 2012 by The American Society for Biochemistry and Molecular Biology, Inc.
*Running title: Alkaline phosphatase inhibition and Francisella survival

novicida (1). While type A and B strains are the In this study, F. novicida lysate protein was
most relevant in terms of human disease, F. subjected to anion exchange chromatography
novicida and the live vaccine strain F. tularensis and electrophoretic separation. Using an in vitro
LVS (derived from holarctica) are attenuated in assay, inhibition of AP was determined. We
humans while retaining virulence in mice (3-5). provide evidence that heat shock protein DnaK
F. novicida exhibits > 95% genetic homology of F. novicida binds to AP reducing enzymatic
and shares biochemical features with type A (6). activity. This is the first report of such a novel
We have previously reported that in a murine mechanism used by a pathogen to evade the
pneumonic tularemia model, F. novicida rapidly host’s defense.
disseminated from the challenge site (lungs) to
liver with a progressive increase in bacterial load EXPERIMENTAL PROCEDURES
by 72 h (7). Liver damage resulting from
pulmonary F. novicida infection was assessed by Bacterial strains-Francisella novicida strain
analyzing liver function enzymes in plasma and U112, F. tularensis subsp. tularensis (type A,
a marked decrease in total alkaline phosphatase SCHU S4 strain), F. tularensis subsp. holarctica
(AP) activity as early as 48 h after pulmonary strains (type B, OR96-0246 and LVS, lot 703-
challenge was observed. This observation of 0303-016), Klebsiella pneumoniae (KPPR
1
decreased AP was unexpected since most strain) (16), and Salmonella typhimurium D
reported pathogen infections give rise to (ATCC, strain 14028) were inoculated in ow
n
increased AP activity. trypticase soy broth (TSB) supplemented with loa
d
e
0.1 % (w/v) L-cysteine hydrochloride, 0.025 % d
Alkaline phosphatase [(AP), orthophosphoric (w/v) sodium pyruvate, 0.025 % (w/v) sodium from
EmCon oe3s.1te.3rp.1h]o phiso hyrdersoploasnes,i blael kalfionre orepmtimovuimng, metabisuilphite, and 0.025 % (w/v) FeSO4. After http
phosphate groups from a wide variety of reaching stationary phase, cells were harvested ://w
by centrifugation, and stored at -80 °C until w
w
molecules. In mice, there are four genes coding used. .jb
for AP: intestinal, placental, germ cell, and c.o
tissue nonspecific (TNAP). The latter form is Preparation of Plasma-Female BALB/c mice brg/
y
post-translationally modified to differentiate the (5-8 weeks) were obtained from the National gu
bone, liver, and kidney isoforms. There is Cancer Institute (Frederick, MD). All animal est o
growing evidence to suggest that AP may play n
care and experimental procedures were A
an important role in host defense. Within p
primary sites of infection, such as the lung, AP performed in compliance with the Institutional ril 3
Animal Care and Use Committee (IACUC) , 2
is expressed at a high level and may be produced 0
guidelines. Mice were challenged intranasally 19
in pulmonary surfactant particles by type-II
(i.n.) with 100 cfu of either F. tularensis type A
pneumocytes (8). Alkaline phosphatase has
(LD < 10 cfu) or F. tularensis type B (LD =
50 50
been shown to detoxify Gram-negative LPS by
10 cfu) in 25 µl phosphate buffered saline (PBS)
the removal of terminal phosphate groups (9-
or with 400 cfu F. novicida (LD = 10 cfu),
50
11), and AP synthesized by hepatocytes has
LVS (LD = 2800 cfu), or K. pneumoniae (LD
50 50
been reported to play a protective role during
< 100 cfu). Mice were bled at 0, 24, 48, and 72 h
liver damage by the neutralization of endotoxin
post challenge, and plasma prepared using
(12,13). However, the LPS of F. tularensis
plasma collection tubes containing lithium and
exhibits an unusual lipid A structure that does
heparin sulfate (Fisher Scientific, NJ).
not contain exposed phosphate groups and
Respective plasma samples were centrifuged for
generally exhibits low endotoxicity (14,15).
5 min at 5000 rpm, and aliquots frozen at -20 ºC
Moreover, in our studies, purified LPS from F.
until used.
novicida and F. tularensis LVS demonstrated no
measurable effect on host AP activity, indicating
Plasma biochemical assays-Plasma albumin
that LPS was not involved further suggesting
content, as well as alanine aminotransferase
involvement of other bacterial factors.
(ALT), aspartate aminotransferase (AST), and
alkaline phosphatase (AP) levels were measured
2
*Running title: Alkaline phosphatase inhibition and Francisella survival

at the University of Texas Health Science Center Bacterial lysate preparation-F. novicida, K.
at San Antonio using an Olympus AU640e pneumoniae, and S. typhimurium were grown as
Chemistry Immuno Analyzer (Olympus, Center described earlier and cells harvested by
Valley, PA). Plasma from infected mice also centrifugation. Following suspension in 5 ml
was analyzed for AP activity (nmol or chilled 10 mM Tris buffer, pH 7.4, cells were
pmol/min/µl) in 96 well microplates by ruptured using a French Pressure Cell Press
measuring the rate of hydrolysis of (American Instrument Co., Silver Spring, MD).
paranitrophenyl phosphate (PNPP) (Sigma- Ruptured cells were centrifuged at 30,000 x g
Aldrich, St. Louis, MO) or 4-methylumbelliferyl for 30 min, and lysate supernatant material was
phosphate (4-MUP) (Sigma-Aldrich) as stored at -80° C until used. Only minimal AP
previously described (17,18). Briefly, plasma activity was detected in the respective
samples (10 µl) were added to 190 µl of a Francisella, Klebsiella, and Salmonella bacterial
substrate solution containing 1.9 mM PNPP lysates.
dissolved in AP buffer (0.1 M glycine buffer, pH
7.4 containg 1 mM MgCl and 1 mM ZnCl ). AP inhibition assay-The effect of F. novicida
2 2
Microplates were incubated at 37 ºC, and lysate (Fn lysate) on exogenously added TNAP
substrate hydrolysis monitored from bovine Kidney unless specified otherwise
spectrophotometrically at 410 nm every 10 min (all AP preparations procured from Sigma
D
for 1 h using a µQuant Microplate Chemical Co., St. Louis, MO) was determined ow
n
Spectrophotometer (Biotek, Winooski, VT). For using 4-MUP as substrate. Briefly, TNAP assay lo
a
fluorometric analyses, plasma samples (10 µl, reaction mixtures contained 90 µl 10 mM Tris ded
1:10 diluent) were added to 190 µl of a substrate buffer (pH 7.4), 7 µl TNAP (25 µg), and 3 µl fro
m
solution containing 5 mM 4-MUP dissolved in crude lysate (100 µg protein). Reaction h
AwPit hb umffoedr.e r aMtei crsohpalkaitnegs , waenrde itnhceu bhayteddro alyt s3i7s ºoCf m4 ihx taufrteers wwheirceh s thimakee nre csopnectitnivueo uasslsya ya tt u3b7e s° Cw eforer ttp://ww
w
substrate was monitored fluorometrically at 360 transferred to ice slurry. To each reaction .jb
nm (excitation) and 465 nm (emission) every 10 mixture, substrate solution (900 µl AP assay c.o
min for 1 h using a Synergy HT Multidetection buffer containing 5 mM 4-MUP) was added brg/
y
Plate Reader (Biotek). Quantitation of substrate followed by incubation at 37 °C for 15 min. g
u
hydrolysis was determined using either a linear Reactions were stopped by addition of 2 ml 0.1 est o
paranitrophenol (PNP; 0-60 nmol) or 4- M dibasic potassium phosphate, and n A
mstaenthdyalrudm cbuerlvlief egroennee rate(4d- MunUd;e r i0d-e6n0ti0c al pamssoaly) fflluoourreosmceetnecre (Tmheeramsuor eSdc ienutsiifnicg, Rao ckQfouradn, teIcLh, pril 3, 2
conditions but in the absence of PNPP or 4- filter settings λexcitation = 345 nm, λemission = 440 019
MUP, respectively. nm). The assay was linear with respect to time
and protein for at least 20 min. Control assay
Detection of AP by zymogram analysis- mixtures were carried out in identical fashion as
Samples were loaded onto 4-15 % gradient described above except 3 µl 10 mM Tris buffer
polyacrylamide gels (BioRad Laboratories, pH 7.4 was added in lieu of Francisella cell
Carlsband, CA), and run under native conditions lysate supernatant material. Quantitation of
at 180V for 2 h after which time the gel was hydrolysis of 4-MUP was achieved using a 4-
washed 3 times with 10 mM Tris buffer, pH 7.4. MU standard curve. One unit inhibitory factor is
Following washing, gels were incubated with defined as a 1 % reduction of TNAP activity
substrate solution (5 mM 4-MUP dissolved in 25 following incubation with 100 µg bacterial
ml of AP buffer) for 15 min. The reaction was lysate protein.
stopped by addition of 25 ml 0.10 M NH OH,
4
pH 10.4, and protein bands associated with Fractionation of bacterial lysate by native
hydrolyzed 4-MUP, i.e., 4-MU were observed PAGE and AP inhibition assay-Bacterial lysate
and photographed under UV light. protein (100 µg) was loaded onto 4-15 % Tris-
glycine gradient gels, and run under native
conditions for 2 h at 180 V. The gel was cut into
3
*Running title: Alkaline phosphatase inhibition and Francisella survival

2-mm segments from top to bottom, and each protein assignments by Scaffold, Proteome
segment was resuspended in 100 µl 10 mM Tris Software, Portland, OR). Significance thresholds
buffer, pH 7.4, homogenized on ice using a for peptide and protein assignments were 95 and
micro-glass tissue homogenizer, and centrifuged 99 % respectively, with a minimum of two
at 30,000 x g to sediment the acrylamide. Eluted peptides required for protein identification.
protein was transferred to a clean assay tube for Summarized in Table 1 are mass spectrometric
determnination of AP inhibition as previously data derived in this study.
described but modified as below. The AP
inhibition assay was carried out in triplicate, Immunoprecipitation and western blot
each assay tube containing 25 µl gel eluate, 25 analysis- Amine activated resin (Thermo
µg TNAP (7 µl) and brought to a final volume scientific, Rockford, IL) was coupled with 10 µl
of 100 µl with 10 mM Tris buffer, pH 7.4. anti-alkaline phosphatase antibody (R and D
Eluate from gel segments from a gel run under Systems, Inc., Minneapolis, MN). Francisella
identical conditions but with no bacterial lysate lysate (360 µl, 500 µg protein) and TNAP (40
served as control. µl, 142 µg) were mixed together with antibody
coupled beads and left at 5 °C for 6-8 h. Beads
DEAE anion exchange chromatography- were washed five times with IP/lysis (Thermo
Anion exchange chromatography was carried Scientific) wash buffer followed by elution with
D
out at 5 °C. Francisella (Fn) lysate (500 µl, 7.8 IP elution buffer (Thermo Scientific). Co- ow
n
mg total protein) was added to DEAE slurry immunoprecipitated proteins were separated on lo
a
equilibrated in 10 mM Tris buffer, pH 7.4, 4-15 % SDS polyacrylamide gels (BioRad de
d
thoroughly mixed (end-over-end) for 4 h at 5 °C Laboratories) and visualized either by coomassie fro
m
after which time the resin was transferred to a blue staining or western blotting using anti-AP h
0w.a2s hxe d7 wcimth g2la scso lcuomlunm vno. lu mThese ecqouluilmibnr awtinags ((Rki&ndDly Spyrsotevmid,e dM binyn eDapr.o lJiso,r gMe NB)e, naacnhti,- DSntaatKe ttp://ww
w
buffer (approximately 2.5 ml breakthrough University New York, Stony Brook), anti- .jb
material), and bound protein batch eluted using GroEL (kindly provided by Dr. Daniel Clemens, c.o
50-200 mM NaCl in equilibrating buffer. University of California at Los Angeles), and brg/
y
Breakthrough material (100 µl), and respective anti-HtpG (kindly provided by Dr. Carol Gross, g
u
salt eluates (100 µl) were diluted to 1 ml with 10 University of California San Francisco) est o
mM Tris buffer, pH 7.4, and concentrated to 100 antibodies. Following transfer of proteins to a n A
µ(3l0 0u0s ingd aalt oCne ntrmicoolnec uCleanr trifwuegiaglh Ft iltecru td evoifcfe, p(Aolmyverinshyalimde nHe ybdoinfldu-oPri;d eG E( PHVeDaFlt)h camree,m bLriattnlee pril 3, 2
Millipore, Billerica, MA). Chalfont, UK), membranes were probed with 01
9
anti-AP, -dnaK, -groEL, and -Htpg antibodies.
Mass spectrometry analysis-DEAE Blots were developed following incubation with
breakthrough material and respective NaCl batch appropriate dilutions of horseradish peroxidase-
eluates were analyzed by native-PAGE (4-15 % conjugated secondary antibody using ECL
gradient gel) using the method of Laemmli (19). Western blotting reagents (GE Healthcare, Little
Coomassie blue-stained bands were excised, Chalfont, UK). Chemiluminescence was
digested in situ with trypsin (Trypsin Profile measured by autoradiography using Kodak XAR
IGD Kit; Sigma Chemical Co.), and resulting film (Eastman Kodak, Rochester, NY).
peptides analyzed by capillary HPLC-
electrospray ionization tandem mass Effect of antibodies and ATP on alkaline
spectrometry (HPLC-ESI-MS/MS) using a phosphatase inhibitory activity. Francisella
Thermo Fisher LTQ mass spectrometer lysate was incubated with anti-DnaK (2.5, 5,
(Department of Biochemistry, The University of 12.5, and 25 μg), anti-GroEL (25 μg), and anti-
Texas Health Science Center at San Antonio). HtpG (25 μg) antibodies for 2 h at room
Mass spectra were searched against the Swiss- temperature with continuous shaking.
Prot database by means of Mascot (Matrix Following incubation, AP inhibition was
assessment of probabilities of peptide and determined as described above. Effect of ATP
4
*Running title: Alkaline phosphatase inhibition and Francisella survival

on F. novicida-mediated AP inhibition was lung tissue, respectively (7). Dissemination of
determined in triplicate, each assay tube organisms from the lungs (initial site of
containing 3 µl crude lysate, 25 µg TNAP (7 µl), exposure) to secondary tissues, i.e., liver, the
and brought to a final volume of 100 µl primary source of AP synthesis, is apparent by
with/without 90 μl 4mM ATP-MgSO (dissolved the bacterial burden increasing from 102 cfu/gm
4
in 10 mM Tris buffer, pH 7.4). A 20 µl aliquot tissue at 24 h post challenge to 106, and 108
was removed from above reaction mixtures for cfu/gm tissue at 48, and 72 h, respectively. Ray
zymogram and western blot analyses. et al. observed similar Schu S4 dissemination
from the lungs to the liver by 72 h post
Statistical Analysis-The Student’s t test was challenge to that observed here using F.
used to determine statistical significance. All novicida (21). Consistent with the original
data are presented as mean values ± the observations of Hambleton and coworkers
respective standard deviation. (22,23), plasma AP activity was observed
significantly reduced, i.e., ~40 and 70 % at 48
RESULTS and 72 h, respectively, in mice challenged i.n.
with human virulent Francisella (Fig 1B).
In vivo and in vitro inhibition of alkaline
phosphatase activity-We have previously There are 3 major AP isozymes in
D
reported that F. novicida rapidly disseminated to mammals. In order to determine which o
w
liver following intranasal (i.n.) challenge (7). isoenzyme was affected following Francisella nlo
a
We further assessed acute damage to this organ infection, tissue nonspecific AP (TNAP) de
d
by analyzing a panel of liver function proteins in enriched from bovine liver and kidney as well as fro
the plasma following Francisella infection. As calf intestinal AP (CIP), and human placental hm
shown in Fig. 1A, while albumin amount AP (PLAP) were assayed following incubation ttp
remained unaffected, aspartate aminotransferase with Francisella (Fn) lysate. As shown in Fig. ://w
w
(AST) activity was elevated 4-fold by 72 h 1C, Fn lysate inhibited less than 6 % CIP and w.jb
compared to uninfected mice (time = 0 hr). PLAP activities but significantly inhibited c.o
Alanine aminotransferase (ALT) activity was TNAP (27 % liver and 38 % kidney isoforms), brg/
observed to increase gradually as the infection strongly suggesting that TNAP is the major AP y g
u
progressed consistent with increased bacterial isozyme inhibited by Francisella infection. es
burden in the liver (20). Interestingly, plasma t o
n
A
AP activity was significantly reduced as early as Specificity of Inhibition of Plasma Alkaline p
48 h post challenge and the enzymatic activity Phosphatase-In order to determine if the ril 3
decreased from 200 international units per liter reduction of AP activity was specific to , 20
1
(IU/l) at 24 h to 50 IU/l by 72 h post challenge Francisella infection, we also examined plasma 9
in contrast to most bacterial and viral infections prepared from mice challenged intranasally with
which are associated with increased plasma AP 400 cfu of a related pneumonic Gram-negative
activity. organism, K. pneumoniae. As shown in Fig. 2A,
plasma AP activity was relatively unchanged up
Under identical conditions used in this to 72 h after K. pneumoniae challenge. In
study, enumeration of F. novicida bacteria in contrast, there was a marked reduction (70 %) of
cell free plasma indicated the presence of few if AP in plasma from both F. novicida (400 cfu),
any organisms at 24 h post challenge, but 1.0 x and F. tularensis LVS (400 cfu) infected mice
103 and 3.5 x 103 cfu/ml blood at 48 and 72 h, by 72 h post challenge (Fig. 2A). Alkaline
respectively (7). These burdens are consistent phosphatase enzymatic assays correlated with
with little to no drop in AP activity reported here zymogram analysis showing no significant
at 24 h, and 50 and 75 % decreased plasma AP change in band intensity associated with
activity at 48, and 72 h post challenge, hydrolysis of 4-MUP to 4-MU during the course
respectively (Fig. 1A). Furthermore, the of the K. pneumonia infection. In contrast, a
bacterial burden in the lungs 24, 48, and 72 h marked reduction of AP intensity in LVS, and
post challenge were 6, 8, and ~5 log cfu/gm of Francisella (Fn) infected plasma was observed
5
*Running title: Alkaline phosphatase inhibition and Francisella survival

as the infection progressed (Fig. 2B) suggesting No band was observed in the absence of TNAP
reduction of plasma AP activity may be specific (Fig. 3C). Inclusion of a protease inhibitor
to Francisella infection. Western blot analysis cocktail (Roche diagnostics, Indianapolis, IN)
of plasma prepared from PBS mock treated mice was inconclusive since the cocktail inhibited
after 72 h using anti-AP antibody revealed a TNAP in the absence of lysate (data not shown).
single dark band at ~ 70 kDa which decreased in
intensity in the plasma of LVS infected animals Fractionation of Francisella novicida
(Fig. 2C) indicating decreased plasma AP inhibitory factor(s) by DEAE anion exchange
protein following LVS infection may account chromatography-In order to further purify and
for the observed reduction of AP enzymatic characterize the inhibitory component(s),
activity. Francisella cell lysate (7.80 mg protein
equivalent to 1,700 starting Units inhibitory
Gel electrophoretic characterization of factor) was loaded onto a DEAE anion exchange
inhibitory factor in bacterial lysate-In an column. Of the protein applied to the column,
attempt to identify the protein specific to 1.80 mg did not exchange with the resin coming
Francisella responsible for AP inhibition, through in the column breakthrough. Elution of
lysates from F. novicida, a related pneumonic bound protein using increasing concentrations of
Gram negative organism, i.e., K. pneumoniae, NaCl resulted in the removal of 3.76 mg total
D
and an unrelated Gram negative enteric, i.e., S. protein with a total recovery of protein inclusive o
w
n
typhimurium were coomassie blue stained of breakthrough protein of 5.54 mg (~70 %). lo
a
following PAGE under non-denaturing Assay of breakthrough material and salt eluates de
d
conditions as shown in Fig. 3A. Although some for inhibition of TNAP activity revealed the fro
m
differences in protein profiles were observed inhibitory factor to elute from 50 through 200 h
comparing the respective lanes, an identical gel mM NaCl with no inhibitory factor found in ttp
was loaded with 100 µg Fn lysate protein (37 either the breakthrough or 25 mM NaCl eluate. ://w
w
w
inhibitory units), K. pneumonia, and S. Following removal of salt by Centricon .jb
typhimurium, and run under identical conditions. filtration/concentration, the maximum number of c.o
The gel was cut in 2 mm segments, and the inhibitory units (62.4) was observed to elute in brg/
y
respective segment eluates evaluated for the presence of 150 mM NaCl corresponding to g
u
inhibition of TNAP activity. As shown in Fig. ~39 % of the total amount recovered following es
t o
3B, TNAP inhibitory proteins electrophoresed as elution but only ~7 % of the starting inhibitory n
A
a broad, heterogeneous peak ranging in units (Fig. 4A). Analysis of the respective p
molecular weight from approximately 72 to 170 eluates by PAGE under non-reducing conditions ril 3
, 2
kDa (closed circles) with majority of inhibition indicated enrichment of a band of approximate 0
1
9
at ~130 kDa. Although the gel was cut from top 130 kDa in the 150 mM eluate (Fig. 4B). The
to bottom, the profile shown in Fig. 3B 130 kDa band was excised, subjected to mass
represents only the inhibitory species eluted spectrophotometric analysis, and identified as
from the respective gel segments. Summation of the molecular chaperone heat shock protein
inhibitory units across the profile accounted for DnaK (Hsp70)(cf. Table 1). A dark staining
approximately 31.5 of the 37 inhibitory units band at approximately 150 kDa was observed in
applied resulting in ~85 % recovery. No starting Francisella lysate material (Fn),
inhibition of TNAP activity was observed for gel breakthrough (BT), and salt eluates (100 and
segment eluates from K. pneumoniae and S. 150 mM) but was not analyzed since assay of
typhimurium. Consistent with decreasing breakthrough material exhibited no AP
intensity of AP activity band observed in the inhibitory activity.
zymogram and western blot analyses of plasma
(Fig. 2B and 2C), western blot analysis of TNAP Interaction of Francisella DnaK with TNAP –
incubated with Francisella (Fn) lysate resulted To confirm DnaK interaction with TNAP, a
in a band of considerable less intensity (TNAP + TNAP-Francisella (Fn) lysate complex was
Fn) compared to the control (TNAP - Fn) with pulled down using anti-AP antibody coupled to
no observable degradation products (Fig. 3C). AminoLink Coupling Resin (αAP:ACR).
6
*Running title: Alkaline phosphatase inhibition and Francisella survival

Protein complex captured by αAP:ACR was concentration to 75 μg/ml resulted in little (3-5
analyzed by PAGE under denaturing conditions, %) reduction of TNAP inhibition (data not
and 3 distinct protein bands ranging from ~60 to shown). Additionally, zymogram analysis (Fig.
~90 kDa were visible after coomassie blue 6B) of anti-DnaK antibody incubation with
staining (Fig 5A, middle lane). Binding of these TNAP and lysate was shown to be protective (25
3 proteins to TNAP was specific since none of μg/ml) in a dose-dependent manner (2.5 μg/ml
these F. novicida proteins were captured by being less protective) of TNAP in comparison to
αAP:ACR when the resin was incubated with no anti-DnaK antibody (0 μg/ml). Collectively,
Francisella lysate in the absence of TNAP (Fig. F. novicida heat shock proteins (DnaK, GroEL,
5A, right lane). To identify these 3 TNAP and HtpG) appear to form a complex that binds
binding proteins, bands were excised from the to TNAP; however, only DnaK plays a role in
gel, trysin digested, and subjected to mass AP inhibition.
spectroscopic proteomic analysis. As expected,
DnaK (~90 kDa) was identified in the upper Effect of ATP on inhibition of TNAP by
band along with the identification of GroEL Francisella lysate-DnaK has a N terminal
(~70 kDa), and HtpG (~60 kDa) in the middle, ATPase, and C terminal substrate binding
and lower band, respectively (proteomic data domains (24,25). Given that binding of ATP
summarized in Table 1). Immunoblotting with alters the conformational state of DnaK resulting
D
anti-TNAP, -DnaK, -GroEL, and –HtpG in a low-affinity state, and subsequent release of ow
n
antibodies further confirmed the presence of the the substrate, e.g. TNAP (26), we assessed the lo
a
respective proteins in the TNAP binding effect of ATP on inhibition of TNAP by Fn ded
complex (Fig. 5B). Proteomic analysis did not lysate. As shown in Fig. 7A, TNAP incubated fro
m
identify TNAP; however, western blot analysis in the presence of added ATP and lysate (TNAP h
dαiAdP :AreCveRa lp utlhl ed owprne (sFenigc.e 5 Bo)f. TNAP in the +sa mFen alcytsivatiety +a s AthTaPt )o besxehrivbeidte dfo re sTsNenAtiPal layl otnhee. ttp://ww
w
In contrast, TNAP activity in the presence of Fn .jb
To further characterize inhibition of TNAP lysate but absence of ATP was reduced ~ 32 %. c.o
by these 3 identified proteins, we used This is corroborated in the zymogram analysis. brg/
y
corresponding antibodies (anti-DnaK, -GroEL, Furthermore, addition of ADP and AMP had no g
u
and -HtpG) to compete for TNAP binding and/or effect on TNAP inhibition by Fn lysate (data not es
t o
neutralization of AP inhibition. Specifically, Fn shown). To further examine whether ATP n
A
lysate was pre-incubated with 0, 2.5, 5, 12.5, and reverses TNAP inhibition by Fn lysate is due to p
25 μg anti-DnaK antibody for 2 h followed by 4 reduction of TNAP binding to DnaK by ATP, ril 3
, 2
h reaction with TNAP. Alkaline phosphatase we used αAP:ACR to capture the TNAP 0
1
9
activity was assayed and results indicated pre- complex formed from the Fn lysate and TNAP
incubation of anti-DnaK antibody with Fn lysate mixture in the presence and absence of ATP. As
markedly reduced AP inhibition in a dose- shown in the immuoblots of the resin captured
dependent manner with up to 80 % reduction of TNAP complex (Fig. 7B), an equivalent amount
TNAP inhibitory activity by 25 μg/ml of anti- of TNAP was recovered from the reaction with
DnaK antibody (Fig. 6A, 25 μg/ml α-DnaK). or without ATP; however, less DnaK was
Abrogation of F. novicida mediated AP detected when ATP was present. Collectively,
inhibition by anti-DnaK antibody is specific these results further support DnaK as being the
since Fn lysate pre-incubated with heat Fn lysate component that binds to and inhibits
denatured anti-DnaK antibody (25hi in Fig. 6A) TNAP.
or IgG isotype (data not shown) has essentially
no effect on AP inhibition. Also, anti-DnaK DISCUSSION
antibody alone did not alter TNAP enzymatic
Francisella tularensis is a highly infectious
activity (data not shown). Incubation of TNAP
bacterium, since inhalation of only a few
with 25 μg anti-GroEL (α-GroEL) or anti-HtpG
organisms can cause severe disease and death.
(α-HtpG) antibodies had no significant effect
Despite the high mortality rate in untreated
(Fig. 6A), and increasing the antibody
7
*Running title: Alkaline phosphatase inhibition and Francisella survival

individuals, little is understood regarding F. However, we confirmed by overlap extension
tularensis virulence factors, or the innate and PCR using primers specific for the DnaK gene,
adaptive immune responses operating at the sites and western blotting with anti-DnaK antibody
of primary infection. Because F. tularensis that although the transposon was present, it
colonizes and causes severe disease in the liver, appears not to be in the DnaK gene. An attempt
an important aspect of virulence is related to the to generate a DnaK mutant by homologous
ability of the organism to survive and multiply recombination was not successful suggesting
inside of hepatic cells. DnaK may be essential for Francisella growth at
37 0C.
In this study, we demonstrate that the plasma
of F. tularensis challenged animals exhibited Consistent with the presence of an N-terminal
marked elevation in both AST and ALT enzyme ATPase domain in Hsp70 and the proposed
activity indicative of liver damage by 72 h as DnaK chaperone cycle, ATP appears to
previously reported (20). The temporal increase significantly reduce inhibition of TNAP by
of these enzymes coincided with a significant Francisella lysate. Binding of ATP at the
decrease of AP in the plasma of Francisella ATPase domain of Dnak has shown to trigger
infected mice. This reduction of host AP the release of substrate by the C terminus
expression was observed across all Francisella substrate binding domain (28). This is
D
species and subspecies, but was not apparent consistent with less Francisella DnaK bound to o
w
n
with another Gram-negative pneumonic TNAP in the presence of ATP. Alkaline lo
a
pathogen, K. pneumoniae. Our results are in phosphatase of plasma was observed to be de
d
agreement with those of Hambleton et. al. significantly inhibited (approximately 75 %) 72 fro
m
(22,23), who observed a significant reduction of h post infection; whereas, TNAP inhibition h
circulating AP in rabbits and monkeys infected using the in vitro assay was 40 % maximum. ttp
by aerosolization, or intraperitoneal challenge Because bacterial cell lysate is the source of AP ://w
w
w
with F. tularensis type A (SCHU S4 strain). inhibitory activity, the 40 % inhibitory .jb
maximum could arise from cell lysate c.o
Francisella induced reduction of AP activity endogenous ATP thus reducing binding of brg/
was further characterized using an in vitro assay DnaK. Additionally, the high turnover number y g
u
and commercially available AP preparations. exhibited by mammalian AP gives a greatly es
Francisella lysate significantly inhibited TNAP, exaggerated impression of the amount of t on
A
the major AP isozyme present in mouse plasma. phosphatase protein actually present in a given p
Bacterial lysates prepared from K. pneumoniae tissue (29). Thus, the amount of DnaK present ril 3
and S. typhimurium indicated no such inhibition could significantly exceed that of plasma AP , 20
1
suggesting inhibition to be Francisella specific resulting in greater inhibition than that observed 9
consistent with in vivo observations. for the in vitro assay (25 µg TNAP protein).
Identification of the inhibitory factor was Inorganic phosphate has been shown to inhibit
acheived using PAGE, DEAE anion exchange TNAP (30). However, using the standard
chromatography, proteomic, and immunologic clinical assay for TNAP with high concentration
means revealing the presence of a complex of artificial substrates (PNPP and 4-MUP), and
comprised of heat shock proteins DnaK, GroEL, diluted plasma and bacterial lysate in both in
and HtpG. Incubation of bacterial lysate with vivo and in vitro TNAP assays respectively,
increasing amounts of anti-DnaK resulted in attainment of V is achieved due to dilution of
max
dose-dependent reduction of TNAP inhibition; P well below threshold inhibitory levels (30).
i
whereas, anti-GroEL and -HtpG had no Incubation of ATP, ADP, or AMP alone with
significant effect. TNAP had no effect upon TNAP activity.
In order to assess the role of DnaK in The nature of the interaction of TNAP with
inhibition of plasma AP activity, we initially DnaK was assessed using western blot analysis.
utilized the DnaK mutant FTN_1284 of the Decreased antibody binding as well as
transposon library of Gallagher, et al. (27). enzymatic activity could arise from
8
*Running title: Alkaline phosphatase inhibition and Francisella survival

conformation alteration consistent with DnaK’s Release of DnaK into the extracellular milieu
‘remodeling’ function or simple blocking of the and inhibition of host plasma AP could promote
TNAP epitope and/or catalytic site preventing an inflammatory response advantageous to
binding of substrate. Heat shock proteins do not Francisella. Recently, Fraley and coworkers
appear to have proteolytic activity, but have demonstrated profound effects on cellular
been shown to be associated with degradation of composition and morphology in Pseudomonas
proteins (31,32). Associated with DnaK is aeruginosa polyphosphate kinase mutants (39).
protease La. Although, a Franciscella La The Francisella poly P kinase gene is induced
mutant lysate (prepared using a Francisella intracellularly and is required for intracellular
mutant library kindly provided KK by Dr. Colin growth and virulence (40). Polyphosphate is an
Manoil, University of Washington, Seattle, WA) inorganic, linear polymer of orthophosphate
had no effect on inhibition of TNAP (data not units linked by phosphoanhydride bonds and has
shown), the involvement of other proteases been extensively studied in prokaryotes and
cannot be ruled out. lower eukaryotes where it functions in basic
metabolism, stress responses, and as a structural
In a comparative proteomic profiling of component (41). In like fashion to DnaK, large-
culture filtrate proteins of F. tularensis subsp. scale release into the plasma compartment of
tularensis, strain SCHU S4 and attenuated F. poly P via bacterial cell lysis or secretion could
D
tularenis subsp. holartica, Konecna et al., function as a proinflammatory mediator by ow
n
identified the most abundant group of culture activating the plasma contact activation, or so- lo
a
filtrate proteins, i.e., secreted to include a group called “intrinsic” coagulations system (42). The ded
of heat-shock proteins (GroES, GroEL, and pathology of the pulmonary tularemia sepsis fro
m
DnaK) which have previously been syndrome is characterized by wide h
doef mFo. ntusltararteends itso t ob es uorfv iimvep aonrtda/nocre m fuolrt itphley ainbisliidtye dinisflsaemmminaattiioonn, aonfd npeycorgortaicn ufloomci asw, iatchc ohmisptoalnyiteidc ttp://ww
w
host cells, suggesting that stress responses are of by fibrin deposition, hemorrhage, and vascular .jb
significance for the virulence of Francisella inflammation (43). These pathologic changes c.o
(33,34). These chaperone proteins are in general are consistent with coagulation system brg/
y
cytoplasmic proteins and none of the proteomic activation. Coagulation activation and fibrin g
u
prediction algorithms suggested that they should deposition may be advantageous to Francisella est o
be found in the extracellular space. However, in vivo as a means to isolate foci of infected n A
rmeecmenbt ransteu diaesss ocsiuagtegde st or thaset crethteedy imn ayo thbeer tailslsouwei ngfr omba ctiemrimalu nere psluicravteioilnla ncaen,d asnudr vitvhauls. pril 3, 2
bacteria (35,36). Pierson and coworkers have Inhibition of AP by DnaK would facilitate this 01
9
reported DnaK to be present in outer membrane process by decreasing poly P clearance.
vesicles of F. novicida suggesting yet another Persistence of Francisella in vivo has been
possible role in virulence (37). Interestingly, shown not to correlate with the mere ability to
such altered and unexpected localization of induce a protective immune response (44). Thus,
proteins is often a hallmark of ‘moonlighting’ the release of bacterial proteins/metabolites via
proteins, proteins possessing multiple apparently secretion and/or cell lysis, i.e., cell death may
unrelated functions performed by one constitute in finality an orchestrated advantage.
polypeptide chain (38). Since the diverse
functions of a protein are frequently associated
with it its cellular location, a protein’s function
in the cytosol may differ from that if located on
the cell envelope, in vesicles, and/or the
secretome, and could be implicated in virulence,
i.e., altering of the extracellular environment.
The mechanism by which DnaK mediates the
inhibition of TNAP remains to be elucidated.

9
*Running title: Alkaline phosphatase inhibition and Francisella survival

BIBLOGRAPHY
1. Ellis, J., Oyston, P.C., Green, M., and Titball, R.W. (2002) Tularemia. Clin. Microbiol. Rev. 15,
631-646
2. Tarnvik, A. (1989) Nature of protective immunity to Francisella tularensis. Rev. Infect. Dis. 11,
440-451
3. Chong, A., Wehrly, T.D., Nair, V., Fischer, E.R., Barker, J.R., Klose, K.E., and Celli, J. (2008)
The early phagosomal stage of Francisella tularensis determines optimal phagosomal escape and
Francisella pathogenicity island protein expression. Infect. Immun. 76, 5488-5499
4. Cong, Y., Yu, J.J., Guentzel, M.N., Berton, M.T., Seshu, J., Klose, K.E., and Arulanandam, B.P.
(2009) Vaccination with a defined Francisella tularensis subsp. novicida pathogenicity island
mutant (DeltaiglB) induces protective immunity against homotypic and heterotypic challenge.
Vaccine 27, 5554-5561
D
5. Conlan, J.W., Shen, H., Golovliov, I., Zingmark, C., Oyston, P.C., Chen, W., House, R.V., and ow
n
Sjostedt, A. (2010) Differential ability of novel attenuated targeted deletion mutants of lo
a
d
Francisella tularensis subspecies tularensis strain SCHU S4 to protect mice against aerosol e
d
challenge with virulent bacteria: effects of host background and route of immunization. Vaccine fro
m
28, 1824-1831 h
6. Titball, R.W., and Petrosino, J.F. (2007) Francisella tularensis genomics and proteomics. Ann. N. ttp://w
Y. Acad. Sci. 1105, 98-121 w
w
7. Yu, J.J., Raulie, E.K., Murthy, A.K., Guentzel, M.N., Klose, K.E., and Arulanandam, B.P. (2008) .jb
c
The presence of infectious extracellular Francisella tularensis subsp. novicida in murine plasma .o
rg
after pulmonary challenge. Eur. J. Clin. Microbiol. Infect. Dis. 27, 323-325 b/
y
8. Edelson, J.D., Shannon, J.M., and Mason, R.J. (1988) Alkaline phosphatase: a marker of alveolar g
u
e
type II cell differentiation. Am. Rev. Respir. Dis. 138, 1268-1275 s
t o
9. Beumer, C., Wulferink, M., Raaben, W., Fiechter, D., Brands, R., and Seinen, W. (2003) Calf n A
idnitseesatsiensa,l aatltkeanluinaete ps hLoPsSph taotxaiscei,t ya inno mveilc eth aenradp peiugtliect sd.r Ju.g P fhoar rlmipaocpooll.y Esaxcpc. hTahriedre. 3(L07P,S 7)3-m7-e7d4i4at ed pril 3, 2
10. Koyama, I., Matsunaga, T., Harada, T., Hokari, S., and Komoda, T. (2002) Alkaline phosphatases 01
9
reduce toxicity of lipopolysaccharides in vivo and in vitro through dephosphorylation. Clin.
Biochem. 35, 455-461
11. Poelstra, K., Bakker, W.W., Klok, P.A., Hardonk, M.J., and Meijer, D.K. (1997) A physiologic
function for alkaline phosphatase: endotoxin detoxification. Lab. Invest. 76, 319-327
12. Verweij, W.R., Bentala, H., Huizinga-van der Vlag, A., Miek van Loenen-Weemaes, A., Kooi,
K., Meijer, D.K., and Poelstra, K. (2004) Protection against an Escherichia coli-induced sepsis by
alkaline phosphatase in mice. Shock 22, 174-179
13. Xu, Q., Lu, Z., and Zhang, X. (2002) A novel role of alkaline phosphatase in protection from
immunological liver injury in mice. Liver 22, 8-14
14. Conlan, J.W., Vinogradov, E., Monteiro, M.A., and Perry, M.B. (2003) Mice intradermally-
inoculated with the intact lipopolysaccharide, but not the lipid A or O-chain, from Francisella
tularensis LVS rapidly acquire varying degrees of enhanced resistance against systemic or
aerogenic challenge with virulent strains of the pathogen. Microb. Pathog. 34, 39-45
15. Dreisbach, V.C., Cowley, S., and Elkins, K.L. (2000) Purified lipopolysaccharide from
Francisella tularensis live vaccine strain (LVS) induces protective immunity against LVS
infection that requires B cells and gamma interferon. Infect. Immun. 68, 1988-1996
10