Killing of gram-negative bacteria by lactoferrin
and lysozyme.
R T Ellison 3rd, T J Giehl
J Clin Invest.
1991;
88(4)
:1080-1091.
https://doi.org/10.1172/JCI115407
.
Although lactoferrin has antimicrobial activity, its mechanism of action is not full defined.
Recently we have shown that the protein alters the Gram-negative outer membrane. As this
membrane protects Gram-negative cells from lysozyme, we have studied whether
lactoferrin's membrane effect could enhance the antibacterial activity of lysozyme. We have
found that while each protein alone is bacteriostatic, together they can be bactericidal for
strains of V. cholerae, S. typhimurium, and E. coli. The bactericidal effect is dose
dependent, blocked by iron saturation of lactoferrin, and inhibited by high calcium levels,
although lactoferrin does not chelate calcium. Using differing media, the effect of lactoferrin
and lysozyme can be partially or completely inhibited; the degree of inhibition correlating
with media osmolarity. Transmission electron microscopy shows that E. coli cells exposed
to lactoferrin and lysozyme at 40 mOsm become enlarged and hypodense, suggesting
killing through osmotic damage. Dialysis chamber studies indicate that bacterial killing
requires direct contact with lactoferrin, and work with purified LPS suggests that this relates
to direct LPS-binding by the protein. As lactoferrin and lysozyme are present together in
high levels in mucosal secretions and neutrophil granules, it is probable that their
interaction contributes to host defense.
Research Article
Find the latest version:
Killing of
Gram-negative
Bacteria by
Lactofernn
and Lysozyme
Richard T. EllisonIII*"and Theodore J. Giehl*Medical and
tResearch
Services, Department of VeteransAffairs
Medical Center, and Division ofInfectious Diseases, Department ofMedicine, University ofColorado School ofMedicine, Denver, Colorado 80220Abstract
Although
lactoferrin
hasantimicrobial activity,
itsmechanism
of action is
notfully defined. Recently
we haveshown that
theprotein alters
theGram-negative
outermembrane.
As thismembrane protects Gram-negative
cells fromlysozyme,
we havestudied whether lactoferrin's membrane effect could
en-hancethe
antibacterial
activity of lysozyme. We
havefound
that while each
protein
alone is bacteriostatic, together they
canbe
bactericidal
for strains of
V.cholrae, S.
typhimurium,
and E.
coli.
The
bactericidal
effect is dose dependent, blocked
by iron
saturation
of
lactoferrin,
and inhibited by high calcium
levels,although
lactoferrin
does notchelate calcium. Using
dif-fering media,
theeffect of lactoferrin and lysozyme
canbe
par-tially
orcompletely inhibited; the degree of inhibition
correlat-ing with media
osmolarity. Transmission
electron microscopy
showsthat
E.coli cells exposed
tolactoferrin and lysozyme
at40 mOsm
becomeenlarged
and
hypodense,
suggesting
killing
through osmotic
damage. Dialysis
chamber studies indicate
that
bacterial
killing
requires
direct
contactwith
lactoferrin,
and work with
purified
LPS suggests that this relates
todirect
LPS-binding
by
theprotein.
Aslactoferrin
and
lysozyme
arepresent together in high levels in mucosal secretions and
neutro-phil granules, it is probable that their interaction contributes
tohost defense. (J. Clin. Invest. 1991.
88:1080-1091.) Key words:Escherichia coli
-Salmonella typhimurium*
Vibriocho-lerae
*lipopolysaccharide
*transferrin
* ironIntroduction
The
proteins
lysozyme and lactoferrin
aremajor components
of
the
specific
granules
of
human
polymorphonuclear
leuko-cytesand
arealso
found
in
high
concentration
in human
mu-cosal
secretions, including
those
ofthe
eye,the oropharynx,
thebreast, the
respiratory
tract,and the vagina (1-3). Several lines
ofevidence
suggest that
their
localization
atthese sites is related
toroles in the human host
defense
system. First, during the
inflammatory response both
areactively secreted
by thepoly-morphonuclear
leukocyte into the external environment
asthis cell releases the
contentsofits
specific
(secondary) granules
Portions ofthis workwerepresented 18September 1989atthe 29th
Interscience Conference Antimicrobial Agents and Chemotherapy, Houston, TX(Abstr.No. 174)andon6May 1990,atthe National
Meeting, American Federation for Clinical Research, Washington,
DC, and werepublishedin abstract form(1990. Clin.Res.
38:392a).
Received
for
publication17August 1990andinrevised form16April1991.
(4). Second, each of
theproteins
has been found to haveinde-pendent
antimicrobial
activity (5-7).
Human lysozyme is
a1,4-fl-N-acetylmuramidase
thatenzy-matically degrades
aglycosidic
linkage
of bacterial
peptidogly-can(8).
Proteins
with comparable activity have
beenidentified
in
essentially
all vertebrates
suggesting
that this is
ahighly
con-served
enzyme.Acting alone human lysozyme lyses and kills
several
Gram-positive microorganisms
by
damaging their
sur-face-exposed peptidoglycan.
Incontrast,
mostGram-negative
organisms
areresistant
tokilling by the
protein.
Inthese
organ-isms
an outermembrane
shields
the
peptidoglycan murein
sac-culus from the
external
environment
(9,
10).
Aslysozyme
can-noteasily penetrate the
outermembrane, the
organisms
areresistant
toits
effects
and the
protein
has
routinely
been
consid-ered
tohave
at mostasecondary
function
in the host
defense
against
these
pathogens
(1
1).
Lactoferrin
wasfirst
recognized
as ahigh-affinity
iron
che-lator in human milk,
butin the 1960s
wasfound
to havebacte-riostatic
properties (2,
12).
Becausethese
effects
onbacteria
could be markedly reduced
oreliminated by
iron
saturation
of
the
protein,
it
washypothesized
that
lactoferrin
actstodeprive
microorganisms
of the
essential
nutrient iron
(13).
Whereas
subsequent work has
continued
toconfirm
this
theory,
several
studies
have
indicated
that
lactoferrin
maybe
directly
bacteri-cidal and that it
canalso
markedly
enhance the
activity
of
secretory
IgA
(sIgA) (14-17).
Theseobservations
suggestthat
lactoferrin
hasadditional mechanisms
ofaction towards
bacte-ria
beyond
those related
toiron
deprivation.
Recent
work in
ourlaboratory has
confirmed
such
activity.
We have
found
thatlactoferrin,
and the related iron chelator
transferrin,
candamage the
outermembrane of
enteric
Gram-negative bacilli
(18-20).
To
varying
degrees the
twoproteins
cause
release
of
LPS molecules from the
bacterial
cell, sensitize
organisms
toantibiotics
such
asrifampin
and
cefotaxime,
and
enhance
susceptibility
tothe
detergent
deoxycholate.
This
con-stellation
of
properties
is
similar
tothose
of
anumber of
differ-entagents
that
permeabilize
the
Gram-negative
outermem-brane,
including
metal chelators such
asEDTAand
polycat-ionic
compounds
such
aspolymyxin
Bnonapeptide,
poly-l-lysine,
the
defensins,
and
thebactericidal
permeability
increasing
protein
of
neutrophils (BPI)
(21-24).
Inthat these
agents canenhance the
susceptibility
of
Gram-negative
organ-isms
tolysozyme by
increasing
penetration
of the
protein
through the
outermembrane,
wehave undertaken
studies
todetermine
if
lactoferrin
and
transferrin
could
havecomparable
effects.
Additionally,
aslactoferrin
and
lysozyme
almost
al-wayscoexist in vivo,
demonstration
of
aninteraction is ofboth
physiological
and
mechanistic
relevance.
Methods
Lactoferrin,
transferrin,
andlysozyme.
Human milklactoferrin waspurchased commercially
(Sigma
ChemicalCo.,
St.Louis, MO,
orCal-biochem-Behring
Corp.,LaJolla, CA),
aswashumanserumtrans-J.Clin.Invest.
© The American Society forClinical Investigation,Inc.
0021-9738/91/10/1080/12
$2.00
ferrin (Sigma Chemical Co.) and human placental lysozyme
(Calbio-chem-Behring Corp.). Iron-saturated lactoferrin was obtained both commercially and prepared according to the method ofWest and Spar-ling (25). For selected experiments further purification of the commer-ciallysozyme was performed on an FPLC system (Pharmacia Fine Chemicals, Piscataway, NJ) using a Mono S column (Pharmacia Fine Chemicals) and0.0I M, pH 7.0, imidazolebuffers(bufferAwasimidaz-ole alone, and buffer B imidazimidazolebuffers(bufferAwasimidaz-ole with 1 M NaCl) in a series of linear gradients with the following parameters: flow rate 1 ml/min, 0-5 min 0%bufferB,8 min 17% bufferB,55min 22% bufferB,60min 100% buffer B. Eluted fractions were tested for lysozyme lytic activity on Micrococcusluteusagar plates (26). Immunologicalidentitywas con-firmed by double immunodiffusion using goat anti-human lysozyme (Kallestad, Austin, TX) and subsequent SDS-PAGE. Material from two runs was pooled, concentrated, and rechromatographed under identical conditions. This material was concentrated before use in time-kill assays.
Analyticprocedures. Proteinconcentrationswere determined ei-therby the biuretorthebicinchoninic acid-copper sulfate techniques (27, 28). The levels of iron in individual samples were measured using flameless atomic absorption spectrophotometry (model 5000 spectro-photometer
[Perkin-Elmer
Corp., Norwalk, CT] equipped with a model 500 programmer and an AS-Iautosampler) in the laboratory of Dr. Allen Alfrey (Department of Veterans Affairs Medical Center[DVAMC],
Denver, CO). The osmolarity of solutions was determined byfreezing point depression(Laboratory Wide-Range Model 3WII Os-mometer;Advanced Instruments, Needham Heights, MA). Determina-tion of concentraDetermina-tionsofsodium, potassium, chloride, bicarbonate, calcium, andmagnesium wereperformedin theClinicalChemistryLaboratoryof the Denver DVAMC. Analysis of carbohydrate fermen-tation by bacteriawasperformed usingmodifiedchromogenicassays inamicrotiter system(MicroScan,WestSacramento, CA) (29). Mea-surement of LPS concentration was performed usingaquantitative,
kinetic, chromogenic limulus amebocyte lysate assay (Whittaker M.A.
Bioproducts, Walkersville, MD) with a kinetic ELISA reader and soft-ware system(Molecular Devices, Menlo Park, CA) using purified E.
coli055 B5endotoxinas astandard. Discontinuous SDS-PAGE analy-sis ofproteinswasperformedinamodified Laemmeli systemusing
1.5-mmslabgels with 4.5% stacking and 10% resolvingsections,and subsequentanalysis by silverstain (30-32). The methodsof Perez-Perez and Blaser were used forSDS-PAGE of bacterial LPS (33). Briefly,pelleted cellswereresuspended in 200
Al
solubilizingbuffer and the supernatantslyophilized andresuspendedin 100 ulsolubilizingbuffer. Allsamplesweretreated with
proteinase
Kfor 1 hat60°Cand boiledfor10 min.DiscontinuousSDS-PAGEusing
the Laemmelisys-tem wasperformed with 4.5% stackingand 15%resolving gelsand
analysisby silver stain(34).
Bacterialstrains. Vibrio cholerae3083 was obtained fromDr.Mary Boesman-Finkelstein (University ofMissouri, Columbia, MO); the
0111 B4 UDPgalactoseepimerasedeficientEscherichia coliCL99-2 strain from Dr. KeithA. Joiner (NationalInstitutes of
Health,
Be-thesda, MD); and Salmonellatyphimurium
strainSL696 fromDr.Ilkka M. Helander(National Public Health
Institute, Helsinki,
Fin-land)(16,35-38).E.coli 5448 isahumangastrointestinal
isolatefrom theUniversityof Colorado Health Sciences Center Clinical Microbiol-ogy Laboratory(19). SDS-PAGEanalysis of bacterial LPS demon-strated that all four strains hada"smooth"wild-type
LPSphenotype(datanotshown).
Time-kill studies. Peptone media were obtained
commercially
(Difco, DetroitMI) and 1%wt/volsolutionsprepared. WMS,Davis andLuria brothwereprepared accordingtopublished recipes (18, 39-41). Bacteriaweregrowntostationary phase,the cells
centrifuged
and washed. A5 X l0 CFUbacterial inoculumwasaddedto500 ML of media withorwithout transferrin(2.0 mg/ml),lactoferrin(2.0mg/ml),
lysozyme(0.5 mg/ml), orEDTA (8X 10-5 M). The mixtureswere
incubatedat37°C,aliquots removed,serially diluted,andplated
over-nightat37°Consoybean caseindigestagar(SigmaChemicalCo.).For studies with V.cholerae3083 the strainwasgrownto
stationary phase
inproteosepeptone3with 0.3mMCaCl2and thentested in proteose
peptone 3. For allother strains, bacterialcells were grown and tested in the same media. For osmolarity studies, 1% bacto-peptone was supple-mented withmyo-inositol (SigmaChemical Co.). Forselected experi-ments E.coli 5448 cells were grown for 24 h in 1% bacto-peptonewith 0.05 mg/mllysozyme, washed, and then incubated with lactoferrin and lysozyme as above. Dialysis cell studies were performed with 1% bacto-peptone in 1 mlequilibrium dialysis chambers with 6,000 D exclusion regenerated-cellulose membranes (Bel-Art, Pequannock, NJ).
Transmissionelectron microscopy (TEM). 5 X 10 CFU inocula of E.coli 5448 were added to 500 Ml of 1% bacto-peptone with or without lactoferrin (2.0 mg/ml) or repurified lysozyme (0.005 mg/ml). The mixtures wereincubated at370Cfor 24 h and the bacterial cells pel-leted. The pelletswere resuspended in 2% glutaraldehyde in 0.1 M cacodylic acid, pH7.4, for 1 h at 40C. Samples were postfixed in buff-ered1% osmium tetroxide, dehydrated through a graded series of eth-anols, and embedded in poly/bed 812-araldite (Mollenhauer medium; Polysciences, Inc., Warrington, PA). For electron microscopy, 70-nm thin sections were obtained with diamond knives and stained routinely with aqueoussolutions of uranyl acetate and lead citrate. Sections were examined with a Philips CM-12 transmission electron microscope at 60 kV.
Equilibrium dialysis studies. Binding of calcium was studied by adding 750Mlof a solution of45CaC12in pH 7.4 HBSS lacking calcium and magnesium (HBSS-CM) (Whittaker M. A. Bioproducts) to one side of acrylic equilibrium dialysis cell chambers with 6,000 D exclu-sion regenerated-cellulose membranes. To the opposite side ofthe dialy-sis chambers were added solutions of either 2 mg/ml of BSA (Sigma Chemical Co.), transferrin,lactoferrin, apo- or iron-saturated lacto-ferrin, or 260 ul of 20% Chelex 100 (Bio-Rad Laboratories, Inc., Rich-mond, CA)in HBSS-CM. The cells were allowed to equilibrate at room temperature andserial aliquots sampled over 96 h.
LPSbinding studies. To study the ability of the iron-binding pro-teins to bind LPS; lactoferrin, transferrin, BSA,poly-l-lysine(Sigma Chemical Co.), and soybean trypsin inhibitor (Sigma Chemical Co.) were coupled to cyanogen bromide-activated Sepharose 4B beads (Pharmacia FineChemicals) at a concentration of 1X
10-'
mol/ml gel (molecular weightrange of poly-l-lysine is 1,000-4,000, and was esti-mated at 2,000).After protein coupling the beads were blocked in pH 8.0Trisbuffer, and stored in pH 7.2, 0.03 M barbital-acetate, 0.1 16 M NaCI buffer (BABS) with 0.02% thimerosol. To control for nonspecific binding, Sepharose beads were also prepared that were not reacted with protein, but instead simply blocked with Tris.Tritium-labeled LPS was prepared by growing E. coliCL99-2in modified WMS broth supplemented with
D-[6-3H]
galactose (Amer-shamCorp.,Arlington Heights,IL), andextractingLPS either by wash-ing the cells in pH 8.0 barbital-acetatebufferorby thephenol-water methodof Westphal(18,42). When this strain is grown in the presence of[3H]-galactose,theradiolabelis almostexclusivelyincorporatedinto theLPS O-polysaccharide (35).Forexperiments requiring highLPS concentrations the[3H]-LPS
was supplemented with similarly pre-pared unlabeled LPS.Bindingof the LPS was determined byincubating0.1 ml of the protein-Sepharose or Tris-Sepharose beads withvarying concentra-tionsof[3H]-LPSin pH 7.2 BABS for 5 min with gentleshaking.The beadswerethenpelleted bycentrifugation,washed twice withBABS,
and the beads andpooledBABS wash material countedby liquid scin-tillation.
Results
Interaction
oflactoferrin
and
lysozyme.
Initial studies
wereper-formed
addressing
the effects of lactoferrin andlysozyme
against
V.cholerae 3083
based on apreliminary
observation ofBoesman-Finkelstein
and Finkelstein
thatthis
strain could
bekilled
byhuman milk
lactoferrin and lysozyme (16). We found
that over 24 h this strainconsistently
demonstrated over a1,000-fold increaseinCFU/ml when grown either in proteose peptone 3 broth alone or with 0.5
mg/ml
human
lysozyme
A
blJ
Ln
LLI
z
D
0
z
A:
0
710
0
00
LUJ
LLJ
Co LUJ
10
IL
LOJ
ud
B
0 2 4 6 8 20 24
TIME
(hours)
.LU
2 4 6 8 20 24
TIME
(hours)
Figure1.(A)Effects of lysozyme (LYS) (0.5 mg/ml) andhumanapo-andiron-saturated lactoferrin(LF)(2.0 mg/ml)onthe growthofV.
cho-lerae 3083 in 1%proteose peptone3(mean+SEM,sevenexperiments; 6-, 8-, and20-hfindingsrepresentthree, three, andtwoobservations respectively).(B) Effects oflysozyme(LYS) (0.5 mg/ml),transferrin(TF)(2.0 mg/ml), and 8X
10'
M EDTAonthe growthofVcholerae 3083in 1%proteose peptone3(mean±SEM,sevenexperiments; 6-, 8-, and 20-h findingsrepresentthree, three,andtwoobservations, respectively).
(Fig. 1A). Incontrast,2 mg/ml(a 2X
10-5
M) humanlacto-ferrin wasbacteriostatic alone, and the combination of lacto-ferrinandlysozymewasconsistently bactericidal. Iron
satura-tion oflactoferrin inhibited both the bacteriostatic and syner-gistic bactericidal effect of lactoferrin.' Parallel studies performed with 2 mg/ml (- 2 x
10-5
M) human transferrinand 8 X
10-5
M EDTA indicated that both of these chelatorscould also enhance the activity of lysozyme, but neithertothe degree oflactoferrin (Fig. 1 B).
Experiments werethen performed to determine if
lacto-ferrinandlysozyme could have similar effectsonother
Gram-negative organisms.
Studies with 1 S. typhimurium and 2 E.coli strains again found that lactoferrin markedly enhanced the activity oflysozyme (Fig. 2). The strains differed in their suscep-tibilitytothe combination of lactoferrin and lysozymeaswell
asintheir susceptibilitytolysozyme combined withtransferrin orEDTA. Such variability had been noted in prior studies of
1. These studieswereperformedwithcommerciallyobtained
iron-sa-turated lactoferrin. Comparable results weresubsequently obtained withiron-saturated lactoferrin prepared bythemethod of West in stud-ies withE.coli 5448.
the effects of lactoferrin, transferrin,and EDTAonLPS release
and antibiotic susceptibility ofthese strains(18, 20).
As E.coli 5448appearedtobe themostsusceptible strainto
the effects of lactoferrin and lysozyme, this isolatewasusedto
define how varying either theconcentration ofthetwoproteins orthe bacterial inoculum sizewould affect the interaction. As
expectedfromprior observations onthe effects oflactoferrin
onLPSrelease( 18),thesynergistic activityof thetwoproteins wasfoundtobedosedependent for lactoferrin (Fig.3A).
Com-parableresultswerefound forlysozyme (Fig. 3B).Instudies withdiffering bacterialinoculatheactivityof lactoferrin and
lysozymewascomparable against5 X
I05
and 5X 106CFU/ml, but diminished whenthe bacterial inoculumwasincreased
to5x
I07
CFU/ml (Fig. 4).To confirm thatanimpurityinthecommercial lysozymewasnotcontributingtothe observed
in-teractions with lactoferrin, transferrin, and EDTA;
experi-mentswererepeatedwith
lysozyme
thatwasrepurified byion-exchange chromatography. The purified preparation again showed a synergistic interaction with lactoferrin (data not
shown).
Influence ofmedia composition. Studies were then
per-formedtoassessthe effect of mediacompositiononthe
lacto-0
LU
LUJ
bI
-z
D
0
z
0
11
7r
0
0
0f
O0
O--Ad ;
tu
2 4 24 0 42T
co
U-~~ ~ ~ ~~~W
Zernlszm
ineacin
WhnoermdawesutiW
0
o
o 0
0 2 4 24 0 2 4 24
TIME
(hours)
ferrin-lysozyme
interaction. When other media weresubsti-tuted
for proteose peptone 3 inparallel experiments,
the activ-ity of lactoferrin andlysozyme towards E. coli 5448 changed significantly (Fig. 5). Although the effects of lactoferrin and lysozyme weresimilar in
proteose peptone3 and
proteose pep-tone,the
activity
wasenhanced
inbacto-peptone.
In contrast, there was no bactericidal effect noted when the strain was tested in either proteose peptone 2 or in the more definedWMS, Luria
andDavis
media (Table I). To determine
whatfactors
inmedia
composition
contributedtothe variation in theeffectsoflactoferrin andlysozyme,
theelectrolyte
composi-tion
andosmolarity
of each of the media was defined. Wenoted
no apparentrelationship between
the lactoferrin-lyso-zymeactivity
andconcentrations
ofcalcium, magnesium,
iron,
sodium, potassium,
orchloride. However,
thereappeared
A
S. typhimurium SL696
Figure
2. Effects oflysozyme,
lacto-ferrin(2.0
mg/ml),
transferrin(2.0
mg/ml), and 8x 10-i M EDTAon thegrowth of E.coliand S. typhi-murium strains.Lysozyme was testedat0.05
mg/ml
for studies with E.colistrains and 0.5mg/mlfor S. typhimurium. (Opencircles)Pro-D3
teosepeptone3; (open squares)lac-toferrin;
(opentriangles)
transferrin;
(open diamonds)EDTA; (solidcir-cles)
lysozyme;
(solid
squares)
lacto-0 2 4 24
ferrin
+ lysozyme; (solid triangles) transferrin + lysozyme; (soliddia-monds)EDTA+lysozyme. tobe a consistent inverse correlation between the degree of killing and media osmolarity. Lactoferrin-lysozyme activity wasabsent in media above 60 mOsm.
To test the hypothesis that mediaosmolarity influenced lactoferrin-lysozyme killing, parallel time-kill studieswerethen performed in 40 mOsmbacto-peptone media thatwas supple-mented with graded concentrations of ametabolically inactive material. Preliminary studies found that E. coli 5448 didnot
ferment inositol, and this sugarwasusedas anosmotic supple-ment. It wasfound that the addition ofincreasing
concentra-tions of myo-inositol tothe mediadid not alter thebacterial growth curve but did affect thebactericidal effects oflactoferrin and lysozyme (Fig. 6).Bacterialkilling progressively decreased as the media
osmolarity
was increased, and was completely absentwhen theosmolaritywasincreased to 100mOsm.B
-0 2 4
TIME (hours)
Figure 3. (A) Effect ofvaryinglactoferrin concentration on its interaction with lyso-zymeagainst E.coli 5448 (mean±SEM, three
experiments).(Opencircles)Proteose pep-tone3; (open triangles) 2mg/mllactoferrin; (open squares) 0.025mg/mllysozyme; (solid
circles)4mg/ml lactoferrin + lysozyme; A (solidtriangles) 2 mg/mllactoferrin+
lyso-zyme; (solid squares) Img/ml lactoferrin+
lysozyme;(solid diamonds) 0.5 mg/ml lacto-ferrin+
lysozyme.
(B)
Effect ofvarying
lyso-zymeconcentrationonitsinteraction withlactoferrin against E. coli 5448 (mean±SEM, fourexperiments). (Opencircles) Proteose
peptone 3; (open triangles) 2 mg/ml
lacto-fenin;
(open squares) 0.025 mg/ml lysozyme;(solid circles) lactoferrin+0.05mg/ml
lyso-zyme;(solid triangles) lactoferrin+0.025
mg/ml lysozyme;(solid squares)lactoferrin
24 + 0.0 125mg/mllysozyme; (solid diamonds)
lactoferrin+0.0063mg/ml lysozyme.
z
D
0 z
0
U-z
0 0
0
ci
00
0o
E
(I)
z D
0 z 0
LL
z
0 0 0
2 4
TIME (hours)
C/)
z
0~~~~~~~~~~~~~~I
LLi.
z
o oo.
0 0
0
0 2 4 24 -0 2 4 24
TIME
(hours)
As
consistent
early killing and subsequent regrowth of E.coli 5448 cells
wasobserved
with exposure to lysozyme alone, todetermine ifthis represented
the emergence ofresistant cells,BACTOPEPTONE PROTEOSEPEPTONE2
hi
z 0 4024 2
I. g
:D
0~~~~~~~
Z 00
z
~0 2 4 24 0 24 24
0
LL_
PROTEOSE PEPTONE
PROTEOSE PEPMNEHK
ai
0 2 4 24
TIME
(hours)
Figure 5. Evaluation oflactoferrin-lysozymebactericidalactivity
to-wardsE.coli5448 in differing peptone media (mean±SEM, three
experiments). (Open circles)Mediaalone; (opentriangles)2mg/ml lactoferrin;(open squares) 0.05 mg/ml lysozyme; (opendiamonds)
lactoferrin+lysozyme.
hi 4
\<Figure
4. Effectof bacterial inocu-_ lumsizeontheactivity
of lactoferrinI_______________
and lysozymeagainst E. coli 54480.z* * * *(mean±SEM, three experiments).*
* * *
0 2 4 24 (Open circles)Proteosepeptone 3; (opentriangles)2 mg/mllactoferrin;
(open squares) 0.05 mg/ml lyso-zyme; (opendiamonds)
lactoferrin
+lysozyme.
further time-kill studies
wereperformed using
cells thathad
been grownfor
24 hin
1%bacto-peptone media with 0.05
mg/ml
lysozyme. It
wasfound that these
E.coli
cells
grownin
the
presenceof
lysozyme remained
susceptible
tothe
com-bined
effects
of
lactoferrin
and
lysozyme, although in matched
studies the concentration of lysozyme needed
tobe
increased
fourfold
(0.0125-0.05
mg/ml)
toachieve
adegree of
killing
comparable with that
of
cells
notpreviously
exposed
to lyso-zyme.TEM
studies. One model to explain
the effect of media
osmolarity
onbacterial killing by lactoferrin
and lysozyme would be that thebactericidal
effect is primarily
related todeg-radation of
the
peptidoglycan
matrix. Without
the
protection
of
this
rigid
framework,
under low
osmotic conditions the
bac-terial cells would absorb
fluid allowing
them
toballoon and
explode. Such
aphenomenon would
notoccur athigh
osmo-larity.
To
evaluate this
possibility, bacterial
cells
wereexam-ined by
TEMafter
24 hof growth in 40 mOsm bacto-peptone
with
orwithout lactoferrin
orlysozyme.
It wasfound
thatthe
majority
of bacterial cells
werecylindrical
when
grownin
me-dia alone
(Fig.
7A)
orin the
presenceof either lactoferrin
(Fig.
7
B)
orlysozyme (Fig.
7C)
individually.
Incontrast,in
keeping
with the
hypothesis of
damage tothe
peptidoglycan
matrix,
when cells
wereexposed
tolactoferrin and lysozyme
together
(Fig.
7D) there
was alarge
amountof cell debris noted
andanumber of the
remaining
cells
appeared
tobequite enlarged,
hypodense,
androunded.
Influence of
calcium and magnesium concentration.
Fur-ther work
wasthen
initiated
to moreprecisely
define
howlacto-ferrin damages the
outermembrane and alters bacterial
suscep-tibility
tolysozyme.
Based onprior
work
indicating
that the
calcium and
magnesium
concentration could
significantly
in-fluence
the
ability oflactoferrin
and transferrin
toeither
induce
LPS release
orchange bacterial
susceptibility
todeoxycholate
(18,
20),
theeffects
of these cations
onlactoferrin-lysozyme
synergy
towards
E.coli
5448
wereevaluated
using
concentra-tions
similar
tothose studied
previously. Although
theaddition
ofeither 1.3 mM
CaCl2
or0.8
mMMgCl2
tobacto-peptone
hadonly minimal effect
onmedia
osmolarity (measured
inhib-TableLAnalysis ofthe BactericidalActivityofLactoferrinand Lysozyme inRelation to MediaComposition
Lactoferrin
Media -lysozyme 24 h
studied LoglOchange Mg+2 Ca+2 Fe Na+ K' a- C02 Osmolarity
mM mM ppb mM mM mm mM mOsmol
Bacto-peptone -3.7 0.1 0.1 35 10 0.7 12 3 41
Proteose peptone -2.8 0.2 0.2 114 18 2.8 15 3 54
Proteose peptone 3 -0.7 0.2 0.2 187 18 3.4 12 3 57
Proteosepeptone 2 1.7 0.1 0.1 60 23 3.3 16 3 79
WMSII 2 <0.01 0.01 20 37 25 170 3 405
WMSIII 2 <0.01 0.01 20 36 24 173 3 444
SupplementedDavis 2 <0.01 0.02 14 189 15 3 321
Luria broth 3 <0.01 0.002 550 22 8 15 3 110
Time-kill studies were performed in triplicate in peptone media and singly in others.
ited the bactericidal effect of lactoferrin and
lysozyme(Fig.
8A). These studies
wereextended
byevaluating
theaddition
of 8 x1O-5
M or 1.6 x10-4
M CaCl2 to the bacto-peptone media.With these lower calcium concentrations, there
waspartial
anddose-dependent inhibition of the lactoferrin-lysozyme
bacteri-cidal interaction
(Fig. 8 B).
Calcium
chelation
studies. A mechanism by which calcium
could interfere with the lactoferrin-lysozyme bactericidal effect
would be through
aninhibition of
theability of ladtoferrin
tochelate membrane stabilizing calcium. As
wewere unawareof
published
data on theability of lactoferrin (or transferrin) toId
- ) PM40+LF(2)
cO 0Pe40W+Y.0125)
L~~l - 1 ^ ^ 50~PO mozm
/ *°EP 10 moem
I\"
L^J -t/>
O- l MP 0mownVPEP80.LF/LYS
z
°J
0-
PEP60+U^AY
0 0
0
2
4
24
TIME
(hours)
Figure 6. Evaluation oflactoferrin-lysozymebactericidal activity
to-wards E.coli5448 inbacto-peptone media supplemented with
in-creasingosmoticconcentrations of
myo-inositol
(mean±SEM,threeexperiments).
chelate
calcium,
equilibrium dialysis
studies
wereperformed
todirectly
define
WCa+
binding by
the
proteins.
HBSS-CMwaschosen
asthe
buffer for these studies
asprior
work had shownthat in this
solution
the
outermembrane effects of
lactoferrin
and transferrin
wereboth demonstrable
andcalcium-depen-dent
(20).
Whereas the
synthetic
chelator
Chelex caused theexpected
migration
of the
isotope
acrossthe
dialysis chamber,
neither
lactoferrin
ortransferrin
had
anydemonstrable effect
ondistribution
of
"Ca"++
within the
dialysis chamber,
there-sults
with lactoferrin
wereidentical with
apo- andiron-satu-rated
preparations
(Table II). Thus,
these studies indicate thatthe
twoproteins
do
not haveanysignificant
ability
tochelate
calcium under
conditions
where
they have consistent
outermembrane
effects.
Dialysis chamber
studies. To extend the studies on the
mechanism
bywhich lactoferrin
enhancesbacterial
susceptibil-ity
to lysozyme,dialysis
chamberstudies with
a6,000-Dexclu-sion membrane
werethen
performed
todefine
whether
direct
cell
contact wasrequired
for lactoferrin's
effects
(Fig. 9).
Asexpected, lysozyme
wasnotactive unless it
wasin
direct
bacte-rial cell
contact.The
ion exchange resin, Chelex,
wasfound
tobe
moreactive
when in
direct
cell
contact,but also enhanced
lysozyme's
effects
when
added
tothe
opposite
side
of
the
dialy-sis chamber
(Fig.
9
B).
In contrast,lactoferrin
only
enhanced
the
effects
oflysozyme when it
wasin direct
contactwith
bacte-rial
cellswhether tested
atconcentrations
of
1 or 2mg/ml (Fig.
9
A). These
observations
are moreconsistent with
lactoferrin
augmenting lysozyme's
effects through
adirect interaction
with the
bacterial
outermembrane than with
theprotein acting
through its
chelating properties.
LPS
binding
studies.
Polycationic
agents thatalter
outermembrane
permeability
appear todirectly interact with LPS
molecules
(10,
24, 43). Several previous
studies
haveindicated
that
latoferrin
will
bind
tobacterial cells, although the
mecha-nism ofbinding
maybe
related
tothe
presenceof
specific
bacte-rial
lactoferrin
receptors(44-46).
To morecarefully elucidate
whether lactoferrin might be altering the
outermembrane in
a mannersimilar
topolycationic
agents, westudied
whetherlac-toferrin
could
directly
bind LPS. For these
studies
equimolar
concentrations of
humanlactoferrin,
human
transferrin, BSA,
poly-l-lysine,
and
soybean
trypsin
inhibitor
(previously
shown
not tobind
LPS[47])
werelinked
toTris-blocked
Sepharose
beads. These
protein
beads werethen
incubated with
varying
"d
w:wi
,:)p
_ _
I
r.
AY.
I
\(
_V6,X W/, h , 0 0
Figure 7. Transmission electron microscopy of F. coili 5448 cells incubated for 24 h in (A) bacto-peptone, (B) bacto-peptone with 2
mg/mi
lactoferrin, (C) bacto-peptone with 0.005 mg/ml lysozyme, (D) bacto-peptone with lactoferrin and lysozyme (markers indicates 1
nm).
A
n
Cf)
z D 0 z
:2
0
U-z
0 0
C-)
0 2 4
TIME (hours)
24
B
cn
z
D
CD0 z 0
LL
z
0
Jo
0
C)
o 2 4
TIME
(hours)
Figure8.Effectof calcium andmagnesiumon
lactoferrin-lysozyme bactericidal activity
to-wards E.coli 5448 inbacto-peptonemedia
(mean±SEM,
threeexperiments). (A) (Opencircles)
mediaalone; (open triangles)2mg/ml
lactoferrin; (open squares) 0.05 mg/ml lyso-zyme;(solid
circles)lactoferrin +lysozymeinmedia alone; (solid squares)lactoferrin +
lyso-ail, zymeinmedia supplementedwith 1.3X
10-3
MCaCl2;
(solid diamonds)
lactoferrin+ lyso-zymein media supplementedwith 8X 10-4MMgCl2.(B)(Opencircles)media alone; (open
triangles)
2mg/ml lactoferrin;(open squares) 1 0.05mg/ml lysozyme; (solid circles) lactoferrin+lysozymeinmedia alone; (solid triangles) lactoferrin+lysozymeinmedia supplemented
with 8x
IO'5
MCaCl2; (solid squares)lacto-24 ferrin+lysozymeinmediasupplementedwith
1.6 x
10-4
MCaCl2.concentrations of
[3H]-LPS
prepared from
E.coli strain
CL99-2
(Fig. 10). The binding
curvesobtained indicate
thatlacto-ferrin
andpoly-l-lysine
haveasimilar
ability
tobind
LPS,
and eachhas
adistinctly
greaterability
tobind LPS
thandoes
theother
proteins
orthe
Tris-blocked control beads.
Anexactcal-culation of the number
ofLPS
binding
sites and
theKd for
theinteractions with lactoferrin and
poly-l-lysine
is
limited
bothbecause
of the
inability
todefine
themolar
concentration
ofLPS (due
tosize
variability)
and because
ofthe
capacity
for
freeLPS
molecules
to aggregatein solution
(48).
Discussion
The
presentstudies demonstrate that
lactoferrin and
lysozyme
synergistically
kill
Gram-negative
bacteria. This
activity
is
de-monstrableagainst multiple
different
"smooth"bacteria,
isdose
dependent
andinfluenced
by
bacterialinoculum
size and basalgrowth media. The relative
activity
of
lactoferrin
andlysozyme
appears tocorrelate with
media
osmolarity,
and
canbe
inhibited
by
increasing
theosmolarity
ofapermissive
mediawith
ametabolically inert material.
Morphological analysis
ofbacterial cells
by
TEMindicates
thatthebactericidal
effect is associated with theformation
ofballooned spheroplasts.
Theseobservations
appear consistent withlactoferrin acting
toin-creasethe
penetration
oflysozyme
into theGram-negative
or-ganism
where the enzymecanthendisrupt
theglycosidic
link-ages of therigid
murein sacculus. Thatlactoferrin
canenhancelysozyme activity
in thisfashion
is consistentwith
other obser-vationsontheoutermembraneeffects
oflactoferrin. Previous
studies havefound
that theprotein
causes LPS release andenhances
bacterialsusceptibility
toantibiotics
suchasrifampin
and
cefotaxime
(18-20).
Enteric
Gram-negative bacteria
areprotected from theex-ternal
environment by
theircomplex
outermembrane struc-ture(9, 10).
The membrane hasanasymmetric lipid
bilayer
with
negatively charged
LPS moleculesprimarily
localizedontheouterleaflet and
stabilized by
the presence of divalentcat-ions. The
polysaccharide sidechain
of the LPS moleculepro-vides
ahydrophilic
surfacetothe bacterial cell, and thesatu-rated
fatty
acid chains make this membrane relativelyrigid
ascompared
with otherphospholipid
structures(49).
The dualhydrophilic/hydrophobic
natureofthe outer membrane Table II.Chelation
of 5Calcium by
Proteinsand Chelex100Determinedby Equilibrium Dialysis
Percentmigration of4"Caacrossdialysismembraneafter differingincubation times
Chelator Oh 2 h 18 h 24h 96h
BSA (I mg/ml) 0.03 24.9±2.3 51.7±0.3 52.3±0.6 47.0±4.8
Human
transfernin
(1 mg/ml) 0.03 27.9±3.4 48.4±0.4 50.5±0.6 51.3±0.5Humanlactoferrin
(1
mg/ml) 0.03 27.3±3.2 48.9±0.1 49.6±0.3 45.9±4.6Iron saturatedhuman
lactoferrin
(1
mg/ml) 0.03 27.4±3.3 49.9±1.4 50.6±0.8 52.2±1.9Chelex resin(20%) 0.03 21.6±1.9 56.6±1.9 74.8±1.8 82.8±15.7
0, 2,and 24hresultsare
mean±SEM,
threeexperiments;
18 and 96hresultsarefromtwoexperiments.
A
E
Un zz
CD
z
0
U-z
0 0
C-B
or
cn
z D CD F-z
:E 0
0
0
-j
0 2 4 24
TIME
(hours)
TI
thereby provides
apermeability barrier
thatlimits penetration
of both
hydrophobic and hydrophilic molecules.
Incompensa-tion
for
this
barrier, protein porins allow for diffusion of
smallmolecules into
thebacterial
cell through transmembrane pores,and
inducible
specific
transport systemsprovide the
transportof
sugars,vitamin
B12,
and
iron
acrossthe
membrane (43).
Several
groupsof
agentshave been
found
todamage the
Gram-negative
outermembrane making the
structure morepermeable
tohydrophobic
compounds
(10).
These
include
metal
chelators
such as EDTAand EGTA which
removestabi-lizing
cations
andcauserelease of
LPS molecules; andpolycat-ions such
aspolymyxin
Bnonapeptide, polylysine,
and afam-ily
of cationic
defense proteins
of
neutrophils
which
appear tobind
tothe anionic
corecomponentsof
LPS molecules
thereby
altering
their
relationship within the membrane (50).
Ineach
instance these
agentsdamage the membrane
structureand
en-hance bacterial susceptibility
tohydrophobic
substances, such asantibiotics
and
lysozyme, that have limited ability
to pene-tratethe
outermembrane. Thus, the collected observations
that
lactoferrin
causesLPS release
andenhances bacterial
sus-ceptibility
torifampin
and lysozyme strongly
suggestthat
lac-toferrin
hasmembrane
permeabilizing
activity.
Three
major mechanisms
canbeproposed
toexplain how
lactoferrin
might
alter thepermeability
barrier of
the outer membrane.First, through its
metalchelating capacity
the
pro-tein could
removestabilizing
cations in
afashion similar
toEDTA
(18).
Evidence
supporting
this
hypothesis
comesfrom
inhibition
of
themembrane effects
by saturation of the
protein
with iron,
and theability of calcium and magnesium
tomodu-late the
activity
(20). However, several of the
presentstudies
arguestrongly
against
this
hypothesis.
Specifically,
both the
inability
of
lactoferrin
toeither
chelate45calcium
or toenhance
lysozyme activity in the absence of direct cell
contact arein-consistent with divalent cation chelation
being
themechanism
of action.
A
second
possibility is
thatlactoferrin
could altermem-brane
permeability through
stimulation of bacterial
sidero-IME
(hours)
Figure 9. Evaluation of the role of direct cell contactin theability of lactoferrin or the synthetic chelator Chelex to enhance lyso-zyme activity against E. coli 5448 in dialysis chamber (6,000 D exclusion membrane) (mean±SEM, three experiments). (A) (Open
circles)Bacterial cells and bacto-peptone me-diaalone;(opensquares)cells and lactoferrin
(1 mg/ml) incontact,lysozyme(0.125 mg/ ml) on opposite side of membrane; (open
diamonds)cells andlysozymein contact, lactoferrin on opposite side of membrane;
(closed circles)cells, lysozyme,and lacto-ferrin in contact.(B)(Opencircles)Cells and
media alone; (open squares) cells and lyso-zyme (0.125 mg/ml) in contact; (open trian-gles)bacterial cells and Chelex (0.00692 mEq/ml) in contact, lysozyme on opposite sideofmembrane; (solid squares) cells and lysozyme in contact, Chelex on opposite side of membrane;(solid triangles) cells, lyso-zyme, and Chelex in contact.
phore
production.
Bydepleting
theenvironment of available
iron,
lactoferrin
has beenfound
toinduce the bacterial
produc-tion of
iron-regulated
outermembrane
transportproteins (13).
The
specificity of these
transportproteins is
still
being
defined
andit is
possible
that an indiscriminate transport systemmight
allow for the
uptake of
othermolecules
aside from iron. Such
amechanism has recently been proposed
toexplain
the
en-hanced
activity of
aspecific
class
of
catechol-cephalosporins
in
low
iron
conditions (51, 52).
Inconsidering
the
presentstudies,
the
inhibition
of lactoferrin
activity
by iron saturation would
supportthis
hypothesis. Still,
it
would beexpected
that this
mechanism
would notrequire
direct
interaction with thebacte-rial cell, and
theinability
of lactoferrin
toenhance
lysozyme
activity
across adialysis
membrane
arguesagainst
it.
Another
potential
mechanism for the membrane effects of
lactoferrin is through
adirect
interaction with the bacterial
outermembrane. Such
apossibility
is
supported by
the work of
Valenti
andco-workers
correlating
theability
of
henovotrans-ferrin
andbovine
lactoferrin
tobind
tobacterial strains
with
thebacteriostatic effects of the
proteins against
thestrains (44,
53). The
dialysis chamber time-kill studies
weperformed
simi-larly
support adirect
interaction,
and the
ability of lactoferrin
tobind
LPS suggests amechanism of action.
Recently, the
crystallographic
structureof
humanlactoferrin
has been solved toaresolution of
2.8A
(54). This analysis indicates
thatonesurface subunit of lactoferrin contains nine
arginine
and
lysine
residues
in closeproximity. This cationic region
onthe
protein
surface could function
asthebinding
site for LPS
through
aninteraction with
theanionic
corestructureof the
lattermole-cule. This
would allowlactoferrin
toactsimilarly
tothe
well-defined
polycationic
membrane-active
agentswhere the
physi-cal
presenceof
thebound
protein
thenalters normal
mem-brane
physiology (21, 24).
Still this
hypothesis
must alsoexplain
how theeffects of
lactoferrin
arealteredby changing either
thecalcium
ormagne-sium concentrations
in the assayortheiron
saturation of the
14 250(
150'
1001
50
o
O-O
N.TRIS 0 STI
A BSA..
00HUO HTFF
* PLL <+ a
00
0 10000 20000 30000 40000 50000 TOTAL LPS LOADED (ng)
12
10
0
0
x
8
6
4
2
0
o BSA
o TRANSFERRIN
v LACTOFERRIN
* POLY-L-LYSINE 0 500 1000 1500 2000
BOUND (ng LPS)
2500
Figure 10. Binding of E. coli [3H]-LPStohuman lactoferrin(HLF), human transferrin (HTF), poly-l-lysine(PLL),soybean trypsin inhibitor
(STI), and bovineserumalbumin (BSA) boundtoSepharose, andtoTris-blockedSepharose (TRIS)(mean±SEM, three experiments).(A) The
bindingcurvewith second-order linear regression.(B)CorrespondingScatchard plotperformedbysubtracting nonspecific bindingto
Tris-blockedSepharose beads; for lactoferrinr=-0.937; for poly-l-lysiner=-0.898.
ofcalcium, and thismayinterfere with its biologic activity (55). Additionally, changes in calciumor magnesiumconcentration intheenvironmentmayalter theamountof the cations incor-porated into the outermembrane and thereby influence the membrane susceptibility to membrane-active agents (9). Fi-nally, and most importantly, the divalent cations
Ca2"
andMg2"
have beenshowntoblock the activity of well-character-ized polycationic membrane active agents including poly-myxin, poly-l-lysine, and aminoglycosides (22, 56, 57). Studies with BPI have shown thatexposure of bacterial cellstoeither 40 mMCa2"
orMg2"
blocks thebactericidal activity of the protein against Gram-negative bacteria, apparently by causing therelease ofprotein from the bacterial cell surface (58-60). Thus,the inhibition of lactoferrin's membrane effects by thesetwodivalentcations is consistent with the protein actingas a polycationicagent. Inconsideringthe effectsof ironsaturation
oflactoferrin, both electrophoretic and crystallographic studies indicate that iron binding
significantly
altersthestructural con-formation of the protein (61, 62). Such a conformational change maytheninfluencean interaction with thebacterialmembrane. Clearly each of these hypotheses requires further evaluation.
In these studies lactoferrin was consistently foundto be bacteriostaticandnotbactericidal. This contrasts with the
stud-ies by Arnold and co-workers that describeadirectbactericidal effect of the proteinwhen tested in distilledwater (63, 64). However, the observed lactoferrin-lysozyme bactericidal effect
appearssimilartotheresultsoftheseinvestigators.It is conceiv-ablethatinthestudies of theseotherinvestigators, the
bacteri-cidaleffectascribedtolactoferrinmayhave beenrelatedin part
toinadvertent contamination ofthe lactoferrin
preparations
withlysozyme.Thelactoferrin-lysozyme effectmustbeconsidered in the
context of the natural environment. The in vivo concentra-tionsoflactoferrin andlysozymearehighly heterogeneous.
Re-centworkontheconcentration of lactoferrin in mucosal fluids hasnotedlevels ranging from 12
gg/ml
in mixedrespiratorysecretionsto6.7 mg/mlin preterm colostrum(65, 66).
Lyso-zyme varied from 0.77 sg/mlto 0.06 mg/ml in these same fluids.Beyond this variability, the packaging and release of the proteins within granulessuggeststhatthere isprobably
signifi-cantmicroheterogeneity of protein concentrations within the individual secretions.
Additionally, precise quantitation
oftwoproteins within the phagolysosome of polymorphonuclear leu-kocytes hasnotyetbeen accomplished. Beyond these consider-ationsonprotein concentration, therearealsohigh
concentra-tions ofcalcium andmagnesium inmostbiological fluids that could limit lactoferrin's membrane activity. However, the in vivodistribution of thecations is alsonot homogeneous; the calcium concentrationinthehumanmacrophage phagolyso-somehas been found tobe < 100
,M
(67). This is thenone microenvironmentwherelactoferrincould enhance the activ-ity of lysozyme. Similarly, the osmolaractiv-ity ofmostbodyfluids is higher than that where the combination oflactoferrin and lyso-zymehasdirect bactericidal activity (68). Yetconcurrent dam-agetoboth theoutermembraneandthe murein sacculus pro-duced by the two proteins should continue to occur, and should enhance bacterial susceptibilitytootherhostdefensesystems suchas
immunoglobulins,
the complement cascade, andphagocytic cells. Thus,aslactoferrin andlysozyme havea similar distributionwithinmucosalsecretionsandareprimary constituents of thespecific granules of polymorphonuclear leu-kocytes ( 1-4), it is likely that the observed synergistic interac-tion is relevanttotheir host defenseproperties.Acknowledgments
The authors wishtothankDrs. AllenAlfreyand Arnold Schultz for
measuringmetalconcentrations,Dr.PhilipAisenforadviceon
evaluat-ingcalciumbindingby lactoferrin,andMr.Vincent Buric for assis-tancewith the transmissionelectronmicroscopystudies.
This workwassupported bytheDepartmentof Veterans Affairs ResearchService.Dr.Ellison is therecipientofaDepartmentof
Vet-eransAffairsResearchAssociate CareerDevelopmentaward.
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c
z
3
0
m LI)
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