antimicrobial components of vaginal fluid

561

The human vagina supports certain commensal mi-crobes but, at the same time, resists colonization by ex-ogenous microbes and presents a barrier to microbialentry into deeper tissues. The vaginal surface is lined by amoist noncornified stratified squamous epithelium and iskept moist by a fluid that is, in part, secreted as a plasmatransudate through the vaginal wall,1,2 with additionalcontributions from the cervical and vestibular glands.The volume of the fluid in the sexually unstimulatedvagina3 has been reported to be between 1 and 4 mL.

Several mechanisms of vaginal defense against colo-nization by exogenous microbes have been proposed.The squamous epithelial layer continuously sloughs off,removing attached microbes. The commensal microbes,among which lactobacilli normally predominate, com-pete with exogenous microbes by using nutrients, estab-lishing and maintaining a low pH,2 and generatingantimicrobial bacteriocins, peroxides, and organic acids.The presence of epithelia-derived antimicrobial proteinsin the vaginal fluid was also noted.4 However, detailedanalysis of its antimicrobial components has not been re-

ported. Antimicrobial polypeptides that are abundant inother epithelial fluids5 include lysozyme, lactoferrin, secretory leukoprotease inhibitor (SLPI), calprotectin,human α-defensins human neutrophil peptides 1through 3 (HNP1-3) that are released from the neu-trophils, and the β-defensins human β-defensins 1 and 2(HBD-1, HBD-2) from epithelial cells. The objective ofthis study was to identify antimicrobial polypeptides andother antimicrobial components that are present in vagi-nal fluid, to determine whether these components areregulated during the menstrual cycle, and to determinethe effect of vaginal fluid on the growth of selected resi-dent and exogenous microorganisms.

Material and methods

Sample collection. The study protocol was approved bythe UCLA institutional review board. Preweighed tam-pons (rayon fiber with cotton cord; Tampax, Proctor andGamble, Cincinnati, Ohio) were inserted in the vaginafor 8 to 10 hours and then were reweighed to determinethe amount of fluid that had collected. The tamponswere processed on the day of collection or, in some cases,stored at –20°C until extraction. Freezing did not appearto affect the integrity and recoverability of the proteinsthat were present in the sample, as determined by proteinprofiles that were observed after separation by acid ureapolyacrylamide gel electrophoresis (AU-PAGE) beforeand after freezing (data not shown).

Vaginal fluid extraction. Fifty milliliters of extractionfluid was added to each tampon and incubated, with ro-tation at room temperature for 3 hours. The bulk of theextraction fluid was then transferred to a centrifuge tube;

From the Department of Medicine and Pathology, School of Medicine,University of California, Los Angeles.Supported by National Institutes of Health grants No. AI 37945 andAI46514.Received for publication November 15, 2001; revised February 26,2002; accepted March 28, 2002.Reprint requests: Tomas Ganz, PhD, MD, UCLA Department of Medi-cine, CHS 37-055, Los Angeles, CA 90095-1690. E-mail: [email protected]© 2002, Mosby, Inc. All rights reserved.0002-9378/2002 $35.00 + 0 6/1/125280doi:10.1067/mob.2002.125280

Antimicrobial components of vaginal fluid

Erika V. Valore, MS, Christina H. Park, BS, Sorina L. Igreti, BS, and Tomas Ganz, PhD, MD

Los Angeles, Calif

OBJECTIVE: We examined the antimicrobial activity and composition of vaginal fluid.STUDY DESIGN: Vaginal fluid from preweighed tampons was assayed for pH, lactic acid, and antimicrobialpolypeptides. The fluid was also fractionated by molecular filtration. Antimicrobial activity of whole fluid wasdetermined against representative resident and exogenous microbes, and its fractions were tested againstEscherichia coli.RESULTS: Vaginal fluids (5/5 donors) were permissive for Lactobacillus crispatus and vaginalis and Candidaalbicans, but not for Escherichia coli, Streptococcus group B, and Lactobacillus jensenii in three of fivedonors. The antimicrobial activity against E coli was predominantly in a <3-kd fraction and correlated withboth low pH and high lactic acid content. Compared with a matched pH buffer, lactic acid markedly sup-pressed the growth of E coli. Concentrated 2- or 5-fold, the protein-rich fraction was active against E coli.CONCLUSION: Vaginal fluid exerts selective antimicrobial activity against nonresident bacterial species. Theactivity is mediated by lactic acid, low pH, and antimicrobial polypeptides. (Am J Obstet Gynecol2002;187:561-8.)

Key words: Lactic acid, antimicrobial polypeptides, vaginal microflora

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562 Valore et al September 2002Am J Obstet Gynecol

the fluid retained in the tampon was squeezed out intothe same centrifuge tube by compression in a large sy-ringe. This method recovered 96% of the extractionfluid, as determined by comparing the weight of a tam-pon prewetted with 1 mL of water (to mimic vaginal fluidvolume) before and after extraction with 50 mL of water.Epithelial cells and tampon debris were removed by cen-trifugation, and the fluid was further processed for pro-tein analysis or antimicrobial testing. The extraction fluidfor protein analysis was 0.1% or 5% acetic acid. For an-timicrobial and physiologic studies, the tampons werefirst extracted with water, followed by a second extractionwith an equal volume of 0.1% acetic acid to obtain pro-teins that had not been extracted by the first water ex-traction. For studies of the activity of the protein fraction,the second extract was combined with the first extract.

Cationic protein extraction from vaginal fluid. The ex-tract was first neutralized to pH 7 with ammonium hy-droxide. After neutralization, 1.5 mL of a 50% slurry ofMacroprep CM resin (Bio-Rad, Hercules, Calif) in 25mmol/L ammonium acetate pH 7.5 was added and incu-bated on a rotator for 3 to 4 hours at room temperature.The resin was allowed to settle, and the supernatant wasdecanted off. The resin was washed several times in am-monium acetate buffer, then proteins were eluted fromthe resin, with one resin volume equivalent of 10% aceticacid followed by two of the same volume elutions with 5%acetic acid. The eluates were pooled, lyophilized to dry-ness, and dissolved in 0.1% acetic acid.

Processing for antimicrobial assays. The water extractwas lyophilized and resuspended in sterile water to theoriginal volume of vaginal fluid, as determined by weight(1 g = 1 mL). The pH was determined by a micro pH elec-trode (Corning, New York, NY) in a tube that contained a100-µL aliquot of water-extracted vaginal fluid.

Vaginal fluid fractionation. The water and acid extractswere separately resuspended in water to the original vol-ume by weight. A portion of the water extract (250 µL)was centrifuged through a filter spin unit (Microcon 3KYM; Millipore Corporation, Bedford, Mass) that had beenprewashed with water to remove membrane stabilizers.Fluid that passed through the membrane (the <3-kd frac-tion) contained no protein, as determined by silverstained AU-PAGE (Fig 1) and was used for the colony-forming unit (CFU) assay. To increase protein recoveryfrom the tampon, 250 µL of a second tampon extract(with acetic acid, as described earlier) was added to the>3-kd fraction and then subjected to centrifugation on the3K membrane to remove the acid and residual small mol-ecules. The high molecular weight fraction was thenwashed on the filter with equal volumes of water, followedby two washes with genital tract (GT) buffer (20 mmol/Lpotassium phosphate, 60 mmol/L sodium chloride, pH4.5), which was designed to mimic the electrolyte compo-sition and pH of vaginal fluid.1 The >3-kd fraction was re-

covered by the filter unit was turned over, and the fractionwas centrifuged into a new tube (as per the manufac-turer’s instructions) then brought to its original 250-µLvolume with pH 4.5 GT buffer and used for the CFU assay.

CFU assay. Microbial suspensions (3 µL in nutrient so-lution) were added to either vaginal fluid or GT buffer toa final volume of 30 µL. The final nutrient concentrationcorresponded to either �0.01 trypticase soy broth (TSB)or �0.1 Lactobacillus MRS broth (Difco, Becton Dickin-son, Sparks, Md). An aliquot was removed and immedi-ately plated in triplicate on TSB plates (or MRS plates forLactobacillus) to determine the initial microbe concentra-tion (T = 0); the remaining mixture was incubated in anenvironmental shaker at 37°C for the times indicated,under conditions that minimized evaporation and con-densation. Test strains that represented the endogenousflora of the vagina6 included gram-positive Lactobacillusjensenii, L crispatus, and L vaginalis (ATCC No. 25258, No.33197, and No. 49540, respectively) and the yeast Can-dida albicans. Exogenous bacteria were represented bythe gram-negative Escherichia coli ML-35p and a gram-pos-itive clinical isolate of β-hemolysing Streptococcus group B.L crispatus was grown under anaerobic conditions withthe use of a BBL GasPak pouch (Becton Dickinson),which generated an anaerobic carbon dioxide–enrichedenvironment. The other Lactobacillus species were grownunder aerobic condition in MRS broth.

Gel overlay assay. The gel overlay assay was accom-plished as previously described.7 Briefly, 35 µL of vaginalfluid was lyophilized, resuspended in loading buffer, andsubjected to AU-PAGE. The gel was washed three times for4 minutes each in 100 mL of 10 mmol/L sodium phos-phate (pH 7.5) and then placed on a plate that containedbacteria which were embedded in agarose (4 � 105 bacte-ria/mL in 1% low electroendo osmosis (EEO) agarose[Sigma Chemical Company, St Louis, Mo], GT buffer, pH4.5, �0.01 TSB) and incubated for 3 hours to allow diffu-sion of protein bands into the bacteria/agarose layer. Thegel was then removed; nutrient agarose (�2 TSB; 1%agarose) was poured over the top of the lawn, and the sur-viving bacteria were allowed to grow overnight. Areas thatcorresponded to antimicrobial peptides that were trans-ferred from the AU-PAGE gel were visualized as zones ofclearance in the microbial lawn.

Lactate assay. Total lactate in the vaginal fluid sampleswas determined with a microtiter plate diagnostic kit (cat-alog No. 735; Sigma Chemical Company). The amount oflactic acid present in the vaginal fluid was calculated bythe Henderson-Hasselbach formula: [HA] = [HA]0 ·10pK/(10pH + 10pK), where [HA]0 is the molar concen-tration of lactate plus lactic acid, [HA] is the molar con-centration of lactic acid, and the pK of lactate is 3.83.

Bacterial growth inhibition assay. The influence of pHand lactic acid on the growth of E coli ML-35p was assessed.Culture medium was prepared in the following manner:


Volume 187, Number 3 Valore et al 563Am J Obstet Gynecol

lactic acid was dissolved in GT (pH 6.5) buffer that con-tained �0.1 TSB to concentrations of 5, 10, 20, and 40mmol/L, and the pH was adjusted with sodium hydroxideor hydrochloric acid, as needed, to attain the desired con-centration of lactic acid (1, 2, 3, 5, and 7 mmol/L), as de-termined by the Henderson-Hasselbach formula. Culturemedium (198 µL) was placed in triplicate wells, and 2 µLof �100 concentrated bacteria was washed in GT buffer,pH 4.5, and was added to each well with a calibratedpipette, to a final concentration of 2 � 106 bacteria/well.The optical density at 620 nm (OD620) of the plate wasread on a Spectra Max enzyme immunoassay (EIA) reader(Molecular Devices, Sunnyvale, Calif) to determine theinitial value. The plate was then incubated in an environ-mental shaker at 37°C, and the OD620 was read at 3, 5, 7,and 20 hours to determine bacterial growth. The controlmedium contained GT buffer (20 mmol/L KHPO4, 60mmol/L NaCl) with �0.1 TSB, adjusted to the same pH asits lactate-containing counterpart.

Semiquantitative Western blot densitometry. The lyophi-late of the acid extract from tampons was resuspended inloading buffer, analyzed by AU-PAGE, and transferred toImmobilon-P (Millipore Corporation), as previously de-scribed.8 Antibodies that were used for Western blotanalysis were as follows: rabbit anti–HBD-1, anti–HBD-2,anti–HD-5, and anti–HNP-1 through 3 were generated inour laboratory8-11; rabbit anti-calprotectin (MRP8 andMRP10) was a gift from Dr Kenneth Miyasaki (UCLA De-partment of Dentistry), anti-lysozyme and anti-lactoferrinwere purchased from DAKO (Glostrup, Denmark); anti-SLPI was purchased from R&D Systems (Minneapolis,Minn); anti-LL-37 was obtained from Dr Robert Lehrer(UCLA Department of Medicine), and anti-histone H2Bwas purchased from Serotec (Washington, DC). Secondantibody conjugated to alkaline phosphatase was pur-chased from Pierce (Rockford, Ill). Vaginal fluid Westernblots were analyzed and compared with standard Westernblots that contained known amounts of the appropriate

protein, with the use of a densitometer and the Image-Quant program (both from Molecular Dynamics Inc, Sun-nyvale, Calif). Donor panel samples that were used forWestern blot analysis consisted of 4 samples that were col-lected approximately 7 days apart during one menstrualcycle, with the exception of the histone and LL-37 West-ern blots, in which case a single sample that was collectedapproximately on day 20 was used from each donor.

HBD-1 quantification by enzyme-linked immunosor-bent assay. The 96-well Maxisorp plates (Nunc, Roskilde,Denmark) were coated with a 100-µL volume of a 1:5000dilution of HBD-1 mouse ascites fluid (IB5A; produced incollaboration with Dr Beverly A. Dale, University of Wash-ington, Seattle, Wash) in coating buffer (BupH ready mixbuffers; Pierce) and incubated overnight. All steps of theprotocol were performed at room temperature; stan-dards and samples were analyzed in triplicate and dupli-cate, respectively. Wells were washed (0.1% bovine serumalbumin, 0.05% Tween 20, in phosphate-buffered salinesolution), incubated with 150 µL blocking buffer (1%bovine serum albumin in BupH–phosphate-bufferedsaline solution, 0.01% CETAB, 0.01% thimerosal) for 1hour; then samples in 100 µL volume were added to theappropriate well and serially diluted in the wells of theplate and allowed to incubate for 1.5 hour with mixing.Plates were washed and incubated with 100 µL rabbit

Table I. Lactate concentration and pH of vaginal fluid of the donor panel

Total lactate Calculated concentration Log change in E coli Donor panel pH (mmol/L) of lactic acid (mmol/L) CFU

Set 11 5.3 0.7 0.02 02 4.2 36 10.8 –33 4.9 11 0.9 04 4.3 21 5.3 –35 4.3 41 10.4 –3

Set 21 5.5 0.7 0.01 +12 4.2 37 11.1 –33 5.4 33 0.9 +14 4.7 27 3.2 05 4.4 18 3.8 –3

Samples were collected during two different menstrual cycles (sets 1 and 2). Total lactate and pH were determined, and the lactic acidconcentration was calculated. The corresponding antimicrobial activity is presented as a change in the log CFU of E coli after 3-hour in-cubation in vaginal fluid.

Table II. Antimicrobial polypeptides in vaginal fluid ofthe donor panel (n = 20; 5 donors, 4 samples each)

Protein Mean concentration ± SE (µg/mL)

Calprotectin 34 ± 7Lysozyme 13 ± 2Lactoferrin 0.9 ± 0.2SLPI 0.7 ± 0.1HBD-2 0.57 ± 0.13HNP1-3 0.35 ± 0.07HBD-1 0.04 ± 0.02



anti–HBD-1 polyclonal antibody (diluted 1:2000 in block-ing buffer) for 45 minutes, followed by washing and 1-hour incubation in 100 µL of a 1:2000 dilution ofhorseradish peroxidase that was conjugated to goat anti-rabbit antibody (Pierce). After being washed with water,the plate was developed with orrthophenylenediamine(OPD) solution (Sigma Chemical Company); the reac-tion was stopped with 2.5N hydrogen sulfate, and theOD620 was read on a spectrophotometer (Spectra-Max;Molecular Devices, Sunnyvale, Calif).

Results

Fluid collections. Vaginal fluid was collected from fivehealthy volunteers, who ranged in age from 25 to 44 yearsand had regular menstrual cycles of approximately 28days. For the analysis of proteins during the menstrualcycle, the five donors collected samples on the followingdays: donor 1, days 6, 12, 17, and 24 of a 25-day cycle (re-spective weights: 1.1, 1.2, 1.6, and 0.9 g); donor 2, days 8,12, 19, and 23 of a 27-day cycle (respective weights: 1.9,1.5, 2.4, and 1.5 g); donor 3, days 7, 14, 20, and 26 of a 26-day cycle (respective weights: 0.7, 1.2, 1.2, and 0.8 g);donor 4, days 7, 14, 21, and 25 of a 35-day cycle (respec-tive weights: 1.2, 1.8, 1.2, and 1.7 g); and donor 5, days 9,15, 19, and 25 of a 29-day cycle (respective weights: 2.0,1.5, 1.3, and 1.6 g). For antimicrobial assays, single sam-ples were collected at day 20 through 25 of the cycle.

Antimicrobial activity of vaginal fluid. The vaginal fluidfrom all of the donors was permissive for the growth of L

crispatus, L vaginalis, and C albicans (Fig 2), organisms thatare considered to be endogenous flora of the vagina.3

However, donors 2 and 5 exhibited strong antimicrobialactivity against L jensenii (3 log decrease after 2 hours),donor 4 exhibited moderate activity (3 log decrease after 8hours), and donor samples 1 and 3 allowed growth. Thesame three donors (2, 4, and 5) whose fluids killed Ljensenii also exhibited strong/moderate killing capabilitiesagainst group B Streptococcus and E coli. Samples fromdonors 1 and 3 allowed moderate growth of all the organ-isms that were tested, similar to growth in the GT buffercontrol.

Effects of high- and low-molecular-weight fractions. Todetermine whether the antimicrobial activity of vaginalfluid samples was due to small or large molecules, thefluid was separated into two fractions by passing througha 3-kd molecular weight cutoff membrane. The fractionthat was retained by the membrane contained virtually allof the protein (as can be seen in the lower right panel ofFig 1 [lane C]), whereas the fraction that passed throughthe membrane contained no protein (as visualized by sil-ver stained AU-PAGE [lane B]). Nearly all the protein inthe vaginal fluid was recovered from the membrane (ascan be seen by comparing lane A [whole fluid] to lane C[>3 kd protein fraction]). Interestingly, when a CFU assaywas done in the whole and fractionated fluid, virtually allof the antimicrobial activity against E coli could be attrib-uted to the low molecular weight fraction (Fig 1). For allbut one of the donors, the protein-containing fractionwas permissive for bacterial growth that was similar to thebuffer control, with the exception of a sample fromdonor 2 that exerted a bacteriostatic effect on E coli.

Fig 1. The antimicrobial activity of vaginal fluid fractions againstE coli ML35p. Vaginal fluid was fractionated into two parts by pas-sage through a 3-kd molecular weight cutoff membrane. A silver-stained AU-PAGE. A, 5 µL vaginal fluid. B, 5 µL of the <3-kdfraction. C, 5 µL of the protein fraction (>3 kd). D, 0.3 µglysozyme. E, 0.5 µg HBD-2. The antimicrobial activity of wholevaginal fluid (closed circles), protein-rich >3-kd fraction (closed in-verted triangles), <3-kd fraction (open circles), and GT buffer con-trol (open inverted triangles) from each donor is shown. Bacteriawere added to fluid that was supplemented with �0.01 TSB for anutrient source and incubated at 37°C for 0, 1, 3, and 24 hours.Aliquots were plated onto TSB plates, and CFUs were deter-mined after overnight incubation.

Fig 2. The antimicrobial activity of vaginal fluid. Microbes (Ljensenii, L crispatus, L vaginalis, and C albicans, group B Streptococ-cus, and E coli ML-35p) were added to vaginal fluid and incu-bated at 37°C. Aliquots were taken at 0, 1, 4, and 8 hours andwere plated in triplicate on TSB agar plates; the CFUs were de-termined after overnight incubation. Samples included GTbuffer control (closed circles), donor 1 (open circles), donor 2 (opensquares), donor 3 (open triangles), donor 4 (open diamonds), donor5 (open inverted triangles).



The antibacterial effect of lactic acid. In previous studies,vaginal fluid was reported to contain a mixture of organicacids, among which lactic acid predominated.12,13 To ana-lyze the role of lactic acid in antimicrobial activity, total lac-tate of the water-extracted vaginal fluid was determined,and the amount of lactic acid that was present in each sam-ple was calculated from the measured pH. The pH of thesamples ranged from 4.2 to 5.5, and the concentration oflactic acid ranged from 0.7 to 11.0 mmol/L (Table I). Thecorresponding antimicrobial activity against E coli is pre-sented as a change in the CFU (expressed in log) from theinoculum after incubation in the vaginal fluid and shows asignificant correlation between the lactic acid concentra-tion and the log change of CFU (Pearson product momentcorrelation coefficient, R = –0.839; P = .002; n = 10 samplesfrom 5 donors) and between the pH and the log change inCFUs (R = 0.941; P = .00005).

To determine the separate effects of pH and lactic acidon the growth of E coli, we performed a growth inhibitionassay. In each graph, bacterial growth in the lactate-con-

taining medium is compared with growth in lactate-freemedium at the same pH (Fig 3). At a pH of ≤4.1 (regard-less of lactic acid concentration), the growth of E coli wassignificantly inhibited. At >pH 4.1, there is a marked ad-ditional growth-suppressive effect of lactic acid concen-trations of ≥3 mmol/L. At 1 and 2 mmol/L lactic acid,there was inhibition of growth at the early time points,with recovery and even enhanced growth at 20 hourslikely to be due to the opposing effects of lactic acid (in-hibition) and lactate (enhancement).

Antimicrobial effects of the high molecular weight fraction.It is likely that the high molecular weight components (eg,proteins) of vaginal fluid are more concentrated on and inthe epithelial surface than in the fluid layer. To determinewhether antimicrobial activity increases with the concentra-tion of the high molecular weight solutes, the protein-con-taining high molecular weight fraction (donor 5) wasconcentrated 2- and 5-fold and then tested in the CFU assay.The unconcentrated high molecular weight fraction (�1)initially decreased the viability of bacteria (Fig 4), but by 24hours the CFU rose to match the buffer control. The highmolecular weight fraction that was concentrated 2- or 5-foldexerted a microbicidal effect throughout the assay.

Gel overlay antimicrobial assay. To estimate whetherone or more antimicrobial proteins in vaginal fluid exertpredominant activity, a gel overlay assay was performed.As can be seen by the zones of clearance, vaginal fluid con-tains many proteins that kill E coli (Fig 5). Similar resultswere also seen for group B streptococci (data not shown).

Protein composition of vaginal fluid. Proteins from sam-ples that were collected throughout the menstrual cyclefrom five donors were analyzed by semiquantitative West-

Fig 3. The effects of pH and lactate/lactic acid on the growth of Ecoli. GT buffer (20 mmol/L potassium phosphate, 60 mmol/Lsodium chloride) with �0.1 TSB that contained 5, 10, 20, or 40mmol/L lactate was adjusted to the appropriate pH with sodiumhydroxide or hydrochloric acid so that the calculated amount oflactic acid form was 1, 2, 3, 5, or 7 mmol/L (closed circles). The con-trols at the matched pH contained no lactate (open circles). A vol-ume of 198 µL culture medium was placed in triplicate wells of a96-well plate and 2 µL bacteria (2 � 109 bacteria/mL) were addedto each well, and the plate was placed in an environmental shakerat 37°C. The bacterial concentration represented by the OD620reading is shown at 0, 3, 5, 6, 7, and 20 hours.

Fig 4. The antimicrobial effect of the protein-rich fraction ofvaginal fluid. The vaginal fraction retained by a 3-kd molecularweight cutoff membrane was dissolved to �1, �2, and �5 origi-nal concentration in GT buffer (pH 4.5; �0.01 TSB). E coli ML-35p was added to each sample; aliquots were taken at 0, 1, 3, and24 hours and plated in triplicate on TSB agar plates, and CFUswere determined after overnight incubation. The symbols repre-sent bacterial CFU in GT or protein-rich vaginal fluid fraction,concentrated as indicated �1-�5 .



ern blot for previously described antimicrobial compo-nents of epithelial secretions (calprotectin, lysozyme, SLPI,and defensins HNP1-3, and HBD-2) and by enzyme-linkedimmunosorbent assay for HBD-1. Overall, there were onlyminor changes in protein concentrations during the men-strual cycle (Fig 6), and the data were pooled therefore tocalculate the mean concentration of each protein (n = 20;Table II). Calprotectin and lysozyme were present at con-centrations of >10 µg/mL; the other proteins were de-tected at concentrations of <1 µg/mL. HD-5 was notdetected in the vaginal fluid by this method (detectionlimit, 20 ng/mL). Subsequently, additional antimicrobialpeptides were sought in donor panel samples that were col-lected in mid menstrual cycle. The human cathelicidin pep-tide LL-3714 was detected in the donor panel, ranging from65 to 1000 ng/mL, with an average of 500 ng/mL (n = 5).Histone15 was detected in only one of the five vaginal fluidsamples (donor 1) and was present at approximately 2µg/mL (detection limit, 1.3 µg/mL).

Comment

In this study, the ability of vaginal fluid from five differ-ent donors to exert an antimicrobial effect on several dif-ferent microbes was assessed. Of the endogenous vaginalmicrobes, two of the three lactobacillus species (L crispa-tus, L vaginalis) and C albicans were able to grow in vaginalfluids from all of the donors. In contrast, L jensenii, groupB Streptococcus, and E coli were killed by three of the fivesamples. Most antimicrobial activity could be attributedto the low-molecular-weight (protein-free) fraction ofvaginal fluid. However, it is possible that the antimicro-bial activity of the polypeptide fraction may have been un-derestimated because of potential losses during tamponextraction or extract storage.

Resident vaginal bacteria can produce hydrogen per-oxide and convert host-derived nutrients into organicacids that include formic, succinic, acetic, propionic, bu-tyric, and lactic acid.12 Hydrogen peroxide is known to beantimicrobial, and several studies have shown thatwomen in whom colonies of hydrogen peroxide-produc-ing lactobacilli have been found are much less prone tosexually transmitted diseases and bacterial vaginosis.16

However, hydrogen peroxide and several of the organicacids are volatile and would likely be removed or greatlyreduced by the extraction procedure used in this study.In contrast, lactic acid, the predominant acid in normalvaginal fluid,12 is nonvolatile and is retained during theextraction procedure. In this study, we found that thevaginal fluid with the highest levels of antimicrobial activ-ity also contained the highest levels of lactic acid (3.8-11mmol/L; Table I). The “permissive” vaginal fluids con-tained ≤3.2 mmol/L lactic acid and had a higher pH thanthe more potently antimicrobial vaginal fluid samples.

It has been widely assumed that lactic acid is an anti-microbial factor in vaginal secretions; however, to ourknowledge, this is the first study to test this hypothesis. Weperformed a checkerboard study of lactate and pH effectson the growth of E coli and surmised that the acid form oftotal lactate is the major determinant of antimicrobial activity. As with vaginal fluids, in our model of lacticacid–dependent antimicrobial activity, approximately 3mmol/L lactic acid represented a transition zone belowwhich we observed no lactic acid–dependent antimicro-bial activity and above which there was complete killing ofE coli (Fig 3). This may explain the paradoxic differencein activity of 2 samples that were collected from donor 4,in which (despite an increase in total lactate from 21 to27 mmol/L between the first and second samples) therewas a pronounced decrease of antimicrobial activity. Inthis donor, a slight increase in pH of vaginal fluid (4.3 vs4.7) resulted in a significant decrease in calculated lacticacid concentration (3.2 mmol/L in the second samplecompared with 5.3 mmol/L in the first sample), whichstraddled the transition zone between killing and no

Fig 5. The antimicrobial polypeptide “fingerprint” of vaginal flu-ids. Vaginal fluids (donors 1-5) were analyzed by AU-PAGE andoverlaid for 3 hours onto a plate of E coli ML-35p which were em-bedded in agarose. With further incubation of the plate, surviv-ing bacteria formed microcolonies. The microcolonies wereabsent from the zones of clearance that indicate multiple activeantimicrobial polypeptides in each sample. The correspondingpattern of AU-PAGE Coomassie blue–stained polypeptides isshown in the lower panel. From top to bottom, lane M1 containsthe human protein markers lactoferrin, HNP-1, and lysozyme,and lane M2 contains histone H2B and SLPI. Arrowheads indicatemarker bands that exhibit antimicrobial activity in this assay.



killing of E coli. Thus, in vaginal fluid, like in the modelsystem, the acid form of total lactate may be a major con-tributor to antimicrobial activity.

This study marks the first time antimicrobial polypep-tides have been assessed in vaginal fluid and observed dur-ing the course of the menstrual cycle. Surprisingly, therewas little change in polypeptide concentration during thecycle (Fig 6). Of the polypeptides that were detected inthe vaginal fluid, calprotectin and lysozyme are present atconcentrations previously found to be antimicrobial,17,18

although the other peptides and proteins could partici-pate in synergistic activity.18 In addition to its role as an an-timicrobial polypeptide, calprotectin has also been foundto inhibit the adherence of exogenous microbes to cul-tured epithelial cells.19 SLPI, which inhibits serine pro-teases such as elastase and cathepsin G, also has beenfound to have antibacterial and antifungal capabilities.20-

22 Both HNP1-3 and HBD-2 concentrations increase in re-sponse to inflammatory stimuli, the former because ofneutrophil influx and the latter by the induction of ep-

ithelial synthesis. Thus, the concentrations of these de-fensins would be expected to increase in response thoseinfections that induce an inflammatory response.

The gel overlay assay (Fig 5) shows that the proteins andpeptides of the vaginal fluid are antimicrobial when placeddirectly on a lawn of bacteria. In addition, we found thatthe protein component of the vaginal fluid was able toexert a significant antimicrobial effect when concentrated2- to 5-fold (Fig 4). This observation could be biologicallysignificant because some of the proteins in the vaginalfluid are produced within the epithelium and are likely tobe present at much higher levels at the surface and in theinterstices of the epithelial cell layers. In addition, somestudies have found that many of these antimicrobial pep-tides can act synergistically. Although Singh et al23 foundonly an additive effect between HBD-2 and lysozyme, theydid find synergism with the following combinations:lysozyme/lactoferrin, lysozyme/SLPI, lactoferrin/SLPI,and lysozyme/LL-37. Thus, even though individually manyof the proteins are present in vaginal fluid at concentra-

Fig 6. Antimicrobial polypeptide levels in vaginal fluid collected throughout the menstrual cycle of the donor panel: donor1 (closed squares), donor 2 (open inverted triangles), donor 3 (open circles), donor 4 (closed circles), and donor 5 (closed invertedtriangles). The solid line indicates the log-linear regression (log trend) of all of the points in each graph.



tions below their active range, in or on the epithelium, andtogether they may constitute an effective barrier to micro-bial invasion of the epithelial surface.

Histone and fragments of histone are cationic polypep-tides with antimicrobial activity.15,24 In our study, histonewas found in one vaginal fluid sample at a concentrationof 2 µg/mL. The limitations imposed by the available an-tibody enabled only a relatively high detection limit; it ispossible that other vaginal fluid samples also contain an-timicrobial concentrations of histone. Epithelial cells innormal vaginal fluid frequently appear lysed25 and may re-lease nuclear histone into the vaginal fluid milieu. There-fore, the role of histones in vaginal fluid must be morecarefully examined in future studies. A potentially impor-tant factor that was not considered in this study, becauseof our focus on innate immunity, was the role of lymphoidpopulations in the vagina and immunoglobulin A and Gin the vaginal fluid. Although such antibodies are not di-rectly microbicidal, they could inhibit the attachment ofmicrobes to the epithelia. The likely contribution of an-timicrobial products that originate from vaginal bacteriaalso was not specifically addressed in this study.

In our study, vaginal fluid was collected on tamponsthat were inserted in the vagina for 8 to 10 hours thenremoved and weighed to determine the volume ab-sorbed over this time period. This method has the ad-vantage that quantitation is precise, and the fluid issampled from most of the vaginal surface. Clearly, thesampled fluid is a mixture that represents both vaginaland cervical sources, but this mixture is physiologicallyrelevant. We presented evidence that vaginal fluid is se-lectively antimicrobial and that lactic acid and, to alesser extent antimicrobial peptides and proteins, con-tribute to the resistance of the normal vagina to colo-nization by exogenous microbes represented in ourexperiments by E coli. Antimicrobial polypeptides couldhave a greater role in vaginal host defense of womenwho have low concentrations of lactate and high vaginalpH. Future studies will be necessary to determinewhether there are differences in the concentration ofantimicrobial polypeptides in the vaginal fluid ofwomen who are “normal” compared with women whohave recurrent problems with abnormal levels of micro-bial colonization, such as bacterial vaginosis and can-didiasis.

We thank Dr Edith M. Porter for her critical review of thismanuscript and her insightful scientific discussions and DrRose Linzmeier for her helpful contributions to this project.

REFERENCES

1. Wagner G, Levin RJ. Vaginal fluid. In: Hafez ES, Evans TN, edi-tors. The human vagina. Vol 2. Amsterdam: Elsevier/North-Hol-land Biomedical Press; 1978. p. 121-37.

2. Kistner RW. Physiology of the vagina. In: Hafez ES, Evans TN, ed-itors. The human vagina. Amsterdam: Elsevier/North-HollandBiomedical Press; 1978. p. 109-20.

3. Eschenbach DA, Thwin SS, Patton DL, Hooton TM, StapletonAE, Agnew K, et al. Influence of the normal menstrual cycle onvaginal tissue, discharge, and microflora. Clin Infect Dis2000;30:901-10.

4. Cohen MS, Black JR, Proctor RA, Sparling PF. Host defences andthe vaginal mucosa: a re-evaluation. Scand J Urol Nephrol Suppl1984;86:13-22.

5. Cole AM, Dewan P, Ganz T. Innate antimicrobial activity of nasalsecretions. Infect Immun 1999;67:3267-75.

6. Antonio MA, Hawes SE, Hillier SL. The identification of vaginalLactobacillus species and the demographic and microbiologiccharacteristics of women colonized by these species. J Infect Dis1999;180:1950-6.

7. Lehrer RI, Rosenman M, Harwig SS, Jackson R, Eisenhauer P.Ultrasensitive assays for endogenous antimicrobial polypeptides.J Immunol Methods 1991;137:167-73.

8. Valore EV, Park CH, Quayle AJ, Wiles KR, McCray PB, Ganz T.Human beta-defensin-1: an antimicrobial peptide of urogenitaltissues. J Clin Invest 1998;101:1633-42.

9. Liu L, Wang L, Jia HP, Zhao C, Heng HHQ, Schutte BC, et al.Structure and mapping of the human β-defensin HBD-2 gene andits expression at sites of inflammation. Gene 1998;222:237-44.

10. Porter EM, Liu L, Oren A, Anton PA, Ganz T. Localization ofhuman intestinal defensin 5 in Paneth cell granules. InfectImmun 1997;65:2389-95.

11. Ganz T, Selsted ME, Szklarek D, Harwig SS, Daher K, Bainton DF,et al. Defensins: natural peptide antibiotics of human neu-trophils. J Clin Invest 1985;76:1427-35.

12. Stanek R, Gain RE, Glover DD, Larsen B. High performance ionexclusion chromatographic characterization of the vaginal or-ganic acids in women with bacterial vaginosis. Biomed Chro-matogr 1992;6:231-5.

13. Bauman JE, Kolodny RC, Webster SK. Vaginal organic acids and hor-monal changes in the menstrual cycle. Fertil Steril 1982;38:572-9.

14. Frohm M, Agerberth B, Ahangari G, Stahle-Backdahl M, Liden S,Wigzell H, et al. The expression of the gene coding for the an-tibacterial peptide LL-37 is induced in human keratinocytes dur-ing inflammatory disorders. J Biol Chem 1997;272:15258-63.

15. Hirsch JG. Bactericidal action of histone. J Exp Med1958;108:925-44.

16. Klebanoff SJ, Hillier SL, Eschenbach DA, Waltersdorph AM.Control of the microbial flora of the vagina by H2O2-generatinglactobacilli. J Infect Dis 1991;164:94-100.

17. Brandtzaeg P, Gabrielsen TO, Dale I, Muller F, Steinbakk M,Fagerhol MK. The leucocyte protein L1 (calprotectin): a puta-tive nonspecific defence factor at epithelial surfaces. Adv ExpMed Biol 1995;371:201-6.

18. Travis SM, Conway BA, Zabner J, Smith JJ, Anderson NN, Singh PK,et al. Activity of abundant antimicrobials of the human airway.Am J Respir Cell Mol Biol 1999;20:872-9.

19. Nisapakultorn K, Ross KF, Herzberg MC. Calprotectin expres-sion inhibits bacterial binding to mucosal epithelial cells. InfectImmun 2001;69:3692-6.

20. Hiemstra PS, Maassen RJ, Stolk J, Heinzel-Wieland R, Steffens GJ,Dijkman JH. Antibacterial activity of antileukoprotease. InfectImmun 1996;64:4520-4.

21. Tomee JF, Hiemstra PS, Heinzel-Wieland R, Kauffman HF. An-tileukoprotease: an endogenous protein in the innate mucosaldefense against fungi. J Infect Dis 1997;176:740-7.

22. Wiedow O, Harder J, Bartels J, Streit V, Christophers E. An-tileukoprotease in human skin: an antibiotic peptide constitu-tively produced by keratinocytes. Biochem Biophys ResCommun 1998;248:904-9.

23. Singh PK, Tack BF, McCray PB Jr, Welsh MJ. Synergistic and addi-tive killing by antimicrobial factors found in human airway surfaceliquid. Am J Physiol Lung Cell Mol Physiol 2000;279:L799-805.

24. Rose FR, Bailey K, Keyte JW, Chan WC, Greenwood D, Mahida YR.Potential role of epithelial cell-derived histone H1 proteins in in-nate antimicrobial defense in the human gastrointestinal tract.Infect Immun 1998;66:3255-63.

25. Donders GG, Bosmans E, Dekeersmaecker A, Vereecken A, VanBulck B, Spitz B. Pathogenesis of abnormal vaginal bacterialflora. Am J Obstet Gynecol 2000;182:872-8.


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