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Infection and Immunity, June 2000, p. 3443-3447, Vol. 68, No. 6
0019-9567/00/$04.00+0
Copyright © 2000, American Society for Microbiology. All rights reserved.
Identification of a Human Lactoferrin-Binding
Protein in Gardnerella vaginalis
Gregory P.
Jarosik* and
Carol Beth
Land
Department of Biological Sciences, Louisiana
State University, Baton Rouge, Louisiana 70803
Received 24 November 1999/Returned for modification 18 January
2000/Accepted 6 March 2000
 |
ABSTRACT |
Previous studies have shown that Gardnerella vaginalis
can utilize iron-loaded human lactoferrin as a sole source of iron. In
this study, G. vaginalis cells were shown to bind
digoxigenin (DIG)-labeled human lactoferrin in a dot blot assay. Using
the DIG-labeled human lactoferrin, a 120-kDa human lactoferrin-binding protein was detected by Western blot analysis of G. vaginalis proteins. The lactoferrin-binding activity of this
protein was found to be heat stable. Competition studies indicated that
this binding activity was specific for human lactoferrin. Treatment of
G. vaginalis cells with proteases suggested that this
protein was surface exposed. An increase in lactoferrin binding by the 120-kDa protein was observed in G. vaginalis cells grown
under iron-restrictive conditions, suggesting that this activity may be
iron regulated.
 |
INTRODUCTION |
Gardnerella vaginalis is
the predominant microorganism associated with bacterial vaginosis (BV),
a common disorder of women of reproductive age. BV is characterized by
a shift in the microbiological flora of the lower vagina in which the
Lactobacillus-predominant flora is replaced by a number of
different microorganisms, including G. vaginalis,
Mobiluncus spp., Peptostreptococcus spp.,
Prevotella spp., Bacteroides spp., and
Mycoplasma hominis (18, 19, 46). Other
characteristics include (i) the presence of a homogenous discharge,
(ii) an amine (fishy) odor, (iii) the presence of vaginal epithelial
"clue cells," and (iv) an increase in the pH of the vagina to >4.5
(11, 46). Although found at low concentrations in healthy
subjects, G. vaginalis is found in higher concentrations in
BV patients. BV is a significant risk factor for upper genital tract
infections (12, 34) in pregnant women, which can result in
adverse outcomes of pregnancy, including preterm delivery and low birth
weight of infants (21), premature rupture of membranes (29), premature labor (22), and impaired fetal
development (13). More recent studies (44)
indicate that BV increases the risk of human immunodeficiency virus
(HIV) infection. Furthermore, it has also been demonstrated that the
microflora associated with BV could activate HIV type 1 (HIV-1)
expression in a promonocytic cell line chronically infected with HIV-1
(1, 32). It is postulated that a BV microflora-associated
HIV-inducing factor may contribute to HIV transmission.
G. vaginalis is a fastidious, nonmotile, beta-hemolytic,
unencapsulated, rod-shaped bacterium (6). Although G. vaginalis cells stain gram variable, this organism possesses a
gram-positive cell wall (37). In addition to being
associated with BV, G. vaginalis has been isolated from or
detected in a number of infections, including intra-amniotic and
chorioamniotic infections (14, 15, 20), intrauterine
infections (26), and urinary tract and bladder infections
(27, 45), as well as pelvic inflammatory disease
(12). However, little is known about the mechanism of G. vaginalis pathogenesis. One potential virulence factor is
a 60-kDa hemolysin that lyses human red blood cells, neutrophils, and
endothelial cells (7). G. vaginalis also
possesses pili and an exopolysaccharide coat that are involved in the
adherence of G. vaginalis to vaginal epithelial cells
("clue cells") and red blood cells (4, 43). However,
their specific roles in the establishment of G. vaginalis
infection remain to be determined. Of great importance, recent work by
Hashemi et al. demonstrated that G. vaginalis cell lysates
could stimulate HIV-1 gene expression in human cell cultures,
suggesting that G. vaginalis may play a role in the
increased rate of HIV transmission in BV patients (17).
Virtually all microorganisms require iron for their survival. For many
bacterial pathogens, the ability to acquire iron is related to their
virulence potential (16, 28, 47). However, in the human
host, free iron is found in limited amounts as a result of being
sequestered in compounds such as heme, ferritin, and hemoglobin or
bound by high-affinity iron-binding proteins such as transferrin or
lactoferrin (33, 47). To overcome this iron-withholding
capacity of the host, bacteria have developed several high-affinity
mechanisms to obtain this essential nutrient. One mechanism is the
utilization of siderophores (8, 28). Siderophores are
low-molecular-weight, high-affinity iron chelators which remove
iron from carrier molecules. After binding iron, siderophores are bound
by outer surface receptors for import of the iron or iron-siderophore
complex into the bacterial cell (8, 28). A second mechanism
is the direct binding of iron-containing compounds (such as heme,
hemoglobin, heme-hemopexin, lactoferrin, and transferrin) by specific
cell surface receptors (28, 33, 47). Other mechanisms
include the production of hemolysins or cytolysins which lyse host
cells, presumably resulting in the release of iron-containing compounds
(28), and the utilization of iron reductases
(25).
Little is known about iron acquisition by G. vaginalis.
Previous studies demonstrated that G. vaginalis could
utilize several iron-containing compounds, including iron salts, heme,
hemoglobin, and lactoferrin, as a sole source of iron (24).
G. vaginalis was also shown to produce siderophores and
express iron-regulated proteins (24). Preliminary work in
our lab (C. B. Land and G. P. Jarosik, Abstr. 99th Gen. Meet.
Am. Soc. Microbiol., abstr. B/D-156, 1999; C. B. Land, M. S. Smith, and G. P. Jarosik, Abstr. 98th Gen. Meet. Am. Soc.
Microbiol., abstr. D-102, p. 230, 1998) suggested that G. vaginalis could directly bind several iron-containing compounds,
including human lactoferrin (hLf). In this study, the interaction
between G. vaginalis strains and hLf was examined. G. vaginalis cells were shown to directly bind hLf. Additionally, we
demonstrate the detection of a G. vaginalis hLf-binding protein.
 |
MATERIALS AND METHODS |
Bacterial strains, reagents, media, and growth conditions.
The G. vaginalis type strain, 594 (ATCC 14018), and G. vaginalis 317 (ATCC 14019), a clinical isolate, were obtained from the American Type Culture Collection (Manassas, Va.). G. vaginalis strains were routinely grown on human blood bilayer
Tween agar plates obtained from Remel (Lenexa, Kans.) or on basal
medium (36) supplemented with 0.3% soluble starch (BMS) but
lacking heme. The G. vaginalis strains were colistin and
nalidixic acid resistant and beta-hemolytic when cultured on human
blood bilayer Tween plates, hydrogen peroxide sensitive, and catalase
negative. G. vaginalis cultures were incubated at 37°C in
an atmosphere of 5% CO2. Culture stocks were stored at
75°C in Proteose Peptone 3 (Difco, Detroit, Mich.) broth with 50%
glycerol. Escherichia coli strain DH5
MCR, which does not
bind lactoferrin (35), was routinely cultured on
Luria-Bertani medium (38) at 37°C. All iron-containing
compounds, the iron chelator 2,2'-dipyridyl, trypsin, proteinase K, and
the protease inhibitor phenylmethylsulfonyl fluoride were purchased
from Sigma Chemical Company (St. Louis, Mo.). All iron compounds were
freshly prepared by dissolving the compounds in distilled water
followed by filter sterilization, with the exception of heme, which was
dissolved in 0.02 N sodium hydroxide prior to filter sterilization.
DIG labeling of hLf.
Labeling of hLf with digoxigenin (DIG)
was performed using the DIG Protein Labeling Kit (Boehringer Mannheim,
Indianapolis, Ind.) according to the manufacturer's instructions.
Briefly, 1 mg of hLf dissolved in phosphate-buffered saline (pH 7.4)
was incubated with 100 µg of DIG
(digoxigenin-3-O-succinyl-
-aminocaproic acid-N-hydroxy-succinimide ester) for 2 h at room
temperature with gentle mixing. Unbound DIG was removed by dialysis in
phosphate-buffered saline buffer followed by ultrafiltration using a
Centricon-30 microconcentrator (Millipore, Bedford, Mass.). The
concentration of DIG-labeled hLf (DIG-hLf) was adjusted to 1 mg/ml, and
the solution was stored at
20°C.
Solid-phase dot blot binding assay.
Cultures of G. vaginalis strains grown on BMS were resuspended in BMS broth to a
concentration of 109 cells/ml, and 10 µl of the cell
suspensions were vacuum blotted onto nitrocellulose filters
(Immobilon-NC; Millipore) using the Bio-Rad (Hercules, Calif.) Bio-Dot
Blot apparatus. After drying for 1 h at 37°C, the filters were
incubated for 1 h with a blocking solution consisting of 3%
(wt/vol) skim milk dissolved in Tris-buffered saline (TBS) (pH 7.4).
DIG-hLf was then added to a final concentration of 1 to 2 µg/ml.
After incubation with DIG-hLf for 1 h at room temperature, the
filters were washed three times with TBS (pH 7.4), followed by a 1-h
incubation with the 3% skim milk blocking solution. Anti-DIG antibody
coupled to horseradish peroxidase (anti-DIG-POD; Boehringer Mannheim)
(150 U/ml) was added to a 1:25,000 final dilution and incubated for
1 h at room temperature. After three TBS buffer washes, the
filters were subjected to chemiluminescence analysis using the ECL
chemiluminescence detection agents (Amersham Pharmacia Biotech,
Piscataway, N.J.) according to the manufacturer's instructions.
Protein analysis.
Except where noted, G. vaginalis cells (109/ml) were routinely lysed by
incubation in lysis buffer (2% sodium dodecyl sulfate [SDS], 50 mM
Tris, pH 6.8) overnight at 4°C. Protein concentrations were
determined using the dotMETRIC protein assay (Gene
Technology, St. Louis, Mo.) or the Bio-Rad DC protein assay kits.
Separation of proteins was routinely performed by SDS-polyacrylamide
gel electrophoresis (SDS-PAGE) using 10% polyacrylamide gels and the Tris-glycine buffer system (38). For Western blot analysis, the separated proteins were then electroblotted onto nitrocellulose filter paper. After blocking for 1 h with 3% skim milk, the
filters were probed with DIG-hLf (1- to 2-µg/ml final concentration), washed with TBS, and then incubated with anti-DIG-POD antibody (1:25,000 final dilution). After the unbound antibody was removed by
washing, the blots were developed utilizing the chemiluminescence detection kit as described above.
Competitive binding assays.
The specificity of the binding
activity of the 120-kDa protein was examined in a competition assay
using Western blot analysis. Briefly, Western blot analysis of G. vaginalis proteins was performed as described above with the
exception that the nitrocellulose filters were preincubated with the
following unlabeled iron-containing compounds for 1 h prior to the
addition of the DIG-hLf: hLf, human transferrin, bovine lactoferrin,
catalase, hemin, or hemoglobin. The final concentration of the
competitor compounds was 100 µg/ml.
Proteolytic treatment of G. vaginalis cells.
One
milliliter of G. vaginalis cells (108/ml) was
incubated with proteinase K (50 µg/ml) or trypsin (50 µg/ml) for 30 min at 37°C. After being washed with BMS broth to remove the
proteases, the cells were resuspended (108/ml) in BMS broth
containing phenylmethylsulfonyl fluoride at a final concentration of 1 mM and lysed with Laemmli SDS-PAGE sample buffer. The cell proteins
were then analyzed by SDS-PAGE and Western blot analysis as described above.
 |
RESULTS |
Binding of hLf by G. vaginalis cells.
We have
previously reported that G. vaginalis could utilize
iron-loaded hLf as a sole source of iron (24). Previous work has demonstrated that some bacteria which utilize lactoferrin as an
iron source can directly bind this iron-containing compound (10,
23, 41, 42). To determine if G. vaginalis cells could bind hLf, a solid-phase dot blot assay was performed utilizing DIG-hLf
as a probe. As shown in Fig. 1,
chemiluminescence was detected from G. vaginalis strains 594 and 317 (Fig. 1, lanes a and b), indicating the binding of DIG-hLf by
these cells. As a negative control, a dot blot assay was performed
using E. coli DH5
MCR, and no lactoferrin binding was
observed (Fig. 1, lane c). In control experiments assaying for
nonspecific binding of the reagents, no chemiluminescence was detected
from G. vaginalis cells incubated with only DIG-hLf or the
anti-DIG-POD antibody (data not shown).

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FIG. 1.
Binding of DIG-hLf by G. vaginalis cells.
G. vaginalis cells were blotted onto nitrocellulose filters
and probed with DIG-hLf as described in the text. Lanes: a, G. vaginalis 594; b, G. vaginalis 317; c, E. coli DH5 MCR.
|
|
Detection of a 120-kDa Lbp.
Many bacteria which utilize
lactoferrin as an iron source express one or more specific receptors
which bind this glycoprotein (41). Since G. vaginalis cells could utilize and bind lactoferrin, we wanted to
identify a potential G. vaginalis protein(s) involved in the
binding of DIG-hLf by utilizing Western blot analysis. Proteins from
G. vaginalis whole-cell lysates separated via SDS-PAGE were
electroblotted onto nitrocellulose filters and probed with DIG-hLf.
Figure 2 shows that an hLf-binding
protein with an estimated molecular mass of 120 kDa was detected from
G. vaginalis strains 594 and 317. In control experiments
assaying for nonspecific binding of DIG-hLf, the 120-kDa
lactoferrin-binding protein (Lbp) was not detected from blots incubated
with only the DIG-hLf or only the anti-DIG-POD antibody (data not
shown). The 120-kDa hLf-binding protein was also observed from cell
lysates heated to 100°C prior to SDS-PAGE, indicating that the
binding activity was heat stable and that the 120-kDa protein was not
comprised of smaller protein subunits (Fig.
3).

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FIG. 2.
Detection of a 120-kDa Lbp via Western blot analysis.
G. vaginalis proteins from whole-cell lysates were
electroblotted onto nitrocellulose and probed with DIG-hLf. Lane 1, G. vaginalis 594; lane 2, G. vaginalis 317. MW,
molecular mass standards in kilodaltons.
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FIG. 3.
Heat stability of the 120-kDa protein
lactoferrin-binding activity. Proteins from whole-cell fractions of
G. vaginalis 594 (lanes 1 and 2) and G. vaginalis
317 (lanes 3 and 4) were not boiled (lanes 1 and 3) or boiled (lanes 2 and 4) prior to SDS-PAGE and Western blot analysis. MW, molecular mass
standards in kilodaltons.
|
|
To determine if the 120-kDa Lbp binding activity could be detected from
G. vaginalis grown under different iron conditions,
Western
blot analysis of
G. vaginalis proteins was performed
utilizing
whole-cell lysates from
G. vaginalis 317 cells
grown under iron-replete
conditions (BMS), iron-supplemented conditions
(BMS supplemented
with 100 µM ferric chloride), or iron-deficient
conditions (BMS
supplemented with 100 µM 2,2'-dipyridyl). The results
are shown
in Fig.
4. An increase in
lactoferrin-binding activity was detected
from
G. vaginalis
317 cells grown under iron-restrictive conditions
(Fig.
4, lane 3)
compared to cells grown under iron-replete or
iron-supplemented
conditions (Fig.
4, lanes 1 and 2), suggesting
that this activity may
be iron regulated.

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FIG. 4.
Western blot analysis of proteins from G. vaginalis cells grown under different iron conditions. Equal
amounts of protein (35 µg) from whole-cell lysates of G. vaginalis 317 grown under iron-replete (lane 1), iron-supplemented
(lane 2), or iron-restrictive (lane 3) conditions (see text) were
probed with DIG-hLf. The arrow indicates the 120-kDa G. vaginalis Lbp.
|
|
Specificity of the 120-kDa Lbp binding activity.
Previous work
in our laboratory (24; Land and Jarosik, Abstr. 99th
Gen. Meet. Am. Soc. Microbiol.; Land et al., Abstr. 98th Gen. Meet. Am.
Soc. Microbiol.) indicated that G. vaginalis could utilize
and directly bind a number of host iron-containing compounds, including
catalase, hemoglobin, and hemin. In order to examine the specificity of
the 120-kDa Lbp, a competitive binding assay utilizing various
iron-containing proteins was performed. Briefly, Western blot analysis
of G. vaginalis proteins was performed as described above
with the exception that the nitrocellulose blot paper was preincubated
with unlabeled iron-containing compounds for 1 h prior to the
addition of the DIG-hLf. The unlabeled iron compounds included human
transferrin and bovine lactoferrin, which are structurally and
functionally similar to hLf, as well as catalase, hemin, hemoglobin,
and hLf. The results are shown in Fig. 5.
Preincubation with unlabeled hemin, hemoglobin, catalase, bovine
lactoferrin, or human transferrin did not affect the ability of the
120-kDa protein to bind DIG-hLf (Fig. 5C, D, E, F, and G), whereas
preincubation with unlabeled hLf inhibited the binding of DIG-hLf by
the 120-kDa protein (Fig. 5B). These results indicated that the binding
activity of the 120-kDa Lbp was specific for hLf.

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FIG. 5.
Specificity of the G. vaginalis 120-kDa Lbp.
Equal amounts of protein from whole-cell lysates of G. vaginalis 594 (lanes 1) or G. vaginalis 317 (lanes 2)
were electroblotted onto nitrocellulose filters. Prior to exposure to
DIG-hLf, the filters were preincubated with various unlabeled
iron-containing compounds. (A) no pretreatment; (B) hLf; (C) hemin; (D)
hemoglobin; (E) catalase; (F) bovine lactoferrin; (G) human
transferrin. The arrow indicates the 120-kDa G. vaginalis
Lbp.
|
|
Proteolytic treatment of G. vaginalis cells.
To
examine if the 120-kDa Lbp may be surface exposed, G. vaginalis 317 cells were exposed to either proteinase K or trypsin prior to Western blot analysis. The results are shown in Fig. 6. Binding of DIG-hLf by the 120-kDa
protein was detected from cells not treated with the proteases (Fig. 6,
lane 1). However, the 120-kDa Lbp activity was not detected from cells
treated with either proteinase K or trypsin prior to Western blot
analysis (Fig. 6, lanes 2 and 3), suggesting that this protein may be
surface exposed. It is also possible that a second protein sensitive to the proteolytic treatment is required for the hLf-binding activity of
the 120-kDa Lbp.

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FIG. 6.
Sensitivity of the 120-kDa protein lactoferrin-binding
activity to protease treatment. Western blot analysis was performed to
examine the 120-kDa protein hLf-binding activity from G. vaginalis 594 cells treated with proteases prior to cell lysis.
Lane 1, no protease treatment; lane 2, proteinase K treatment; lane 3, trypsin treatment. MW, molecular mass standards in kilodaltons.
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 |
DISCUSSION |
hLf is an extracellular iron-binding glycoprotein which can be
found in mucosal secretions, including those found in the respiratory tract, gastrointestinal tract, and urogenital tract. It is also released by neutrophils at sites of infection. During infection, the
binding of iron by lactoferrin is proposed to reduce the amount of free
extracellular iron. This process, known as the hypoferremia of
infection, is thought to further limit the free iron available to
invading microorganisms. However, a number of bacterial pathogens, including Helicobacter pylori (23),
Neisseria meningitidis (41), Neisseria
gonorrhoeae (41), Moraxella catarrhalis
(5), and Staphylococcus aureus (31),
possess the ability to utilize lactoferrin as an iron source. Some
bacteria, for example, Bordetella pertussis and
Bordetella bronchiseptica (30), utilize
siderophores to capture iron from lactoferrin. However, a number of
bacteria are able to sequester iron from lactoferrin through the use of
specific surface receptors. The lactoferrin receptors found in
Neisseria and Moraxella species have been the
best studied (41, 42). For example, N. gonorrhoeae expresses an Lbp designated LbpA, whereas two Lbps,
LbpA and LbpB, have been identified in N. meningitidis (41, 42). M. catarrhalis possesses three Lbps,
namely, LbpA, LbpB, and CopB (41). However, mutational
analysis indicates that LbpA, but not LbpB, is essential for iron
acquisition from lactoferrin in these two pathogens (3, 41,
42). Lbps have been identified in other bacteria, including a
70-kDa hLf receptor in H. pylori (10), a 40-kDa
hLf receptor in the oral pathogen Prevotella nigrescens
(9), and a 450-kDa Lbp complex in S. aureus
(31). However, whether the Lbps from these organisms play a
direct role in iron acquisition from lactoferrin remains to be determined.
Previous studies demonstrated that G. vaginalis could
utilize hLf as a sole source of iron in vitro (24). In this
study, we began work examining the possible interaction between
G. vaginalis cells and hLf. Using a solid-phase dot blot
assay, it was shown that G. vaginalis cells could directly
bind DIG-hLf (Fig. 1). To determine if this binding could be mediated
by a receptor, proteins from G. vaginalis whole cells were
analyzed using Western blots probed with DIG-hLf. This resulted in the
detection of an hLf-binding protein with an apparent molecular mass of
about 120 kDa (Fig. 2). This activity was also detected from protein
samples heated to 100°C under reducing conditions prior to SDS-PAGE
(Fig. 3), suggesting the hLf-binding activity is heat stable and that the 120-kDa protein is a monomer. The heat-stable activity of this
protein is different from the activity of other Lbps. For example, the
lactoferrin-binding activities of N. meningitidis LbpA and
LbpB and the 40-kDa P. nigrescens Lbp are heat labile (2, 9). The 450-kDa S. aureus hLf-binding protein
complex was found to consist of two protein subunit components of 62 and 67 kDa upon exposure to reducing conditions (31).
In this study, the binding activity of the G. vaginalis Lbp
was shown to be specific for hLf. Competition binding assays utilizing unlabeled iron compounds which G. vaginalis can utilize as
iron sources did not inhibit the hLf-binding activity of the 120-kDa protein, whereas preincubation with unlabeled hLf did inhibit this
activity (Fig. 5). Interestingly, bovine lactoferrin or human transferrin, two compounds which are structurally and functionally very
similar to human lactoferrin, also did not inhibit the hLf-binding activity of the 120-kDa protein. These observations correlate with
studies demonstrating that G. vaginalis cannot utilize human transferrin (24) or bovine lactoferrin (G. P. Jarosik,
unpublished data) as an iron source. This suggests that the 120-kDa Lbp
may interact with amino acid residues or structural motifs unique to
the hLf molecule. Other bacteria express Lbps possessing similar binding specificities. For example, the Lbps of N. meningitidis, N. gonorrhoeae, M. catarrhalis, and H. pylori specifically bind human
lactoferrin (10, 41, 42).
In addition to the evidence suggesting their direct involvement in iron
acquisition, additional studies have suggested that the LbpA and LbpB
proteins of N. meningitidis and M. catarrhalis are surface exposed and that the expression of these proteins is iron
regulated (41, 42). Whether the G. vaginalis
120-kDa Lbp is directly involved in the acquisition of iron from hLf
remains to be determined. Furthermore, it is not known if G. vaginalis siderophores may be involved in the acquisition of iron
from hLf. It is possible that G. vaginalis may utilize two
mechanisms, siderophores and the direct binding of lactoferrin, to
obtain iron from lactoferrin. However, results from this study indicate
that the 120-kDa Lbp is surface exposed (Fig. 6) and that the binding
activity of this protein is iron regulated (Fig. 4), suggesting that
this protein may play a role in iron acquisition. In the gram-positive
bacterium Corynebacterium diphtheriae, the regulation of
several proteins is mediated by the DtxR protein and iron (39,
40). It is possible that the increase in the hLf-binding activity
by the G. vaginalis 120-kDa Lbp under iron-restrictive
conditions, and presumably its expression, is mediated by a DtxR
homolog. The binding activity of the 120-kDa Lbp was also detected from
cells grown under iron-supplemented conditions (Fig. 4). Thus, the
binding activity of the 120-kDa Lbp under iron-replete conditions as
well as an increase in lactoferrin-binding activity under
iron-restrictive conditions could potentially allow G. vaginalis to more effectively compete with other vaginal
microorganisms for lactoferrin-bound iron. Whether this contributes to
the changes in the vaginal microflora, such as those associated with
BV, remains to be determined. Further studies will be required to
determine what role the G. vaginalis Lbp plays in iron
acquisition and to determine the mechanism by which its activity is
iron regulated.
 |
ACKNOWLEDGMENTS |
We thank Alan Biel for critical reading of the manuscript.
This study was supported in part by a grant from the Joe W. and Dorothy
Dorsett Brown Foundation and in part by grant no. LEQSF(1999-02)-RD-A-05 from the State of Louisiana Board of Regents.
 |
FOOTNOTES |
*
Corresponding author. Mailing address: Department of
Biological Sciences, 508 Life Sciences Building, Louisiana State
University, Baton Rouge, LA 70803. Phone: (225) 388-2792. Fax: (225)
388-2597. E-mail: gjarosi{at}unix1.sncc.lsu.edu.
Present address: Invitrogen, Carlsbad, CA 92008.
Editor:
E. I. Tuomanen
 |
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Infection and Immunity, June 2000, p. 3443-3447, Vol. 68, No. 6
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Copyright © 2000, American Society for Microbiology. All rights reserved.
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