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Gardnerella vaginalis is a fastidious, beta-hemolytic, nonmotile, unencapsulated, rod-shaped bacterium (6). Originally named Haemophilus vaginalis by Gardner and Dukes (17), the organism was renamed Corynebacterium vaginale by Zinnemann and Turner (84) on the basis of a Gram stain reaction and cell morphology. Subsequent extensive taxonomic studies using biochemical and DNA hybridization assays and electron microscopy led to the assignment of this bacterium to its current taxonomic designation (23, 58). Although G. vaginalis cells stain gram variable, several studies indicate that G. vaginalis possesses a gram-positive cell wall (28, 54, 62, 65). Furthermore, a recent study which analyzed G. vaginalis proteins, fatty acids, and 16S rRNA gene sequences supported the current taxonomic designation of G. vaginalis and indicated that this bacterium was closely related to the genus Bifidobacterium (82).

G. vaginalis is the predominant microorganism associated with bacterial vaginosis (BV), a common disorder which occurs primarily in women of reproductive age and is characterized by (i) the presence of a milky or gray homogeneous 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 (13, 49, 77). It is also characterized by a shift in the microbiological flora of the lower vagina, where the Lactobacillus-predominant flora is replaced by a number of different microorganisms, including Mobiluncus spp., Peptostreptococcus spp., Prevotella spp., Bacteroides spp., and Mycoplasma hominis (2, 13, 31, 32, 77, 78). Although it is found at low concentrations in healthy subjects, G. vaginalis is found in higher concentrations in BV patients. Recent studies have suggested that BV is a significant risk factor for upper genital tract infections (14, 15, 19, 33, 40, 53, 57) in pregnant women, which can result in adverse outcomes of pregnancy, including preterm delivery and low birth weight of infants (22, 34, 47, 50), premature rupture of membranes (48), premature labor (35, 63), and impaired fetal development (18). Furthermore, a study by Sewankambo et al. suggests that BV may increase susceptibility to infection by human immunodeficiency virus (75). However, with the exception of evidence for a commensal relationship between G. vaginalis and Prevotella bivia (61), very little is known about the interactions between the microorganisms associated with BV or the contributions of G. vaginalis and the other microorganisms to the establishment of BV or upper genital tract infections.

In addition to being associated with BV, G. vaginalis has been detected in intrauterine infections (6, 39, 46), intraamniotic and chorioamniotic infections (19, 20, 33, 40, 52), and pelvic inflammatory disease (14, 15). G. vaginalis has also been isolated in cases of urinary-tract infection and bladder infection (41, 76), and G. vaginalis bacteremia has also been documented (37). However, there is little information concerning the pathogenic mechanisms of G. vaginalis. G. vaginalis secretes a 60-kDa hemolysin which lyses human erythrocytes, neutrophils, and endothelial cells and thus is a potential virulence factor (7, 64). Studies have also indicated that pili and an exopolysaccharide coat are involved in the adherence of G. vaginalis to vaginal epithelial cells and erythrocytes (5, 74), although their specific roles in the establishment of G. vaginalis infection remain unclear.

Iron is an essential growth factor required by virtually all living cells. Furthermore, the acquisition of iron plays an important role in the virulence potential of many bacterial pathogens (24, 44). There are many examples of bacterial virulence factors regulated by iron levels, some of which are directly or indirectly involved in iron acquisition, including toxins, hemolysins, and high-affinity iron uptake systems (42, 44, 83). However, free iron is found in limited amounts in the human body and is sequestered in compounds such as ferritin, heme, and hemoglobin or bound by high-affinity iron-binding proteins such as lactoferrin and transferrin (56, 83). As a result, bacteria have developed high-affinity mechanisms to obtain this essential nutrient. One mechanism is the secretion of high-affinity iron chelators, known as siderophores, which remove iron from carrier molecules and then are bound by outer-surface receptors for import of the iron or iron-siderophore complex into the bacterial cell (9, 44, 45). Another mechanism is the use of cell surface receptors to directly bind iron-containing compounds such as heme, hemoglobin, heme-hemopexin, lactoferrin, and transferrin (44, 56, 83). A third mechanism is the production of hemolysins or cytolysins which lyse host cells, presumably resulting in the release of iron-containing compounds (44). Many bacteria possess more than one iron acquisition system and/or obtain iron from more than one source, presumably to ensure the acquisition of this essential nutrient. For example, Haemophilus ducreyi, a sexually transmitted pathogen which does not produce siderophores, can utilize several heme-containing compounds as iron sources, but not lactoferrin or transferrin (43). H. ducreyi expresses a protein, designated HgbA, which has been shown to bind hemoglobin directly (12). Vibrio vulnificus, which can obtain iron from a number of sources, uses a siderophore-mediated mechanism to acquire iron from transferrin (45).

Virtually nothing is known about iron acquisition by G. vaginalis. It is not known what host iron-containing compounds G. vaginalis may potentially use as a source of iron. Furthermore, although the lysis of host cells by the 60-kDa hemolysin may be one mechanism by which G. vaginalis obtains iron, it is not known if G. vaginalis has the potential to sequester iron by other mechanisms, such as the production of siderophores. In this study, the ability of G. vaginalis strains to utilize various iron-containing compounds as iron sources was examined. The ability of this organism to produce and excrete siderophores was also examined by a universal chemical assay for siderophore production. Finally, experiments were performed to determine if G. vaginalis possesses iron-regulated proteins.

Bacterial strains, reagents, media, and growth conditions. The bacterial strains used in this study are listed in Table 1. G. vaginalis strains were routinely grown on human blood bilayer-Tween (HBT) agar plates (81) obtained from BBL Microbiology Systems (Cockeysville, Md.) or basal medium (58) agar plates supplemented with 0.3% starch. Depending on the experiment, proteose-maltose-dextrose (PMD) or proteose-starch-dextrose (PSD) medium (11) was also used. All G. vaginalis cultures were incubated at 37°C under an atmosphere of 5% CO₂. Permanent frozen stocks were stored at 75°C in Proteose Peptone 3 (Difco, Detroit, Mich.) broth with 50% glycerol. All eight G. vaginalis strains were colistin and nalidixic acid resistant and beta-hemolytic when cultured on HBT plates, catalase negative, and hydrogen peroxide sensitive. No beta-hemolysis was detected when the G. vaginalis strains were cultured on Columbia-colistin-nalidixic acid (BBL Microbiology Systems) agar plates containing 5% sheep blood. Escherichia coli strains were routinely cultured on Luria-Bertani medium (67) at 37°C. All iron-containing compounds, apo-transferrin, the iron chelators 2,2'-dipyridyl and deferoxamine mesylate, Chelex-100 chelating resin, chrome azurol S dye, and piperazine-N,N'-bis(2-ethanesulfonic acid) (PIPES; free acid) were purchased from Sigma Chemical Company (St. Louis, Mo.) or from Fluka Chemical Corporation (Milwaukee, Wis.). All iron-compound solutions were freshly prepared by dissolving the compounds in distilled water (dH₂O) followed by filter sterilization, with the exception of hemin, which was dissolved in a solution of 0.02 N sodium hydroxide prior to filter sterilization.

Iron source utilization assays. For the plate bioassay, fresh overnight cultures of the G. vaginalis strains grown on PSD plates were suspended at a concentration of approximately 10⁸ CFU/ml in PSD broth made low in iron by treatment with Chelex-100. Chelex-100 treatment consisted of stirring 10 g of resin/100 ml of broth for 4 to 6 h prior to autoclaving. Chelex-100-treated medium was supplemented with 0.1 mM magnesium sulfate, 0.1 mM calcium chloride, and 10 µM zinc chloride. Fifty microliters of the cell suspension was spread onto PSD agar plates containing 100 µM deferoxamine mesylate or 100 µM 2,2'-dipyridyl. Following drying, sterile filter disks (7 mm) were placed onto the agar plates and the various iron sources (10 µl of each, except for hemin [2.5 µl]) were spotted onto the filters. After drying, the plates were incubated at 37°C under an atmosphere of 5% CO₂ for 24 to 48 h and were then examined for bacterial growth around the filters. Bacterial growth around the filter disk indicated that the cells could utilize the iron source. The following iron sources were used at the concentrations indicated: ferrous chloride (FeCl₂), 1 mg/ml; ferric chloride (FeCl₃), 1 mg/ml; ferrous ammonium sulfate, 1 mg/ml; ferrous sulfate (FeSO₄), 1 mg/ml; ferric ammonium citrate, 1 mg/ml; hemin (bovine and equine), 1 mM; catalase (bovine), 85 µM; hemoglobin (equine, human, rabbit, and bovine), 80 µM; apo-transferrin and iron-loaded transferrin (33 or 98% iron-saturated), 125 µM; and iron-loaded lactoferrin (90% saturated), 125 µM. All assays were performed in triplicate. The optimal minimal concentration of the iron chelators which inhibited G. vaginalis growth in the plate bioassay was determined by titration experiments in which G. vaginalis cells were inoculated onto PSD agar plates containing varying concentrations (50, 100, 150, or 200 µM) of deferoxamine mesylate or 2,2'-dipyridyl. From these experiments, it was determined that 100 µM was the optimal minimal concentration of the iron chelators which inhibited G. vaginalis growth.

Acquisition of iron from iron-containing compounds by G. vaginalis. G. vaginalis inhabits an environment where it can potentially be exposed to a variety of iron-containing compounds, including heme, lactoferrin, and hemoglobin. However, it is not known which iron-containing compounds G. vaginalis can utilize as potential iron sources. To determine this, six strains were examined by a plate bioassay for their abilities to utilize a variety of compounds, including host iron compounds, as sources of iron. An example of the plate bioassay using PSD plates containing 100 µM deferoxamine mesylate is shown in Fig. 1. Growth of G. vaginalis 594 was detected around the filter disks inoculated with ferrous chloride, ferric chloride, hemoglobin, catalase, or hemin. No growth was detected around the control disk inoculated with sterile dH₂O.

View larger version (120K): [in this window] [in a new window]   FIG. 1.   Utilization of iron sources by G. vaginalis 594 as determined by the plate bioassay. G. vaginalis cells were inoculated onto a PSD plate containing 100 µM deferoxamine mesylate as described in Materials and Methods. Filter disks were spotted with dH2O (a), ferric chloride (b), ferrous chloride (c), catalase (d), bovine hemin (e), or bovine hemoglobin (f).

Detection of siderophore production. Many bacteria produce siderophores as a mechanism for acquiring iron from the environment. To determine if G. vaginalis produces siderophores, the chrome azurol S universal assay for the detection of siderophores was used (73). The advantage of this assay is that it can detect siderophores based on their affinity for iron and not on their specific chemical structures (73). Briefly, fresh overnight cultures of bacterial cells grown on PSD agar plates were patched onto agar plates containing chrome azurol S. Siderophore production, as indicated by the presence of orange halos around the patches, was determined after incubation for 18 to 24 h at 37°C under an atmosphere of 5% CO₂. E. coli strains which were either proficient or defective in siderophore production were used as controls. All assays were performed in triplicate. All eight of the G. vaginalis strains tested produced siderophores, as indicated by the presence of yellow-orange halos around the cell patches (Table 3). Siderophore production was also detected for E. coli HB101, which is wild type for siderophore production, and E. coli H1780 (27), which contains a fur mutation resulting in the derepression of siderophore production (Table 3). Siderophore production was not detected for E. coli RW193 (73), an entA mutant deficient in siderophore production (Table 3).

Protein profile of the membrane fraction of G. vaginalis 594. It has been demonstrated in many bacteria that the synthesis of some proteins is iron regulated such that, characteristically, the expression of these proteins is induced under iron-limiting conditions. To determine if G. vaginalis expressed iron-regulated proteins, the membrane fraction of G. vaginalis 594 cells grown under iron-replete conditions (PMD medium) or iron-restrictive conditions (PMD medium with 100 µM 2,2'-dipyridyl) was isolated and proteins contained within this fraction were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). The membrane fraction of G. vaginalis 594 was isolated by using the method for isolating the membrane fraction of Streptomyces spp. (21). Briefly, G. vaginalis 594 cells were harvested after growth for 24 h on either iron-replete (PMD agar plates) or iron-restricted (PMD agar plates with 100 µM 2,2'-dipyridyl) medium. Harvested cells were resuspended in a 50 mM Tris (pH 8)-10% sucrose buffer solution containing 0.2 mM dithiothreitol (DTT), lysozyme (10 mg/ml), RNase A (0.2 mg/ml), and DNase I (0.2 mg/ml) and then incubated for 1 h at 37°C to form protoplasts. Protoplast formation was monitored microscopically. Following sedimentation, the protoplasts were resuspended in buffer (50 mM Tris [pH 8]-0.2 mM DTT-0.2 M KCl-0.1 mM phenylmethylsulfonyl fluoride [PMSF]) and lysed by mild sonication. Following filtration through a 0.45 µm-pore-size filter to remove unbroken cells, the membrane fraction was harvested by centrifugation (at 260,000 × g) and resuspended in 50 mM Tris-0.1 mM PMSF. Protein concentrations were determined by using the dotMETRIC protein assay kit (Gene Technology, St. Louis, Mo.) according to the manufacturer's instructions. Membrane proteins were separated by SDS-PAGE as described elsewhere (67) by using a 5% (wt/vol) stacking and a 10% (wt/vol) separating gel. Following electrophoresis, the proteins were visualized by silver staining (67). Comparison of the protein profile of cells grown in iron-replete conditions (Fig. 2, lane 1) with the protein profile of cells grown in iron-restricted conditions (Fig. 2, lane 2) revealed several iron-regulated proteins whose molecular masses ranged from 33 to 94 kDa.

View larger version (40K): [in this window] [in a new window]   FIG. 2.   Iron-regulated proteins of G. vaginalis 594. Equal amounts of protein (50 µg) were loaded onto each lane. Membrane protein profiles of G. vaginalis 594 cells grown under iron-replete (lane 1) and iron-restrictive (lane 2) conditions are shown. MW, molecular weight markers (in thousands).