Novel Selective Inhibition of Lactobacillus iners by Lactobacillus-Derived Bacteriocins

Abstract

Lactobacillus iners is often associated with vaginal dysbiosis and bacterial vaginosis (BV), which are risk factors for adverse gynecological and obstetric outcomes. To discover natural inhibitors of L. iners, cell-free culture supernatants (CFSs) from 77 vaginal human Lactobacillus strains and 1 human intestinal strain were screened for inhibitory activity. Three active strains were identified, and Lactobacillus paragasseri K7 (K7), a human intestinal strain, produced the most potent L. iners-inhibitory activity. The active material was purified from the K7 CFS and yielded three active peptides, identified as components of two different class IIb, two-peptide bacteriocins, gassericin K7A (GasK7A) and gassericin K7B (GasK7B). The peptides corresponded to the GasK7A α peptide and the GasK7B α and β peptides. While all three peptides exhibited individual activity against L. iners, GasK7B α was the most potent, with an MIC of 23 ng/ml (4 nM). When combined in equal amounts, the GasK7B α and β peptides showed synergistic inhibition, with an MIC of 2 ng/ml (each peptide at 0.4 nM). Among the four major vaginal Lactobacillus species, the K7 bacteriocins selectively inhibited L. iners All 21 strains of L. iners tested (100%) were inhibited by the K7 bacteriocins, whereas <20% of the vaginal Lactobacillus crispatus, L. jensenii, and L. gasseri strains were inhibited. The combination of the BV treatment metronidazole and K7 bacteriocins completely killed both L. iners and Gardnerella vaginalis in a coculture experiment to mimic BV conditions. In contrast, this treatment did not inhibit L. crispatus cultures.IMPORTANCELactobacillus iners is a prevalent species of the vaginal microbiome, but unlike other major vaginal Lactobacillus species, it is not considered protective against BV and can coexist with BV-associated bacteria. L. iners is generally the first Lactobacillus species to emerge following the treatment of BV with metronidazole, and mounting evidence suggests that it may contribute to the onset and maintenance of vaginal dysbiosis. The discovery of highly potent bacteriocins that selectively kill L. iners while sparing protective vaginal lactobacilli may provide novel pharmacological tools to better understand the roles of this enigmatic bacterium in vaginal ecology and potentially lead to new and improved therapies for dysbiosis-related conditions such as BV.

Keywords: Lactobacillus iners; bacterial vaginosis; bacteriocins; vaginal dysbiosis; vaginal microbiome.

FIG 1

Selectivity of strains from the four major vaginal Lactobacillus species to inhibition by L. paragasseri K7 cell-free culture supernatants. The L. crispatus, L. jensenii, and L. (para)gasseri isolates were all of vaginal origin. L. (para)gasseri represents the total L. gasseri and L. paragasseri strains tested. The number (N) of strains tested is shown above the bar for each species. Sensitive strains were those exhibiting inhibition at ≥40 IU/ml in the critical dilution assay.

FIG 2

Inhibition of L. iners HM-702 growth in coculture with L. paragasseri K7 (a) and L. gasseri 105-1 (b) for 24 h. The limit of detection for L. iners in coculture by plating and colony counting was approximately 10⁶ CFU/ml. Data represent the means ± standard deviations from three independent experiments. NS, not significant.

FIG 3

Isolation of the GasK7B α antimicrobial peptide from the L. paragasseri K7 culture supernatant. (a) GasK7B α purified by reversed-phase chromatography using a 30 to 50% gradient of 2-propanol in water with 0.1% TFA. The blue line depicts the elution of protein from the column by UV detection at 280 nm. The column fractions are shown in red on the x axis. mAu, milli-absorbance units. (b) SDS-PAGE of GasK7B α in fractions 21 to 28 of the chromatogram shown in panel a. A prestained protein ladder (SeeBlue Plus2) was run between fractions 25 and 26 to show the relative size of the peptide.

FIG 4

Analysis of purified antimicrobial peptides from L. paragasseri K7. SDS-PAGE gels show the bacteriocins purified from the L. paragasseri K7 CFS (GasK7A α [Aα], GasK7B α [Bα], and GasK7B β [Bβ]) and from E. coli Shuffle (GasK7A β [Aβ]). (Left) Bacteriocins visualized with SafeEx protein dye migrating between 3 and 6 kDa. (Right) Zones of growth inhibition of L. sakei subsp. sakei ATCC 15521 in a soft-agar overlay to detect the purified bacteriocins directly in the gel.

FIG 5

MICs of purified bacteriocin peptides, individually and in combination, against L. iners HM-702 or L. sakei subsp. sakei ATCC 15521 determined using the critical dilution assay. The MIC values for the individual GasK7A α and GasK7A β peptides against L. iners were 56 ± 39 and >12,500 ng/ml, respectively, while the MIC for the combined peptides could not be determined (ND). The MIC values for the individual GasK7B α and GasK7B β peptides against L. iners were 23 ± 9.5 and 65 ± 28 ng/ml, respectively, and the MIC for the combined peptides was 2.0 ± 0.8 ng/ml. The MIC values for the individual GasK7A α and GasK7A β peptides against L. sakei were 6.6 ± 3.3 and >6,250 ng/ml, respectively, and the MIC for the combined peptides was 0.9 ± 0.5 ng/ml. The MIC values for the individual GasK7B α and GasK7B β peptides against L. sakei were 2.5 ± 0.2 and 27 ± 14 ng/ml, respectively, and the MIC for the combined peptides was 0.3 ± 0.2 ng/ml. The values represent the means ± standard deviations from at least three independent experiments.

FIG 6

Time courses of L. iners HM-702 (a) and HM-126 (b) growth for 24 h in the absence or presence of K7 bacteriocins (40 IU/ml). Data represent the means ± standard deviations from three independent experiments.

FIG 7

Concentration-dependent inhibition of L. iners strains HM-702 and HM-126 (a) compared to L. crispatus strains HM-103 and SJ-3C (b) by K7 bacteriocins. Data represent the means ± standard deviations from three independent experiments.

FIG 8

Comparison of amino acid sequences for the two-peptide bacteriocins GasK7A and GasK7B from L. paragasseri K7 with gassericin T and gassericin S sequence homologs from L. gasseri 105-1, 151-2, EV1461, LA158, SBT2055, LA327, and LF221 and L. paragasseri JV-V03. Shown are the mature peptide sequences downstream of the double-glycine leader sequences. Amino acid sequence variations from the K7 bacteriocin peptides are shown in red. GenBank accession numbers for the sequences are KDA99706, KDB00192, KDA99085, and KDA99086 for L. paragasseri K7; AAP55752, AAP55753, AAP56344, and AAP56345 for L. gasseri LF221; BAA82353 and BAA82354 for L. gasseri SBT2055; BAM09405 and BAM09406 for L. gasseri LA158; ALX37947 and ALX37948 for L. gasseri EV1641; EFJ70596 and EFJ70595 for L. paragasseri JV-V03; BBE52938, BBE52939, BBE52947, and BBE52948 for L. gasseri LA327; MK598475 and MK598476 for L. gasseri 105-1; and MK598477 and MK598478 for L. gasseri 151-2.

FIG 9

Inhibitory effects of metronidazole (42 μg/ml), L. paragasseri K7 bacteriocins (80 IU/ml), or both on the growth of L. iners HM-702 and G. vaginalis HM-133, either alone or in coculture for 24 h. L. crispatus 178-1 was incubated separately under the same conditions. Bacterial growth was determined by the optical density at 600 nm. Data represent the means ± standard deviations from three independent experiments.