Purification and genetic characterization of gassericin E, a novel co-culture inducible bacteriocin from Lactobacillus gasseri EV1461 isolated from the vagina of a healthy woman

Abstract

Background: Lactobacillus gasseri is one of the dominant Lactobacillus species in the vaginal ecosystem. Some strains of this species have a high potential for being used as probiotics in order to maintain vaginal homeostasis, since they may confer colonization resistance against pathogens in the vagina by direct inhibition through production of antimicrobial compounds, as bacteriocins. In this work we have studied bacteriocin production of gassericin E (GasE), a novel bacteriocin produced by L. gasseri EV1461, a strain isolated from the vagina of a healthy woman, and whose production was shown to be promoted by the presence of certain specific bacteria in co-culture. Biochemical and genetic characterization of this novel bacteriocin are addressed.

Results: We found that the inhibitory spectrum of L. gasseri EV1461 was broad, being directed to species both related and non-related to the producing strain. Interestingly, L. gasseri EV1461 inhibited the grown of pathogens usually associated with bacterial vaginosis (BV). The antimicrobial activity was due to the production of a novel bacteriocin, gassericin E (GasE). Production of this bacteriocin in broth medium only was achieved at high cell densities. At low cell densities, bacteriocin production ceased and only was restored after the addition of a supernatant from a previous bacteriocin-producing EV1461 culture (autoinduction), or through co-cultivation with several other Gram-positive strains (inducing bacteria). DNA sequence of the GasE locus revealed the presence of two putative operons which could be involved in biosynthesis and immunity of this bacteriocin (gaeAXI), and in regulation, transport and processing (gaePKRTC). The gaePKR encodes a putative three-component regulatory system, involving an autoinducer peptide (GaeP), a histidine protein kinase (GaeK) and a response regulator (GaeR), while the gaeTC encodes for an ABC transporter (GaeT) and their accessory protein (GaeC), involved in transport and processing of the bacteriocin. The gaeAXI, encodes for the bacteriocin gassericin E (GasE), a putative peptide bacteriocin (GaeX), and their immunity protein (GaeI).

Conclusions: The origin of the strain (vagina of healthy woman) and its ability to produce bacteriocins with inhibitory activity against vaginal pathogens may be an advantage for using L. gasseri EV1461 as a probiotic strain to fight and/or prevent bacterial infections as bacterial vaginosis (BV), since it could be better adapted to live and compete into the vaginal environment.

Keywords: Bacteriocin; Gassericin; Quorum sensing; Three-component regulatory system.

Fig. 1

MALDI-TOF mass spectra of purified Gassericin E (GasE). [M+H]⁺, monoisotopic peak of GasE. a.u., absorbance units. Inset panel: SDS-PAGE (A) and bioassay (B) of purified GasE. L. paracasei C1351 was used as the indicator strain. MWM, molecular weight marker

Fig. 2

Alignment of the amino acid sequences of double-glycine leader peptides and mature peptides of Gassericin E and other similar gassericin bacteriocins. The sequences were aligned with the ClustalW2 software at the EMBL-Ebi online server. The arrow indicates the Gly-Gly cleavage site of the peptide. Asterisks, dots and double dots mean fully, strongly and weakly conserved residues, respectively. The first 18 aa obtained by Edman sequencing of the peptide GasE are underlined. The two deduced amino acid sequences deposited in databases from L. gasseri K7 (A, B) differed in one amino acid at position 11 of mature peptide (VxA). Theoretical molecular weights (MWt) of the mature bacteriocins are shown. The Genebank accession numbers are BAA82353 for Gassericin T, AAP56345 for Acidocin 221B, EFJ70596 for Lactacin-F subunit LafA, AAP73781 and KDA99085for Gassericin K7 B complemental factor (A) and (B), respectively

Fig. 3

Schematic representation of the locus for Gassericin E (GasE) production (a) and detailed analysis of DNA sequences of putative promoters and Rho-independent terminators (b, c, d, e, f). a P1, P2, P3a and P3b are putative promoter sequences, and T1 is a putative Rho-independent transcription terminator. Genes encoding Gassericin E (gaeA), the putative complement peptide GaeX (gaeX) and their putative immunity protein (gaeI), could form an transcriptional unit, driven by two putative alternative promoters (P3a and P3b). Genes encoding the three-component regulatory system formed by the autoinducer peptide (gaeP), the histidine protein kinase (gaeK) and the response regulator (gaeR), as well as the ABC-transporter (gaeT) and the accessory protein (gaeC), seem to form one transcriptional unit; however two different transcripts could be formed, one larger driven by promoter P1 involving gaePKRTC and one shorter driven by promoter P2 involving gaeTC. a, b, c, d The putative promoters P1, P2, P3a and P3b were detected with the Neural Network Promoter Prediction online server [30], with a promoter score cut-off of 0.9 (score are shown in brackets). The typical -35 and -10 boxes and the ribosome binding sites (RBS) are shown; the +1 indicates the putative transcription start. Putative regulatory DNA sequences (Direct L- and R- repeats) are in italics. e Rho-independent terminator; base-pairs are in boldface and apical loop in italics. f Alignment of the direct DNA repeats (L and R) found upstream of the GasE and GaeP putative promoters (P1 and P3b) and the consensus L- and R- repeats involved in quorum-sensing regulation of Blp bacteriocins in Streptococcus thermophilus [34]