Introduction Human commensal Streptococcus salivarius plays an essential role in the balance of the human oral microbiome, in part controlled by bacteriocin activity finding a wide application as an oral probiotic in the prevention of several upper respiratory tract infections such as otitis media. In this study, we show for the first time the genetic organization of the blpU-like bacteriocin cassette in 24SMBc S.salivarius, previously characterized by us for its remarkable ability against AOM pathogens and its use as an oral probiotic. The secondary aims were to determine: i) genomic strain target identification, ii) the expression of all orfs by Real-time qPCR. MATERIALS AND METHODS The 24SMBc S.salivarius genome was sequenced by NGS, obtaining 375 contigs covering 1,893,903 bp; and finding the bacteriocin locus. The preliminary sequence analysis was performed by PRODIGAL and MyRast server. RNA extraction was performed by RNeasy kit (Qiagen) in stationary and exponential phases. RT-qPCR assays were performed. TaqMan primers and probes were designed by Beacon DesignerTM 8.0. The strain target identification was performed by qPCR. Standard curves were generated by dilution series of purified DNA from 24SMBc S.salivarius (107–10 genome copies ). The cross reaction was evaluated with 20 strains belonging to different streptococcal species. Results The blpU-like bacteriocin cassette is 8,023 bp inserted adjacent to the pepX gene at 1,863,862 bp referred to S.salivarius NCTC 8618 genome (GenBank NZ_CP009913.1). The bacteriocin cassette is organized in 11orfs of which orf1 and orf2 are involved in the immunity system encoding blpx_2-like and EntA_Immun protein, respectively; orf8 encoding blpU-like (59 aa), a pore forming peptide belonging to bacteriocin class II showing the consensus and conserved region relative to the comC family; and orf9-10 encoding ABC transporter involved in peptide export. We found the strain-target sequence between pepX and orf1 that shows high sensitivity and specificity able to identify and quantify the presence of our strain from other S.salivarius strains. The expression of all orfs present in the bacteriocin cassette shows their expression in both stationary and exponential phases and only orf 1 and 5 appear to be upregulated in the stationary phase. Conclusion We describe for the first time the genetic characterization of blpU-like bacteriocin region in 24SMBc S.salivarius, to date reported only for S.pneumoniae and S.thermophilus. The blpU-like could be involved in bacteriocin production since our strain lacks salivaricins, bacteriocins commonly produced by S.salivarius. The identification of the strain specific target distinguishing 24SMBc from other strains could find use in clinical applications.

Genetic characterization of blpU-like bacteriocin cassette in oral probiotic 24 SMBc S.salivarius

STEFANI, Stefania;SANTAGATI, Maria Carmela
2016

Abstract

Introduction Human commensal Streptococcus salivarius plays an essential role in the balance of the human oral microbiome, in part controlled by bacteriocin activity finding a wide application as an oral probiotic in the prevention of several upper respiratory tract infections such as otitis media. In this study, we show for the first time the genetic organization of the blpU-like bacteriocin cassette in 24SMBc S.salivarius, previously characterized by us for its remarkable ability against AOM pathogens and its use as an oral probiotic. The secondary aims were to determine: i) genomic strain target identification, ii) the expression of all orfs by Real-time qPCR. MATERIALS AND METHODS The 24SMBc S.salivarius genome was sequenced by NGS, obtaining 375 contigs covering 1,893,903 bp; and finding the bacteriocin locus. The preliminary sequence analysis was performed by PRODIGAL and MyRast server. RNA extraction was performed by RNeasy kit (Qiagen) in stationary and exponential phases. RT-qPCR assays were performed. TaqMan primers and probes were designed by Beacon DesignerTM 8.0. The strain target identification was performed by qPCR. Standard curves were generated by dilution series of purified DNA from 24SMBc S.salivarius (107–10 genome copies ). The cross reaction was evaluated with 20 strains belonging to different streptococcal species. Results The blpU-like bacteriocin cassette is 8,023 bp inserted adjacent to the pepX gene at 1,863,862 bp referred to S.salivarius NCTC 8618 genome (GenBank NZ_CP009913.1). The bacteriocin cassette is organized in 11orfs of which orf1 and orf2 are involved in the immunity system encoding blpx_2-like and EntA_Immun protein, respectively; orf8 encoding blpU-like (59 aa), a pore forming peptide belonging to bacteriocin class II showing the consensus and conserved region relative to the comC family; and orf9-10 encoding ABC transporter involved in peptide export. We found the strain-target sequence between pepX and orf1 that shows high sensitivity and specificity able to identify and quantify the presence of our strain from other S.salivarius strains. The expression of all orfs present in the bacteriocin cassette shows their expression in both stationary and exponential phases and only orf 1 and 5 appear to be upregulated in the stationary phase. Conclusion We describe for the first time the genetic characterization of blpU-like bacteriocin region in 24SMBc S.salivarius, to date reported only for S.pneumoniae and S.thermophilus. The blpU-like could be involved in bacteriocin production since our strain lacks salivaricins, bacteriocins commonly produced by S.salivarius. The identification of the strain specific target distinguishing 24SMBc from other strains could find use in clinical applications.
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Utilizza questo identificativo per citare o creare un link a questo documento: http://hdl.handle.net/20.500.11769/111875
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