Abstract
Streptococcus pyogenes is a globally prominent bacterial pathogen that exhibits strict tropism for the human host, yet bacterial factors responsible for the ability of S. pyogenes to compete within this limited biological niche are not well understood. Using an engineered recombinase-based in vivo expression technology (RIVET) system, we identified an in vivo-induced promoter region upstream of a predicted Class IIb bacteriocin system in the M18 serotype S. pyogenes strain MGAS8232. This promoter element was not active under in vitro laboratory conditions, but was highly induced within the mouse nasopharynx. Recombinant expression of the predicted mature S. pyogenes bacteriocin peptides (designated SpbM and SpbN) revealed that both peptides were required for antimicrobial activity. Using a gain of function experiment in Lactococcus lactis, we further demonstrated S. pyogenes immunity function is encoded downstream of spbN. These data highlight the importance of bacterial gene regulation within appropriate environments to help understand mechanisms of niche adaptation by bacterial pathogens.
Highlights
Bacteriocin-like peptide operon that was very weakly expressed in vitro, but highly induced in vivo
We integrated an engineered 2,976-bp gene cassette into the tsf/pepO intergenic region using the pG+host[5] temperature sensitive integration vector[17]. This gene cassette was flanked by loxP recognition sites for the Cre-recombinase, and contained both a tetracycline resistance gene, as well as a S. pyogenes codon optimized thymidine kinase from Herpes simplex virus (HSV-tk) (Fig. 1A)
The herpes simplex virus-1 thymidine kinase (HSV-tk) gene was engineered initially for use as a counter selection system in conjunction with ganciclovir for the second round of the RIVET procedure; this gene was only intermittently functional in S. pyogenes and HSV-tk was included in the cassette, we instead relied on colony patching for tetracycline sensitivity during the second round of screening
Summary
Bacteriocin-like peptide (blp) operon that was very weakly expressed in vitro, but highly induced in vivo. The correct integration of the loxP-tetR-loxP cassette was verified by PCR and DNA sequencing, and this clone was designated S. pyogenes MGAS8232 Cas[2]. To evaluate the functionality of the loxP-tetR-loxP cassette, we transformed MGAS8232 Cas[2] with the erythromycin resistant pMSP3535 vector containing the nisin inducible promoter[18], and pMSP3535 containing cre in the forward direction (pMSP3535::cre-forward).
Sign-in/Register to view highlights & summary Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a callDisclaimer: All third-party content on this website/platform is and will remain the property of their respective owners and is provided on "as is" basis without any warranties, express or implied. Use of third-party content does not indicate any affiliation, sponsorship with or endorsement by them. Any references to third-party content is to identify the corresponding services and shall be considered fair use under The CopyrightLaw.
