Molecular mechanism of quorum sensing inhibition in Streptococcus by the phage protein paratox

Nicole R Rutbeek,Hanieh Rezasoltani,Trushar R Patel,Mazdak Khajehpour,Gerd Prehna

doi:10.1016/j.jbc.2021.100992

Nicole R Rutbeek, Hanieh Rezasoltani + Show 3 more

Open Access

https://doi.org/10.1016/j.jbc.2021.100992

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Abstract

Streptococcus pyogenes, or Group A Streptococcus, is a Gram-positive bacterium that can be both a human commensal and a pathogen. Central to this dichotomy are temperate bacteriophages that incorporate into the bacterial genome as prophages. These genetic elements encode both the phage proteins and the toxins harmful to the human host. One such conserved phage protein, paratox (Prx), is always found encoded adjacent to the toxin genes, and this linkage is preserved during all stages of the phage life cycle. Within S. pyogenes, Prx functions to inhibit the quorum-sensing receptor-signal pair ComRS, the master regulator of natural competence, or the ability to uptake endogenous DNA. However, the mechanism by which Prx directly binds and inhibits the receptor ComR is unknown. To understand how Prx inhibits ComR at the molecular level, we pursued an X-ray crystal structure of Prx bound to ComR. The structural data supported by solution X-ray scattering data demonstrate that Prx induces a conformational change in ComR to directly access its DNA-binding domain. Furthermore, electromobility shift assays and competition binding assays reveal that Prx effectively uncouples the interdomain conformational change required for activation of ComR via the signaling molecule XIP. Although to our knowledge the molecular mechanism of quorum-sensing inhibition by Prx is unique, it is analogous to the mechanism employed by the phage protein Aqs1 in Pseudomonas aeruginosa. Together, this demonstrates an example of convergent evolution between Gram-positive and Gram-negative phages to inhibit quorum-sensing and highlights the versatility of small phage proteins.

Highlights

The temperate bacteriophage and phage-like elements associated with S. pyogenes are dsDNA phage belonging to the Siphoviridae family [6, 7]
Each individual ComR exhibits a unique profile of SigX inducing peptide (XIP) specificity ranging from only recognizing their specific species XIP (Streptococcus mutans) to being able to be activated by almost any XIP (Streptococcus bovis) [23]
The ComR:Prx complexes elute as a single species with the exception of ComR S. thermophilus and Prx, which appear to form a larger aggregate near the 8 ml void volume of the column (Fig. S2C)

Summary

Introduction

The temperate bacteriophage and phage-like elements associated with S. pyogenes are dsDNA phage belonging to the Siphoviridae family [6, 7] This includes the lysogenic T12 and SF370 phages that can exist as a prophage within a bacterial host. There are often multiple prophages present within the genomes of each strain of S. pyogenes, many of which encode the deadly toxin and virulence genes characteristic of Group A Streptococcus (GAS) infections. For Gram-positive bacteria, natural competence (natural transformation) is a quorum-sensing regulated process in which bacteria control the expression of a number of genes that encode the machinery for both the acquisition and the incorporation of DNA [4, 11, 12]. Prx binds directly to the apo-form of ComR preventing DNA binding in vitro and acts as a repressor of the S. pyogenes competence regulon in vivo [18]. As our past work has shown Prx can interact with apoComR [18], Prx could be exerting its inhibitory effect to block any or multiple steps in the XIP-dependent activation of ComR

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