Stable Neutralization of a Virulence Factor in Bacteria Using Temperate Phage in the Mammalian Gut.

Bryan B. Hsu,Jeffrey C. Way,Pamela A. Silver,Jack A. Gilbert

doi:10.1128/msystems.00013-20

Bryan B. Hsu, Jeffrey C. Way + Show 2 more

Open Access

https://doi.org/10.1128/msystems.00013-20

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Abstract

Elimination or alteration of select members of the gut microbiota is key to therapeutic efficacy. However, the complexity of these microbial inhabitants makes it challenging to precisely target bacteria. Here, we deliver exogenous genes to specific bacteria by genomic integration of temperate phage for long-lasting modification. As a real-world therapeutic test, we engineered λ phage to transcriptionally repress Shiga toxin by using genetic hybrids between λ and other lambdoid phages to overcome resistance encoded by the virulence-expressing prophage. We show that a single dose of engineered phage propagates throughout the bacterial community and reduces Shiga toxin production in an enteric mouse model of infection without markedly affecting bacterial concentrations. Our work reveals a new framework for transferring functions to bacteria within their native environment.IMPORTANCE With the increasing frequency of antibiotic resistance, it is critical to explore new therapeutic strategies for treating bacterial infections. Here, we use a temperate phage, i.e., one that integrates itself into the bacterial genome, to neutralize the expression of a virulence factor by modifying bacterial function at the genetic level. We show that Shiga toxin production can be significantly reduced in vitro and in the mammalian gut. Alternative to traditional applications of phage therapy that rely on killing bacteria, our genetics-based antivirulence approach introduces a new framework for treating bacterial infections.

Highlights

Elimination or alteration of select members of the gut microbiota is key to therapeutic efficacy
We genetically engineer temperate phage ␭ to express a repressor that neutralizes Shiga toxin (Stx) production in E. coli and take advantage of the genetic mosaicism of lambdoid phages to create a hybrid phage that is capable of overcoming phage resistance mechanisms
We found that our antivirulence phage efficiently infects, lysogenizes, and inhibits Stx2 production from E. coli in vitro but is effective at propagating in the murine gut from a single dose to significantly reduce Stx2 production in vivo

Summary

Introduction

Elimination or alteration of select members of the gut microbiota is key to therapeutic efficacy. Alternative to traditional applications of phage therapy that rely on killing bacteria, our genetics-based antivirulence approach introduces a new framework for treating bacterial infections. The human gut microbiota is a collection of microbes colonizing the gastrointestinal tract and has been associated with various aspects of human health [1] While this community typically works in concert with our bodies, substantial perturbations such as antibiotics or infections can disrupt the microbial balance and lead to long-lasting dysbiosis [2]. Antibiotics nonspecifically decimate swaths of gut species [7], dietary changes affect both the overall microbiota and the mammalian host, probiotics poorly engraft due to colonization resistance [8], and even highly specific lytic phages can cause unintended changes in the bacterial community despite targeting specific species [9]. While the principle of antivirulence is attractive, it remains challenging in application

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