Enhancing bacteriophage therapeutics throughin situproduction and release of heterologous antimicrobial effectors

Abstract

Bacteriophages operate via pathogen-specific mechanisms of action distinct from conventional, broad-spectrum antibiotics and are emerging as promising alternatives. However, phage-mediated killing is often limited by bacterial resistance development (1,2). Here, we engineer phages for target-specific effector gene delivery and host-dependent production of colicin-like bacteriocins and cell wall hydrolases. Using urinary tract infection (UTI) as a model, we show how heterologous effector phage therapeutics (HEPTs) suppress resistance and improve uropathogen killing by dual phage- and effector-mediated targeting. Moreover, we designed HEPTs to control polymicrobial uropathogen communities through production of effectors with cross-genus activity. Using a phage-based companion diagnostic (3), we identified potential HEPT responder patients and treated their urine ex vivo. Compared to wildtype phage, a colicin E7-producing HEPT demonstrated superior control of patient E. coli bacteriuria. Arming phages with heterologous effectors paves the way for successful UTI treatment and represents a versatile tool to enhance and adapt phage-based precision antimicrobials.

Construction and Characterization of T7 Bacteriophages Harboring Apidaecin-Derived Sequences

“…Since apidaecin and its analogs inhibit protein translation by targeting the 70S ribosome [ 26 ], we expected an effect on phage production. This effect could reduce phage-mediated lysis but might also reduce the emergence and growth of phage-resistant bacteria, which frequently happens after the initial lysis phase [ 27 , 28 , 29 ]. Phage-mediated lysis started 2 h after phage addition and regrowth of an E. coli Rosetta DE3 pLysS population, which was potentially phage-resistant, was observed with a maximal growth 24 h after the initial lysis in growth experiments with T7select phage without the addition of apidaecin ( Figure 2 B).…”

Engineered reporter phages for rapid detection ofEscherichia coli, Klebsiellaspp., andEnterococcusspp. in urine

“…In this study, we developed technologies to modify our phage candidates and provide the first genome engineering tool for virulent Enterococcus phages. Using this pipeline, we have recently created the next generation of phage-based UTI therapeutics that enhance uropathogen killing through phage-mediated delivery of bacteriocins as antimicrobial effectors (heterologous effector phage therapeutics, HEPTs) [40]. Prior to HEPT treatment, E2 ::nluc was used to identify urine samples from responder patients, showcasing how reporter phage diagnostics could be combined with engineered phage therapy in the future to realize the full potential of phage-based precision medicine.…”

L-form conversion in Gram-positive bacteria enables escape from phage infection

“…On the basis of our observation of phage-induced L-form switching, we asked whether this process may also be relevant under conditions found in a natural environment. Phage therapy is currently developed as a treatment option for several pathogens causing urinary tract infections, including E. faecalis [41][42][43] . It has recently been shown that urine provides the necessary osmoprotection to enable L-form switching and survival 17 .…”