Enhancing bacteriophage therapeutics through in situ production 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 alternative antimicrobials. However, phage-mediated killing is often limited by bacterial resistance development. 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 phage-based companion diagnostics, 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.

CRISPR-Cas9 enables efficient genome engineering of the strictly lytic, broad host-range staphylococcal bacteriophage K

“…Utilizing existing data on genome structure and transcriptomic profiles, we identified a region associated with the major capsid protein ( cps ) as a favorable locus for payload insertion. This area has been previously demonstrated to be highly expressed in phage K [59], and it has been a common choice for payload insertion in other phages [21, 31, 42]. To mediate expression from a strong, endogenous promoter, the reporter gene and a ribosomal binding site were integrated immediately downstream of the cps coding sequence.…”

“…The plasmid pLEB579 (kindly gifted by T. Takala, University of Helsinki, Finland) is a shuttle vector shown to have high transformation efficiencies in both E. coli and S. aureus and was therefore used as a backbone for both the editing template (pEDIT nluc ) and the CRISPR-Cas9-counterselection system (pSELECT CPS ). We used a previously reported, Streptococcus pyogenes -derived Cas9 (SpyCas9)-based CRISPR system [21] and exchanged the two spacers in the repeat-spacer1-repeat-spacer2-repeat (RS1RS2R) region. This was done to allow targeted restriction of wildtype phage K at two distinct loci (8304 bp and 148 bp up- and downstream of the intended insertion site, respectively) designed to contain a PAM-disrupting synonymous mutation in the successful recombinants.…”

“…Through the strategic selection and engineering of the phage backbone, we can finetune crucial characteristics, such as host specificity, to cater to specific clinical applications. These methods have been employed to transition phages to entirely new hosts [16–19], modify the infection cycle for clinical suitability [20], and deliver antimicrobial payload genes directly to the infection site [21]. The field of synthetic biology-based engineering is also expanding, and it now encompasses a variety of sophisticated DNA manipulation techniques.…”

“…The efficacy of this approach has been validated by its successful application in other phage-host systems. [21, 43–48].…”

A bacterial genome assembly and annotation laboratory using a virtual machine

“…High‐throughput methods to understand how bacterial hosts become resistant to phage are currently making good progress in this area 7 . Phage engineering is focused on creating enhanced phages that may be able to infect a broader range of hosts or actively mount counter‐defenses against host defense systems 8–10 …”