Genetic Engineering and Rebooting of Bacteriophages in L-Form Bacteria.

The rapid increase of circulating, antibiotic-resistant pathogens is a major ongoing global health crisis, and arguably, the end of the "golden age of antibiotics" is looming. This has led to a surge in research and development of alternative antimicrobials, including bacteriophages, to treat such infections (phage therapy). Isolating natural phage variants for the treatment of individual patients is an arduous and time-consuming task. Furthermore, the use of natural phages is frequently hampered by natural limitations, such as moderate in vivo activity, the rapid emergence of resistance, insufficient host range, or the presence of undesirable genetic elements within the phage genome. Targeted genetic editing of wild-type phages (phage engineering) has successfully been employed in the past to mitigate some of these pitfalls and to increase the therapeutic efficacy of the underlying phage variants. Clearly, there is a large potential for the development of novel, marker-less genome-editing methodologies to facilitate the engineering of therapeutic phages. Steady advances in synthetic biology have facilitated the in vitro assembly of modified phage genomes, which can be activated ("rebooted") upon transformation of a suitable host cell. However, this can prove challenging, especially in difficult-to-transform Gram-positive bacteria. In this chapter, we detail the production of cell wall-deficient L-form bacteria and their application to activate synthetic genomes of phages infecting Gram-positive host species.

Enhancing bacteriophage therapeutics through in situ production and release of heterologous antimicrobial effectors.

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.

Engineered reporter phages for detection of Escherichia coli, Enterococcus, and Klebsiella in urine.

The rapid detection and species-level differentiation of bacterial pathogens facilitates antibiotic stewardship and improves disease management. Here, we develop a rapid bacteriophage-based diagnostic assay to detect the most prevalent pathogens causing urinary tract infections: Escherichia coli, Enterococcus spp., and Klebsiella spp. For each uropathogen, two virulent phages were genetically engineered to express a nanoluciferase reporter gene upon host infection. Using 206 patient urine samples, reporter phage-induced bioluminescence was quantified to identify bacteriuria and the assay was benchmarked against conventional urinalysis. Overall, E. coli, Enterococcus spp., and Klebsiella spp. were each detected with high sensitivity (68%, 78%, 87%), specificity (99%, 99%, 99%), and accuracy (90%, 94%, 98%) at a resolution of ≥103 CFU/ml within 5 h. We further demonstrate how bioluminescence in urine can be used to predict phage antibacterial activity, demonstrating the future potential of reporter phages as companion diagnostics that guide patient-phage matching prior to therapeutic phage application.

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

At the end of a lytic bacteriophage replication cycle in Gram-positive bacteria, peptidoglycan-degrading endolysins that cause explosive cell lysis of the host can also attack non-infected bystander cells. Here we show that in osmotically stabilized environments, Listeria monocytogenes can evade phage predation by transient conversion to a cell wall-deficient L-form state. This L-form escape is triggered by endolysins disintegrating the cell wall from without, leading to turgor-driven extrusion of wall-deficient, yet viable L-form cells. Remarkably, in the absence of phage predation, we show that L-forms can quickly revert to the walled state. These findings suggest that L-form conversion represents a population-level persistence mechanism to evade complete eradication by phage attack. Importantly, we also demonstrate phage-mediated L-form switching of the urinary tract pathogen Enterococcus faecalis in human urine, which underscores that this escape route may be widespread and has important implications for phage- and endolysin-based therapeutic interventions.

Engineering therapeutic phages for enhanced antibacterial efficacy.

The alarming rise in antimicrobial resistance coupled with a lack of innovation in antibiotics has renewed interest in the development of alternative therapies to combat bacterial infections. Despite phage therapy demonstrating success in various individual cases, a comprehensive and unequivocal demonstration of the therapeutic potential of phages remains to be shown. The co-evolution of phages and their bacterial hosts resulted in several inherent limitations for the use of natural phages as therapeutics such as restricted host range, moderate antibacterial efficacy, and frequent emergence of phage-resistance. However, these constraints can be overcome by leveraging recent advances in synthetic biology and genetic engineering to provide phages with additional therapeutic capabilities, improved safety profiles, and adaptable host ranges. Here, we examine different ways phages can be engineered to deliver heterologous therapeutic payloads to enhance their antibacterial efficacy and discuss their versatile applicability to combat bacterial pathogens.

Engineering of Long-Circulating Peptidoglycan Hydrolases Enables Efficient Treatment of Systemic Staphylococcus aureus Infection.

Staphylococcus aureus is a human pathogen causing life-threatening diseases. The increasing prevalence of multidrug-resistant S. aureus infections is a global health concern, requiring development of novel therapeutic options. Peptidoglycan-degrading enzymes (peptidoglycan hydrolases, PGHs) have emerged as a highly effective class of antimicrobial proteins against S. aureus and other pathogens. When applied to Gram-positive bacteria, PGHs hydrolyze bonds within the peptidoglycan layer, leading to rapid bacterial death by lysis. This activity is highly specific and independent of the metabolic activity of the cell or its antibiotic resistance patterns. However, systemic application of PGHs is limited by their often low activity in vivo and by an insufficient serum circulation half-life. To address this problem, we aimed to extend the half-life of PGHs selected for high activity against S. aureus in human serum. Half-life extension and increased serum circulation were achieved through fusion of PGHs to an albumin-binding domain (ABD), resulting in high-affinity recruitment of human serum albumin and formation of large protein complexes. Importantly, the ABD-fused PGHs maintained high killing activity against multiple drug-resistant S. aureus strains, as determined by ex vivo testing in human blood. The top candidate, termed ABD_M23, was tested in vivo to treat S. aureus-induced murine bacteremia. Our findings demonstrate a significantly higher efficacy of ABD_M23 than of the parental M23 enzyme. We conclude that fusion with ABD represents a powerful approach for half-life extension of PGHs, expanding the therapeutic potential of these enzybiotics for treatment of multidrug-resistant bacterial infections.IMPORTANCE Life-threatening infections with Staphylococcus aureus are often difficult to treat due to the increasing prevalence of antibiotic-resistant bacteria and their ability to persist in protected niches in the body. Bacteriolytic enzymes are promising new antimicrobials because they rapidly kill bacteria, including drug-resistant and persisting cells, by destroying their cell wall. However, when injected into the bloodstream, these enzymes are not retained long enough to clear an infection. Here, we describe a modification to increase blood circulation time of the enzymes and enhance treatment efficacy against S. aureus-induced bloodstream infections. This was achieved by preselecting enzyme candidates for high activity in human blood and coupling them to serum albumin, thereby preventing their elimination by kidney filtration and blood vessel cells.

Reporter Phage-Based Detection of Bacterial Pathogens: Design Guidelines and Recent Developments.

Fast and reliable detection of bacterial pathogens in clinical samples, contaminated food products, and water supplies can drastically improve clinical outcomes and reduce the socio-economic impact of disease. As natural predators of bacteria, bacteriophages (phages) have evolved to bind their hosts with unparalleled specificity and to rapidly deliver and replicate their viral genome. Not surprisingly, phages and phage-encoded proteins have been used to develop a vast repertoire of diagnostic assays, many of which outperform conventional culture-based and molecular detection methods. While intact phages or phage-encoded affinity proteins can be used to capture bacteria, most phage-inspired detection systems harness viral genome delivery and amplification: to this end, suitable phages are genetically reprogrammed to deliver heterologous reporter genes, whose activity is typically detected through enzymatic substrate conversion to indicate the presence of a viable host cell. Infection with such engineered reporter phages typically leads to a rapid burst of reporter protein production that enables highly sensitive detection. In this review, we highlight recent advances in infection-based detection methods, present guidelines for reporter phage construction, outline technical aspects of reporter phage engineering, and discuss some of the advantages and pitfalls of phage-based pathogen detection. Recent improvements in reporter phage construction and engineering further substantiate the potential of these highly evolved nanomachines as rapid and inexpensive detection systems to replace or complement traditional diagnostic approaches.

Import of Aspartate and Malate by DcuABC Drives H2/Fumarate Respiration to Promote Initial Salmonella Gut-Lumen Colonization in Mice.

Initial enteropathogen growth in the microbiota-colonized gut is poorly understood. Salmonella Typhimurium is metabolically adaptable and can harvest energy by anaerobic respiration using microbiota-derived hydrogen (H2) as an electron donor and fumarate as an electron acceptor. As fumarate is scarce in the gut, the source of this electron acceptor is unclear. Here, transposon sequencing analysis along the colonization trajectory of S. Typhimurium implicates the C4-dicarboxylate antiporter DcuABC in early murine gut colonization. In competitive colonization assays, DcuABC and enzymes that convert the C4-dicarboxylates aspartate and malate into fumarate (AspA, FumABC), are required for fumarate/H2-dependent initial growth. Thus, S. Typhimurium obtains fumarate by DcuABC-mediated import and conversion of L-malate and L-aspartate. Fumarate reduction yields succinate, which is exported by DcuABC in exchange for L-aspartate and L-malate. This cycle allows S. Typhimurium to harvest energy by H2/fumarate respiration in the microbiota-colonized gut. This strategy may also be relevant for commensal E. coli diminishing the S. Typhimurium infection.

Engineered Reporter Phages for Rapid Bioluminescence-Based Detection and Differentiation of Viable Listeria Cells.

The pathogen Listeria monocytogenes causes listeriosis, a severe foodborne disease associated with high mortality. Rapid and sensitive methods are required for specific detection of this pathogen during food production. Bioluminescence-based reporter bacteriophages are genetically engineered viruses that infect their host cells with high specificity and transduce a heterologous luciferase gene whose activity can be detected with high sensitivity to indicate the presence of viable target cells. Here, we use synthetic biology for de novo genome assembly and activation as well as CRISPR-Cas-assisted phage engineering to construct a set of reporter phages for the detection and differentiation of viable Listeria cells. Based on a single phage backbone, we compare the performance of four reporter phages that encode different crustacean, cnidarian, and bacterial luciferases. From this panel of reporter proteins, nanoluciferase (NLuc) was identified as a superior enzyme and was subsequently introduced into the genomes of a broad host range phage (A511) and two serovar 1/2- and serovar 4b/6a-specific Listeria phages (A006 and A500, respectively). The broad-range NLuc-based phage A511::nlucCPS detects one CFU of L. monocytogenes in 25 g of artificially contaminated milk, cold cuts, and lettuce within less than 24 h. In addition, this reporter phage successfully detected Listeria spp. in potentially contaminated natural food samples without producing false-positive or false-negative results. Finally, A006::nluc and A500::nluc enable serovar-specific Listeria diagnostics. In conclusion, these NLuc-based reporter phages enable rapid, ultrasensitive detection and differentiation of viable Listeria cells using a simple protocol that is 72 h faster than culture-dependent approaches.IMPORTANCE Culture-dependent methods are the gold standard for sensitive and specific detection of pathogenic bacteria within the food production chain. In contrast to molecular approaches, these methods detect viable cells, which is a key advantage for foods generated from heat-inactivated source material. However, culture-based diagnostics are typically much slower than molecular or proteomic strategies. Reporter phage assays combine the best of both worlds and allow for near online assessment of microbial safety because phage replication is extremely fast, highly target specific, and restricted to metabolically active host cells. In addition, reporter phage assays are inexpensive and do not require highly trained personnel, facilitating their on-site implementation. The reporter phages presented in this study not only allow for rapid detection but also enable an early estimation of the potential virulence of Listeria isolates from food production and processing sites.

Corrected and Republished from: Identification of Peptidoglycan Hydrolase Constructs with Synergistic Staphylolytic Activity in Cow's Milk.

Peptidoglycan hydrolases (PGHs) have been suggested as novel therapeutics for the treatment of bovine mastitis. However, activity in the presence of cow's milk is an important requirement for drugs administered into the bovine udder. We have used a microtiter plate-based protocol to screen a library of >170 recombinant PGHs, including engineered bacteriophage endolysins, for enzymes with activity against Staphylococcus aureus in milk. Eight suitable PGH constructs were identified by this approach, and their efficacies against S. aureus in heat-treated milk were compared by time-kill assays. The two most active enzymes (lysostaphin and CHAPK_CWT-LST) reduced S. aureus numbers in milk to undetectable levels within minutes at nanomolar concentrations. Due to their different peptidoglycan cleavage sites, these PGH constructs revealed synergistic activity, as demonstrated by checkerboard assays, spot assays, and time-kill experiments. Furthermore, they proved active against a selection of staphylococcal mastitis isolates from different geographical regions when applied individually or in synergistic combination. The PGH combination completely eradicated S. aureus from milk: no more bacteria were detected within 24 h after the addition of the enzymes, corresponding to a reduction of >9 log units from the level in the control. Efficacy was also retained at different inoculum levels (3 log versus 6 log CFU/ml) and when S. aureus was grown in milk as opposed to broth prior to the experiments. In raw cow's milk, CHAPK_CWT-LST showed reduced efficacy, whereas lysostaphin retained its activity, reducing bacterial numbers by >3.5 log units within 3 h.IMPORTANCE Staphylococci, and S. aureus in particular, are a major cause of bovine mastitis, an inflammation of the mammary gland in cows that is associated with high costs and risks for consumers of milk products. S. aureus-induced mastitis, commonly treated by intramammary infusion of antibiotics, is characterized by low cure rates and increasing antibiotic resistance in bacteria. Therefore, alternative treatment options are highly desirable. PGHs, including bacteriophage endolysins, rapidly and specifically kill selected pathogens by degrading their cell walls and are refractory to resistance development; thus, they have promise as novel antibacterial agents. This study employed a screening approach to identify PGH constructs with high staphylolytic activity in cow's milk among a large collection of enzymes. Our results suggest that the most promising enzymes identified by this strategy hold potential as novel mastitis therapeutics and thus support their further characterization in animal models.

Identification of Peptidoglycan Hydrolase Constructs with Synergistic Staphylolytic Activity in Cow's Milk.

Peptidoglycan hydrolases (PGHs) have been suggested as novel therapeutics for the treatment of bovine mastitis. However, activity in the presence of cow's milk is an important requirement for drugs administered into the bovine udder. We have screened a library of >170 recombinant PGHs, including engineered bacteriophage endolysins, for enzymes with activity against Staphylococcus aureus in milk, using a microtiter plate-based protocol. Nine suitable PGH constructs were identified by this approach and further compared in time-kill assays for their efficacy against S. aureus in heat-treated milk. The three most active enzymes (lysostaphin, Ami2638A, and CHAPK_CWT-LST) reduced S. aureus in milk to undetectable numbers within minutes at nanomolar concentrations. Due to their different peptidoglycan cleavage sites, these PGH constructs revealed synergistic activity in most combinations, as demonstrated by checkerboard assays, spot assays, and time-kill experiments. Furthermore, they proved active against a selection of staphylococcal mastitis isolates from different geographical regions when applied individually or in synergistic combination. The most effective PGH combination completely eradicated S. aureus from milk, with no more bacteria being detected within 24 h after addition of the enzymes, corresponding to a reduction of >9 log units compared to the control. Efficacy was also retained at different inoculum levels (3 versus 6 log CFU/ml) and when S. aureus was grown in milk as opposed to broth prior to the experiments. In raw cow's milk, CHAPK_CWT-LST showed reduced efficacy, whereas both Ami2638A and lysostaphin retained their activity, reducing bacterial numbers by >3.5 log units within 3 h.IMPORTANCE Staphylococci and S. aureus in particular are a major cause of bovine mastitis, an inflammation of the mammary gland in cows associated with high costs and risks for consumers of milk products. S. aureus-induced mastitis, commonly treated by intramammary infusion of antibiotics, is characterized by low cure rates and increasing antibiotic resistance in bacteria. Therefore, alternative treatment options are highly desirable. PGHs, including bacteriophage endolysins, rapidly and specifically kill selected pathogens by degrading their cell wall and are refractory to resistance development, therefore holding promise as novel antibacterial agents. This study employed a screening approach to identify PGH constructs with high staphylolytic activity in cow's milk within a large collection of enzymes. Our results suggest that the most promising enzymes identified by this strategy hold potential as novel mastitis therapeutics and support their further characterization in animal models.