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

Abstract

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.

Keywords: CRISPR-Cas editing; bacterial detection; bacteriophage; genetic engineering; luciferase; reporter phage.

Figure 1

Overview of phage-based pathogen detection. Capture-based detection: The limited host ranges of phages towards a given genus, species or subspecies of bacteria make them ideal candidates for development into detection bio-probes. (A) The high binding affinity of whole-phage particles has led to their use as bio-probes in biosensors [28,29,30], or by conjugation with radioactive tracers [43] fluorophores [44], magnetic nanoparticles [31,45] or a combination of both [46] to label and enrich bacteria for detection. (B) Host specificity of phages is mediated by specialized receptor-binding proteins (RBPs) that provide equivalent binding capabilities as whole phages, but at a fraction of the size. Recently, RBPs have been applied in biosensors [47], ELISA-based assays [38,40], and for glycotyping Salmonella [39] and Listeria [32]. (C) Alternatively, cell wall-binding domains (CBDs) of phage endolysins have proven highly effective at detecting Gram-positive pathogens. CBDs have recently been used to detect B. cereus using biosensors [34] lateral flow assays [48] and magnetic enrichment-based detection [49]. In addition, Listeria-targeting CBDs were used to glycotype and identify Listeria serovars [42] and Clostridium tyrobutyricum-targeting CBDs have been employed for spore detection during cheese spoilage [35,50]. Infection-based detection: Infection of a bacterial host by lytic phage leads to rapid progeny phage production and ensuing cell lysis. (D) Released progeny phages [51,52] or bacterial cell content (e.g., ATP, DNA, RNA and bacterial proteins) provide excellent markers for downstream detection of the original bacterial host [53,54,55]. Alternatively, genetically engineered phages encoding reporters such as fluorescent proteins (E) [56,57,58], luciferases (F) [59,60,61,62] or hydrolyzing enzymes (e.g., β-galactosidase) (G) [63,64] are used. These phages express the reporter proteins during host infection to produce an amplifying fluorescent or bioluminescent signal upon the addition of a substrate. The rapid and sensitive nature of reporter phage-based systems has made them ideal tools for detecting low levels of viable, contaminating bacteria in many matrices, including foods, water and clinical samples.

Figure 2

Overview of engineering strategies for reporter phage generation. (A) Heterogeneous reporter genes can be inserted into dual function shuttle phasmids capable of plasmid replication in E. coli and phage replication in a target host, e.g., Mycobacterium spp. (B) Reporter genes can also be directly inserted into phage genomes, e.g., T7Select^®(Novagen). (C) Homologous recombination (and recombineering with bacteriophage-encoded homologous recombination systems, such as the coliphage λ Red system) involves the swapping of a reporter gene containing homology arms featured on an editing plasmid with the homologous region located within a phage genome. Homologous recombination can be combined with CRISPR-Cas counter selection to facilitate the removal of wild-type phages to improve identification of recombinant phages. (D) PCR products of a phage genome and a reporter gene insert are transformed alongside a linearized yeast replicon fragment from a yeast artificial chromosome (YAC) into yeast cells. The phage genome is assembled in the YAC vector by gap-repair cloning. YAC–phage DNA is extracted and directly transformed into the host bacterium leading to the production of recombinant phages. (E) A synthetic genome can be in vitro assembled (e.g., using Gibson assembly) from PCR fragments featuring a reporter gene insert. The synthetic genome can be transformed into L-form bacteria leading to genome activation and the release of viable phage for subsequent propagation on the phage host.