You will be presented with the abstract, introduction, and discussion section from a biomedical research article. Your task is to create a summary that a layperson can understand, capturing the essence of the research article. Keep essential scientific terms, but ensure that the language remains clear and the concepts are explained in an uncomplicated manner.

Title: Mapping the functional landscape of the receptor binding domain of T7 bacteriophage by deep mutational scanning}
Article:
Abstract:
The interaction between a bacteriophage and its host is mediated by the phage's receptor binding protein( RBP). Despite its fundamental role in governing phage activity and host range, molecular rules of RBP function remain a mystery. Here, we systematically dissect the functional role of every residue in the tip domain of T7 phage RBP( 1660 variants) by developing a high-throughput, locus-specific, phage engineering method. This rich dataset allowed us to cross compare functional profiles across hosts to precisely identify regions of functional importance, many of which were previously unknown. Substitution patterns showed host-specific differences in position and physicochemical properties of mutations, revealing molecular adaptation to individual hosts. We discovered gain-of-function variants against resistant hosts and host-constricting variants that eliminated certain hosts. To demonstrate therapeutic utility, we engineered highly active T7 variants against a urinary tract pathogen. Our approach presents a generalized framework for characterizing sequence–function relationships in many phage–bacterial systems.
Introduction:
Bacteriophages( or ‘phages’) shape microbial ecosystems by infecting and killing targeted bacterial species. As a result, they are promising tools for treatment of antibiotic-resistant bacterial infections and microbiome manipulation( Canfield and Duerkop, 2020; Chen et al., 2014; Clokie et al., 2011; Dedrick et al., 2019; Kilcher and Loessner, 2019; Kutter et al., 2015; Mizuno et al., 2020; Sausset et al., 2020; Schooley et al., 2017; Shen et al., 2015; Shkoporov and Hill, 2019; De Sordi et al., 2019). Interaction of phages with their bacterial receptors is a key determinant of their host range and virulence( Bertozzi Silva et al., 2016; de Jonge et al., 2019; Rousset et al., 2018). This interaction is primarily mediated by the receptor binding proteins( RBPs) of the phage( Nobrega et al., 2018). RBPs enable phages to adsorb to diverse cell surface molecules, including proteins, polysaccharides, lipopolysaccharides( LPS), and carbohydrate-binding moieties. Phages exhibit high functional plasticity through genetic alterations to RBPs and by natural and laboratory-guided evolution, which can modulate activity and host range to different hosts and environments( Ando et al., 2015; Chen et al., 2017; Dedrick et al., 2019; Dunne et al., 2019; Garcia et al., 2003; Gebhart et al., 2017; Holtzman et al., 2020; Lin et al., 2012; Meyer et al., 2012; Yehl et al., 2019; Yosef et al., 2017). In essence, survivability of a phage is intimately linked to the adaptability of its RBP. The challenge now is to understand the molecular code of RBPs in sufficient depth to enable predictable manipulation of host range and virulence. We sought to do so by combining deep mutational scanning( DMS) of the RBP with powerful selections on multiple hosts. Although RBPs remain the focus of many mechanistic, structural, and evolutionary studies and are a prime target for engineering, we currently lack a systematic and comprehensive understanding of how RBP mutations influence phage activity and host range. Though insightful, directed evolution enriches only a small group of ‘winners’, which makes it difficult to glean a comprehensive mutational landscape of the RBP( Holtzman et al., 2020). Random mutagenesis-based screens generate multi-mutant variants whose individual effects cannot be easily deconvolved( Dunne et al., 2019; Yehl et al., 2019). Other approaches including swapping homologous RBPs lead to gain of function; however, the underlying molecular determinants of function can be difficult to explain( Ando et al., 2015; Chen et al., 2017; Gebhart et al., 2017; Yosef et al., 2017). In summary, despite the extraordinary functional potential of phage RBPs, how systematic changes to their sequence shape the overall functional landscape of a phage remains unknown. Here, we carried out DMS, a high-throughput experimental technique, of the tip domain of the T7 phage RBP( tail fiber) to uncover molecular determinants of activity and host range. The tip domain is the distal region of the tail fiber that makes primary contact with the host receptor( González-García et al., 2015; Molineux, 2001; Qimron et al., 2006). We developed ORACLE( Optimized Recombination, Accumulation, and Library Expression), a high-throughput, locus-specific, phage genome engineering method to create a large, unbiased library of phage variants at a targeted gene locus. Using ORACLE, we systematically and comprehensively mutated the tip domain by making all single amino acid substitutions at every site( 1660 variants) and quantified the functional role of all variants on multiple bacterial hosts. We generated high-resolution functional maps delineating regions concentrated with function-enhancing substitutions and host-specific substitutional patterns, many of which were previously unknown. We discovered T7 variants with far greater virulence than wildtype T7, demonstrating that even those natural phages that are well adapted to a host can be engineered for higher efficacy. However, many variants highly adapted to one host performed poorly on others, underscoring a tradeoff between activity and host range. This functional screening highlights ideal regions of the tip domain for engineering host range. Furthermore, we demonstrated the functional potential of RBPs by discovering gain-of-function variants against resistant hosts and host-constriction variants that selectively eliminate specific hosts. To demonstrate the therapeutic value of ORACLE, we engineered T7 variants that avert emergence of spontaneous resistance in pathogenic Escherichia coli causing urinary tract infections( UTIs). Our study explains the molecular drivers of adaptability of the tip domain and identifies key functional regions determining activity and host range. ORACLE provides a generalized framework to describe sequence–function relationships in phages to elucidate the molecular basis of phages, the most abundant life form on earth.
Discussion:
In this study, we used ORACLE to create a large, unbiased library of T7 phage variants to comprehensively characterize the mutational landscape of the tip domain of the tail fiber. Our study identified hundreds of novel function-enhancing substitutions that had not been previously characterized. We mapped regions of function-enhancing substitutions on to the crystal structure to rationalize how sequence and structure influence activity and host range. Several important insights emerged from these results. Cross-comparison between different hosts and selection on resistant hosts allowed us to map key substitutions, leading to host discrimination and gain of function. Single amino acid substitutions are sufficient to enhance activity and host range, including some that confer dramatic increases in activity or specificity. The functional landscape on each host is unique, reflecting both different molecular preferences of adsorption and the fitness of wildtype T7 on these hosts. For instance, hydrophilic substitutions were enriched in 10G while hydrophobic substitutions were enriched in BW25113. Notably, substitutions on 10G( an E. coli K-12 derivative lacking LPS components) mirrored substitutions that recovered function on BW25113 mutants with truncated LPS, which shows convergence of selection. Function-enhancing substitutions were densely concentrated in the exterior loops, indicating an orientational preference for receptor recognition. However, they were also found on other surface residues, albeit less frequently, suggesting alternative binding modes of the tip domain for host recognition, and several intolerant residues were located in exterior loops. Taken together, these results highlight the extraordinary functional potential of the tip domain and rationalize the pervasive use of this structural fold in nature for molecular recognition. Comparison of these functional profiles precisely reveals the regions that are ideal engineering targets for customizing host range and activity and identifies intolerant residues that should be avoided when engineering synthetic phages. These results also highlight the power of deep sequencing to detect and resolve small functional effects over traditional low-throughput plaque assays. This is best illustrated in the case of truncated variants visible only to deep sequencing, but incapable of plaque formation without a helper plasmid. The truncated variants are likely not experimental artifacts as some ability to replicate is required to survive multiple rounds of selection on the host. Truncation of the tip domain may misorient the phage relative to the receptor, likely resulting in slower growth and deficiency in plaquing, while still capable of replicating. Since plaque formation is a complex process, inability to plaque may not imply a functionally incompetent phage. ORACLE is designed as a foundational technology to elucidate sequence–function relationships in phage genes. On T7, ORACLE can be used to investigate the function of several important genes, including the remainder of the tail fiber and tail structure, capsid components, or lysins and holins. Together, these will provide a comprehensive view of the molecular determinants of the structure, function, and evolution of a phage. Once the phage variants are created, scaling up ORACLE to investigate potentially tens of hosts merely scales up sequencing volume, not experimental complexity. Such a large-scale study will lead to a detailed molecular understanding and adaptability of phage bacterial interactions. Any phage with a sequenced genome and a transformable host capable of maintaining a plasmid library should be amenable to ORACLE because the phage variants are created during the natural infection cycle. This approach can be leveraged to tune activity for known phages with high activity, such as T7, or to identify engineering targets that dramatically increase activity for newly isolated natural phages. The confluence of genome engineering, high-throughput DNA synthesis, and sequencing enabled by ORACLE together with viral metagenomics could transform phage biology. Phages constitute unparalleled biological variation found in nature and are aptly called the ‘dark matter’ of the biosphere. Their sequence diversity and richness are coming to light in the growing volume of viral metagenome databases. However, what functions these sequences encode remains largely unknown. For instance, fecal viromes estimate 108–109 virus-like particles per gram of feces, but less than a quarter of sequence reads align to existing databases( Reyes et al., 2012). While this knowledge gap is daunting, it also presents an opportunity to mine metagenomic sequences to characterize their function and engineer programmable phages. By enabling sequence programmability, we envision ORACLE as a powerful tool to discover new phage ‘parts’ from metagenomic sequences.
Bacteria can cause diseases , but they also battle their own microscopic enemies: a group of viruses known as bacteriophages . For instance , the T7 bacteriophage preys on various strains of Escherichia coli , a type of bacteria often found in the human gut . While many E . coli strains are inoffensive or even beneficial to human health , some can be deadly . Finding a way to kill harmful strains while sparing the helpful ones would be a helpful addition to the medicine toolkit . Bacteriophages identify and interact with their specific target through a structure known as the receptor binding protein , or RBP . However , it is still unclear exactly how RBP helps the viruses recognize which type of bacteria to infect . Here , Huss et al . set to map out and modify this structure in T7 bacteriophage so the virus is more efficient and specific about which strain of E . coli it kills . First , the role of each building block in the tip of RBP was meticulously dissected; this generated the knowledge required to genetically engineer a large number of different T7 bacteriophages , each with a slightly variation in their RBP . These viruses were then exposed to various strains of bacteria . Monitoring the bacteriophages that survived and multiplied the most after infecting different strains of E . coli revealed which RBP building blocks are important for efficiency and specificity . This was then confirmed by engineering highly active T7 bacteriophage variants against an E . coli strain that causes urinary tract infections . These findings demonstrate that even small changes to the bacteriophages can make a big difference to their ability to infect their preys . The approaches developed by Huss et al . help to understand exactly how the RBP allows a virus to infect a specific type of bacteria; this could one day pave the way for new therapies that harness those viruses to fight increasingly resistant bacterial infections .