Engineering bacteriophage construction platform based on T4gp10-like structural domain

Abstract

The invention provides an engineered bacteriophage as well as a construction method and application thereof. The engineered bacteriophage comprises a first receptor binding protein (RBP), wherein the N-terminal of the first receptor binding protein comprises an anchoring domain; and optionally a second receptor binding protein, wherein the N-terminal of the second receptor binding protein is bound to the first receptor binding protein through the anchor domain. According to the modular assembly platform of the RBP, the RBP is freely switched and combined between the bacteriophages, and accurate customization of the host range of the bacteriophages is successfully achieved. Meanwhile, the method is applied to a plug-and-play engineered bacteriophage screening platform for realizing RBP, an engineered bacteriophage mixture modified only on the protein level can be quickly obtained without any editing of a bacteriophage genome, and the host range of customized bacteriophages can be effectively expanded or obtained to cope with multiple drug-resistant bacteria; and a new solution is provided for developing a phage preparation with high biological safety.

Description

An engineered phage construction platform based on T4gp10-like structural domains Technical Field

[0001] This invention relates to the field of bioengineering technology, and in particular to the construction and application of a plug-and-play engineered bacteriophage. Background Technology

[0002] Bacteriophages, as specific antibacterial agents, have a significant advantage over antibiotics in that they do not disrupt the symbiotic bacterial community. However, it is generally believed that although there are abundant bacteriophage resources in the environment, their direct use in clinical treatment still faces insurmountable obstacles. The diversity of bacterial receptors limits the host range of natural bacteriophages and makes them prone to drug resistance, making it difficult to meet the stringent clinical standards for safety, efficacy, and broad spectrum. Engineered bacteriophages offer an effective solution to these problems. Through the rational design and modification of bacteriophages using molecular biology and synthetic biology techniques, not only can the safety risks of natural bacteriophages be effectively avoided, but they can also be endowed with novel properties such as overcoming host defenses, expanding host range, and efficiently delivering antibacterial payloads.

[0003] The rational design of engineered bacteriophages still relies on the understanding of receptor-binding protein-mediated molecular interactions—interactions that determine the host specificity of bacteriophages by recognizing bacterial surface receptors. Currently, the vast majority of reported bacteriophages contain only a single receptor-binding protein (RBP). However, some members of the Podoviridae family have evolved two distinct RBPs, thus possessing dual receptor recognition specificity. For example, Klebsiella pneumoniae bacteriophages K5-2 and KP32 both carry two RBPs, whose depolymerase domains exhibit differentiated enzymatic characteristics, enabling them to recognize hosts with different capsule types. In structural assembly, typically the N-terminal domain of the first RBP is anchored to the phage substrate, while the second RBP does not directly connect to the substrate but binds to the T4gp10-like domain on the first RBP via its N-terminus, together forming a composite anchoring platform. Theoretically, this unique T4gp10-like domain could serve as an ideal modular connection platform for the assembly and functional demonstration of RBPs from different sources. Current techniques for genetic engineering bacteriophages largely involve whole or partial homologous recombination and mutation between different bacteriophages with high tail structure and gene similarity, thereby altering their host range. This includes complete or partial exchange of tail components and targeted modification of bacteriophage receptor-binding proteins. However, existing techniques have seen almost no exploration or extension into the linker modules on receptor-binding proteins, particularly in-depth research on the T4 gp10-like domain with its anchoring domain structure. Furthermore, there are no reports to date of using this structure as a universal interface for bacteriophage engineering. Summary of the Invention

[0004] To address one of the aforementioned technical problems in existing technologies, this invention focuses on phages of the genus *Przondovirus* within the family Klebsiella pneumoniae. It has been confirmed that the anchoring domain, particularly the T4 gp10-like domain, is not only highly conserved evolutionarily but also widely distributed across phages of this genus, serving as a universal "molecular interface." Utilizing this structural feature, this invention establishes a modular assembly platform for RBPs, successfully achieving precise customization of the phage host range through the free switching and combination of RBPs among phages of the same genus.

[0005] Furthermore, to address the challenges of construction complexity and safety associated with traditional genetically engineered bacteriophages, this invention presents an assembly strategy based on exogenous plasmids providing RBPs. The method of this invention allows for the rapid acquisition of engineered bacteriophage mixtures modified only at the protein level without any editing of the bacteriophage genome.

[0006] In a first aspect, the present invention provides an engineered bacteriophage comprising: a first receptor-binding protein (RBP), the N-terminus of which includes an anchoring domain; and optionally a second receptor-binding protein, the N-terminus of which binds to the first receptor-binding protein via the anchoring domain; wherein the C-terminus of the first receptor-binding protein is the same as or different from the C-terminus of the second receptor-binding protein, and part or all of the first receptor-binding protein and / or part or all of the second receptor-binding protein are engineered to specifically target selected host bacteria.

[0007] In some embodiments, portions of the first receptor-binding protein and / or the second receptor-binding protein are capable of specifically targeting selected host bacteria.

[0008] In some embodiments, the anchoring structural domain is a T4 gp10-type structural domain.

[0009] In some embodiments, the engineered bacteriophage is selected from the Myotail Phage Family, Longtail Phage Family, or Shorttail Phage Family.

[0010] In some preferred embodiments, the engineered bacteriophage is from the family Brachyphageidae.

[0011] In some embodiments, the selected host bacterium is a selected capsular serotype (KL) of Klebsiella pneumoniae.

[0012] In some embodiments, KL is selected from one or more of KL1 to KL82 and KL101 to KL169. In some preferred embodiments, KL is selected from one or more of KL3, KL8, KL11, KL20, KL25, KL30, KL43, KL47, KL57, KL64, KL69, KL71, KL101, KL103, KL104, KL107, KL110, KL111, KL112, KL132, and KL169.

[0013] In some embodiments, the anchoring structural domain comprises a three-layer β-fold structure.

[0014] In some embodiments, the anchoring domain comprises the amino acid sequence shown in SEQ ID No: 1-4 or a variant thereof.

[0015] In some implementations, the variant includes the same three-layer β-fold structure as the anchoring structural domain.

[0016] In some embodiments, the variant comprises an amino acid sequence having at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with the amino acid sequences shown in SEQ ID No: 1-4.

[0017] In some embodiments, the variant comprises an amino acid sequence having a spatial structural similarity™ score of at least 0.70, at least 0.75, at least 0.80, at least 0.85, at least 0.90, at least 0.95, at least 0.96, at least 0.97, at least 0.98, or at least 0.99 with respect to the amino acid sequences shown in SEQ ID No: 1-4.

[0018] In some embodiments, the N-terminus of the second receptor-binding protein contains the amino acid sequence shown in SEQ ID No: 5-6 or an amino acid sequence having at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with the amino acid sequence shown in SEQ ID No: 5-6.

[0019] In some embodiments, the N-terminus of the second receptor-binding protein comprises an amino acid sequence with a spatial structural similarity TM score of at least 0.70, at least 0.75, at least 0.80, at least 0.85, at least 0.90, at least 0.95, at least 0.96, at least 0.97, at least 0.98, or at least 0.99 with respect to the amino acid sequences shown in SEQ ID No: 5-6.

[0020] In some embodiments, the binding force between the N-terminus of the second receptor-binding protein and the anchoring domain is selected from charge complementarity and / or hydrophobic interactions. In this case, the tertiary spatial structure of the N-terminus of the second receptor-binding protein and the anchoring domain is key to their binding. Therefore, the structural similarity TM score of different N-termini of the second receptor-binding protein or the anchoring domain is more important than the sequence identity of their primary amino acid sequences. The anchoring domains involved in this invention comprise amino acid sequences as shown in SEQ ID No: 1-4 or variants thereof, wherein the amino acid sequences shown in SEQ ID No: 1-4 share approximately 50% identity, but their spatial structural similarity TM scores are greater than 0.90, and all can bind to the N-terminus of the second receptor-binding protein. Similarly, the N-terminus of the second receptor-binding protein involved in this invention comprises amino acid sequences as shown in SEQ ID No: 5-6, wherein the amino acid sequences shown in SEQ ID No: 5-6 share approximately 80% identity, their spatial structural similarity TM scores are greater than 0.90, and all can bind to the anchoring domain of the first receptor-binding protein.

[0021] In a second aspect, the present invention provides a method for constructing engineered bacteriophages, comprising: a) providing an initial bacteriophage, the initial bacteriophage comprising an initial first receptor-binding protein, wherein the N-terminus of the initial first receptor-binding protein comprises an anchoring domain; and b) engineering part or all of the initial first receptor-binding protein to specifically target a selected host bacterium.

[0022] In some embodiments, the method includes: a) providing an initial phage comprising an initial first receptor-binding protein and an initial second receptor-binding protein, wherein the N-terminus of the initial first receptor-binding protein includes an anchoring domain, and the N-terminus of the second receptor-binding protein binds to the first receptor-binding protein through the anchoring domain; b) engineering part or all of the initial first receptor-binding protein and / or part or all of the initial second receptor-binding protein to specifically target selected host bacteria.

[0023] In some embodiments, the anchoring structural domain is a T4 gp10-type structural domain.

[0024] In some embodiments, the engineered bacteriophage is selected from the Myotail Phage Family, Longtail Phage Family, or Shorttail Phage Family.

[0025] In some preferred embodiments, the engineered bacteriophage is from the family Brachyphageidae.

[0026] In some embodiments, the anchoring structural domain comprises a three-layer β-fold structure.

[0027] In some embodiments, the anchoring domain comprises the amino acid sequence shown in SEQ ID No: 1-4 or a variant thereof.

[0028] In some implementations, the variant includes the same three-layer β-fold structure as the anchoring structural domain.

[0029] In some embodiments, the variant comprises an amino acid sequence having at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with the amino acid sequences shown in SEQ ID No: 1-4.

[0030] In some embodiments, the variant comprises an amino acid sequence having a spatial structural similarity™ score of at least 0.70, at least 0.75, at least 0.80, at least 0.85, at least 0.90, at least 0.95, at least 0.96, at least 0.97, at least 0.98, or at least 0.99 with respect to the amino acid sequences shown in SEQ ID No: 1-4.

[0031] In some embodiments, the N-terminus of the second receptor-binding protein contains the amino acid sequence shown in SEQ ID No: 5-6 or an amino acid sequence having at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with the amino acid sequence shown in SEQ ID No: 5-6.

[0032] In some embodiments, the N-terminus of the second receptor-binding protein comprises an amino acid sequence with a spatial structural similarity TM score of at least 0.70, at least 0.75, at least 0.80, at least 0.85, at least 0.90, at least 0.95, at least 0.96, at least 0.97, at least 0.98, or at least 0.99 with respect to the amino acid sequences shown in SEQ ID No: 5-6.

[0033] In some embodiments, the selected host bacterium is a selected capsular serotype (KL) of Klebsiella pneumoniae.

[0034] In some embodiments, KL is selected from one or more of KL1 to KL82 and KL101 to KL169. In some preferred embodiments, KL is selected from one or more of KL3, KL8, KL11, KL20, KL25, KL30, KL43, KL47, KL57, KL64, KL69, KL71, KL101, KL103, KL104, KL107, KL110, KL111, KL112, KL132, and KL169.

[0035] Thirdly, the present invention provides a method for constructing a plug-and-play engineered phage, comprising: (a) providing a plasmid containing an exogenous second receptor-binding protein and a receptor bacterium; (b) transforming the receptor bacterium with the plasmid to obtain transformed receptor bacteria; and (c) infecting the transformed receptor bacteria with an initial phage containing a first receptor-binding protein to obtain an engineered phage containing the first receptor-binding protein and the exogenous second receptor-binding protein to specifically target a selected host bacterium, wherein the receptor bacterium and the exogenous second receptor-binding protein target different host bacteria, the N-terminus of the first receptor-binding protein contains an anchoring domain, and the N-terminus of the second receptor-binding protein binds to the first receptor-binding protein through the anchoring domain.

[0036] In some embodiments, the anchoring structural domain is a T4 gp10-type structural domain.

[0037] In some embodiments, the engineered bacteriophage is selected from the Myotail Phage Family, Longtail Phage Family, or Shorttail Phage Family.

[0038] In some preferred embodiments, the engineered bacteriophage is from the family Brachyphageidae.

[0039] In some embodiments, the anchoring structural domain comprises a three-layer β-fold structure.

[0040] In some embodiments, the anchoring domain comprises the amino acid sequence shown in SEQ ID No: 1-4 or a variant thereof.

[0041] In some implementations, the variant includes the same three-layer β-fold structure as the anchoring structural domain.

[0042] In some embodiments, the variant comprises an amino acid sequence having at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with the amino acid sequences shown in SEQ ID No: 1-4.

[0043] In some embodiments, the variant comprises an amino acid sequence having a spatial structural similarity™ score of at least 0.70, at least 0.75, at least 0.80, at least 0.85, at least 0.90, at least 0.95, at least 0.96, at least 0.97, at least 0.98, or at least 0.99 with respect to the amino acid sequences shown in SEQ ID No: 1-4.

[0044] In some embodiments, the N-terminus of the second receptor-binding protein contains the amino acid sequence shown in SEQ ID No: 5-6 or an amino acid sequence having at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with the amino acid sequence shown in SEQ ID No: 5-6.

[0045] In some embodiments, the N-terminus of the second receptor-binding protein comprises an amino acid sequence with a spatial structural similarity TM score of at least 0.70, at least 0.75, at least 0.80, at least 0.85, at least 0.90, at least 0.95, at least 0.96, at least 0.97, at least 0.98, or at least 0.99 with respect to the amino acid sequences shown in SEQ ID No: 5-6.

[0046] In some embodiments, the selected host bacterium is a selected capsular serotype (KL) of Klebsiella pneumoniae.

[0047] In some embodiments, KL is selected from one or more of KL1 to KL82 and KL101 to KL169. In some preferred embodiments, KL is selected from one or more of KL3, KL8, KL11, KL20, KL25, KL30, KL43, KL47, KL57, KL64, KL69, KL71, KL101, KL103, KL104, KL107, KL110, KL111, KL112, KL132, and KL169.

[0048] Fourthly, the present invention provides the use of the engineered phage described in the first aspect, or the engineered phage constructed using the methods described in the second or third aspect, in the preparation of Klebsiella pneumoniae inhibitors, drugs for the diagnosis, treatment or prevention of Klebsiella pneumoniae infection, antibiotic alternatives or disinfectants.

[0049] Fifthly, the present invention provides the method described in the third aspect for rapidly screening or customizing individualized phages that target selected capsular serotypes of Klebsiella pneumoniae.

[0050] In some embodiments, the capsular serotype is selected from one or more of KL1-KL82 and KL101-KL169. In some preferred embodiments, the KL is selected from one or more of KL3, KL8, KL11, KL20, KL25, KL30, KL43, KL47, KL57, KL64, KL69, KL71, KL101, KL103, KL104, KL107, KL110, KL111, KL112, KL132, and KL169.

[0051] Compared with the prior art, the beneficial effects of the present invention are as follows: The modular assembly platform for RBP provided by the present invention can successfully achieve precise customization of the host range of bacteriophages by freely switching and combining RBPs among bacteriophages.

[0052] This invention constructs an engineered phage screening platform that enables "plug-and-play" RBP (Rich Phage Biotechnology). It rapidly yields engineered phage mixtures modified only at the protein level without any editing of the phage genome. This "phenotypic engineering" strategy has dual advantages: firstly, it effectively expands the host range of phages to combat multidrug-resistant bacteria; secondly, because the genome remains wild-type, this expansion of a specific host spectrum has only a "one-off" effect, thus strictly limiting the spillover risk of engineered traits into the environment and providing a new solution for developing highly biosafety-compliant phage formulations. Attached Figure Description

[0053] Figure 1 shows the trimer structures of Prz_RBP1 and Prz_RBP2 predicted by AlphaFold3.

[0054] Figure 2 shows the proportion of T4 gp10-like structural domains in Prz_RBP1 as displayed in the phylogenetic tree.

[0055] Figure 3 shows the N-terminal conservative structure of Prz_RBP2 as displayed by WebLogo sequence alignment.

[0056] Figures 4a-4c show the T4 gp10-like domain-switched phage Prz_RBP2.

[0057] Figure 5 shows the T4 gp10 sample structure domain alignment results of RCIP0109_RBP1 with RCIP0018_RBP1 and PhageP560_RBP1, respectively.

[0058] Figure 6 shows the free combination of phages Prz_RBP1 and Prz_RBP1 based on the T4 gp10-like domain switching.

[0059] Figures 7a-7d illustrate how modular integration of exogenous RBP2 enables the diversification of progeny phage populations.

[0060] Figure 8 shows a schematic diagram of the spatial structure of the T4 gp10 sample domain. Detailed Implementation

[0061] To make the objectives, technical solutions, and advantages of this invention clearer, the invention will be further described in detail below with reference to embodiments. It should be understood that the specific embodiments described herein are for illustrative purposes only and are not intended to limit the invention in any way. Furthermore, descriptions of well-known structures and techniques are omitted in the following description to avoid unnecessarily obscuring the concepts of this disclosure. Such structures and techniques have also been described in many publications.

[0062] definition Unless otherwise defined, all technical and scientific terms used in this invention have the same meaning as commonly used in the field to which this invention pertains. The terminology used to describe this invention is intended only to describe a particular implementation and not to limit the scope of the teachings.

[0063] For the purposes of interpreting this specification, the following definitions will apply, and where appropriate, terms used in the singular will also include the plural form, and vice versa.

[0064] Unless the context clearly indicates otherwise, references to a specific quantity herein include their plural forms. For example, the term "cell" includes one or more such cells and equivalents known to those skilled in the art, etc.

[0065] The term “and / or” as used herein should be understood to mean any one of the options or any combination of two or more of the options.

[0066] As used herein, the term "about" indicates a range of ±20% of the following value. In some embodiments, the term "about" indicates a range of ±10% of the following value. In some embodiments, the term "about" indicates a range of ±5% of the following value.

[0067] The term "phage" as used in this article includes bacteriophages, Klebsiella pneumoniae phages (e.g., those against classical Klebsiella pneumoniae (cKp) and hypervirulent Klebsiella pneumoniae (hvKp)), and any other viruses capable of invading living bacteria and other microbial organisms. Phages have a simple structure, primarily composed of a protein coat and nucleic acid, their genetic material. The vast majority of phages possess a tail structure, and based on tail morphology, they can be divided into three main categories: short-tailed phages, myotailed phages, and long-tailed phages. Long-tailed phages are the most numerous tailed phages in public databases (66%), followed by myotailed phages (20%) and short-tailed phages (14%). Short-tailed phages have a smaller genome, averaging approximately (49±20) kb, and consist of a smaller head and a non-retractable short tail. Different phage morphologies also differ in their ability to infect hosts. Generally, myotail phages have strong lytic ability and a wide host range; short-tailed phages also typically have strong lytic ability, but a narrower host range and are specific to certain hosts; long-tailed phages usually have weaker lytic ability, and their host range falls between that of short-tailed and myotailed phages. The differences in host range are largely determined by the different recognition roles of phage receptor-binding proteins (RBPs) during infection. Although different short-tailed phages share similar structural features, their tail structures and host recognition mechanisms still exhibit significant differences. Based on the consistency of homologous proteins within and between genera (40%), the family Short-tailed Phages is divided into two subfamilies: Autographivirinae and Picovirinae, comprising 11 genera. The heads of these phages are equidistant or elongated icosahedral structures, assembled from scaffold proteins and capsid proteins. A few phages contain head fibers, which facilitate phage attachment to bacteria. The tail of a bacteriophage is connected to a apex of its head and is typically composed of structures such as a tail spike, tail fibers, and tail tube. Although different types of short-tailed bacteriophages have different tail structures and compositions, they all contain RBPs composed of specific protein subunits, which are responsible for binding to receptors on the host surface.

[0068] The term "receptor-binding protein (RBP)" used in this article refers to the collective term for the structures required for bacteriophages to bind to the bacterial surface. A single bacteriophage can possess multiple RBPs, whose main function is to recognize and bind to specific bacterial receptors. RBPs with hydrolytic functions can hydrolyze bacterial surface structures, assisting in the injection of nucleic acids into the host bacteria. According to statistics on RBPs in bacteriophage populations, most RBPs are tail spines, tail filaments, and substrates in the tail structure; a few bacteriophages without tail structures use capsid proteins as adsorption structures. The structure of RBPs is highly templated, consisting of a conserved N-terminal (Amino-terminal), a flexible linker in the middle, and a C-terminal (Carboxy-terminal) that can adsorb and hydrolyze bacterial receptors. High specificity is both an advantage and a disadvantage of bacteriophages as biological agents (acting only on the target, possessing safety). RBPs are key to researching bacteriophage biotechnology and optimizing bacteriophage biological agents: the host specificity of RBPs can be used for bacterial identification and serotype diagnosis; through the editing and domestication of RBPs, the host spectrum of bacteriophages can be artificially selected. In some embodiments, the engineered bacteriophages of the present invention have a wider or broader host range compared to the initial bacteriophage.

[0069] Phage engineering methods mainly include traditional homologous recombination technology, Red system-based recombination, CRISPR-Cas-based phage engineering, and phage reactivation using assembled phage genomic DNA. Applications of genetically engineered phages in bacterial infection include expanding or redirecting the host range of genetically engineered phages. RBP bioengineering is an important potential tool for controlling the host range of phages in biomedical applications. Existing descriptions of engineering methods, as known to those skilled in the art, can be used for the construction of the engineered phages described in this invention, such as those described in Chinese patent application CN109952373A, and are incorporated herein by reference.

[0070] As used herein, the terms "domain" and related expressions, and "region," are generally synonymous, unless otherwise indicated. They refer to a group of amino acids conserved at a specific position along the sequence alignment of an evolutionarily relevant protein. While amino acids at other positions may differ between homologues, highly conserved amino acids at a specific position indicate that they are likely essential for the protein's structure, stability, or function. Identification by their high conservation in the aligned sequences of a protein homologue family can be used to determine whether any protein or polypeptide under discussion belongs to a previously identified group of proteins or polypeptides.

[0071] The term "motif" as used in this article refers to a short, conserved region in the sequence of evolution-related proteins. Motifs are often highly conserved portions of a domain, but they can also include only a portion of the domain, or they can be located outside the conserved domain (if all amino acids of the motif are located outside the defined domain).

[0072] As used herein, the term "primitive phage" can refer to a wild-type phage found in any environment or an engineered phage. In some embodiments, the primitive phage may be specific to at least some desired target host bacteria and may be further modified according to the methods of this application to alter or customize its host range. In some embodiments, the receptor-binding protein of the primitive phage may be replaced to recognize a target host that is different from some or all of the hosts of the primitive phage, thereby producing an engineered phage with a customized host range.

[0073] The term "Klebsiella pneumoniae (Kp)" as used in this article refers to a Gram-negative bacterium belonging to the family Enterobacteriaceae and the genus Klebsiella. It commonly infects the skin, respiratory tract, and intestines of humans and animals. Klebsiella pneumoniae infection can involve multiple organs and sites, potentially causing various types of infectious diseases such as pneumonia, meningitis, urinary tract infections, sepsis, and purulent liver abscesses. It is one of the more common and important pathogens in hospital-acquired infections and community-acquired infections. Based on virulence characteristics, Klebsiella pneumoniae can be divided into two main groups: Classic K. pneumoniae (cKP) and Hypervirulent K. pneumoniae (hvKP). hvKP is generally prevalent in young, healthy individuals in the community, while cKP is mainly seen in secondary infections in immunocompromised individuals within hospitals. hvKP is a highly invasive and pathogenic pathogen capable of causing more severe and disseminated infections. With bacterial evolution and the overuse of some antibiotics, drug-resistant bacterial infections have become one of the major public health challenges of the 21st century. The drug resistance characteristics of Klebsiella pneumoniae are closely related to antibiotic resistance genes (ARGs) encoded by plasmids. Due to plasmid and genetic factors, Klebsiella pneumoniae continues to accumulate ARGs under inappropriate antibiotic use, leading to the emergence of multidrug-resistant (MDR) bacteria, particularly extensively drug-resistant (XDR) bacteria, and "superbugs," posing a serious threat to clinical infection treatment. The emergence and spread of extensively drug-resistant strains is an urgent problem to be solved in the field of Klebsiella pneumoniae control.

[0074] Currently, antibiotics are the primary treatment for Klebsiella pneumoniae infections in clinical practice, including quinolones, β-lactams, and carbapenems. For some drug-resistant strains, combination therapy with structurally diverse drugs is often employed. However, with the continuous evolution of pathogens, multidrug-resistant bacteria are increasingly prevalent, urgently requiring accelerated development of novel antibiotics and exploration of new treatment methods. Bacteriophages, as viruses that obligately infect and cause lysis and death of bacteria, possess certain potential and advantages in treating bacterial infections, especially those caused by drug-resistant bacteria.

[0075] Klebsiella pneumoniae possesses a variety of virulence factors, including siderophores, fimbriae, capsular polysaccharides (CPS), and lipopolysaccharide (LPS). CPS is considered the most important virulence factor in Kp, and there are more than 100 serotypes of Klebsiella pneumoniae capsules. Compared with other serotypes, K1, K2, K5, K20, K54, and K57 are highly virulent strains and are commonly found in pneumonia cases. In some embodiments, the capsular serotype (KL) of Klebsiella pneumoniae described in this invention is selected from one or more of KL1~KL82 and KL101~KL169. As described by Wyres KL et al. (Wyres KL, et al., Identification of Klebsiella capsule synthesisloci from whole genome data. Microb Genom. 2016 Dec 12;2(12):e000102.), is incorporated herein by reference.

[0076] As used herein, the term "host bacterium" and related expressions refer to the microorganisms that a bacteriophage can infect. Bacteriophages can lyse host cells and release new virions upon lysis, transfer genes between hosts, and form lysogenic bacteria, which can alter host function. As used herein, the expression "host range" and related terms and expressions refer to the range or number of hosts that can be infected by a bacteriophage. In other words, "host range" describes the range of organisms (genus, species, strain, or other taxa) that a bacteriophage can infect. Some bacteriophages have a narrow host range, capable of infecting only a few strains within the same species. Other bacteriophages can infect many hosts, sometimes spanning different genera. The breadth of a particular bacteriophage's host range can be attributed in part to the specificity of the bacteriophage's receptor-binding protein (RBP), biochemical interactions during infection, the presence of a relevant prophage or specific plasmid, and host bacteriophage resistance mechanisms. The terms "altered host range" and related expressions are used herein to refer to the artificially altered or engineered host range of a bacteriophage. Such host variability can be achieved through at least some of the methods described in this application, as well as others. In some embodiments, the host range of a bacteriophage is determined by the highly specific RBP it carries, which typically recognizes only one type of host. In Kp, there are many types of capsules (KL), and a bacteriophage typically recognizes only one type of capsule.

[0077] As used herein, "targeting" is a well-known description in the art, referring to the preferential binding of the engineered phage of this invention to the target host bacteria when administered to an organism; or, in other words, the engineered phage of this invention is distributed only in the target host bacteria, i.e., selected host bacteria, such as KL2 or KL20 Klebsiella pneumoniae. In some embodiments, the engineered phage of this invention has multiple targeting properties, meaning that when administered in vivo, the engineered phage specifically acts on multiple KL types of Klebsiella pneumoniae.

[0078] The terms “amino acid” and “amino acid residue” used in this article refer to all naturally occurring L-α-amino acids. These amino acids are identified by single-letter or three-letter names: Asp, D, aspartic acid; Ile, I, isoleucine; Thr, T, threonine; Leu, L, leucine; Ser, S, serine; Tyr, Y, tyrosine; Glu, E, glutamic acid; Phe, F, phenylalanine; Pro, P, proline; His, H, histidine; Gly, G, glycine; Lys, K, lysine; Ala, A, alanine; Arg, R, arginine; Cys, C, cysteine; Trp, W, tryptophan; Val, V, valine; Gln, Q, glutamine; Met, M, methionine; Asn, N, asparagine.

[0079] As used herein, the term "identity" refers to the degree of matching between two or more amino acid sequences. Two or more sequences used for comparison are identical at a site when they share the same amino acid monomeric subunit (e.g., each of the two or more amino acid sequences has a lysine residue at a site). The percentage of identity between two or more sequences is a function of the number of identical sites shared by the two sequences relative to the total number of sites used for comparison × 100. For example, if six out of ten sites in two or more sequences match, then the two or more sequences have 60% identity. Typically, comparisons of two or more sequences are performed in a manner that yields the maximum identity. For example, the percentage identity between two or more amino acid sequences can be determined using the algorithm of E. Meyers and W. Miller, incorporated into the ALIGN program (version 2.0), using a PAM120 weighted residue table, a vacancy length penalty of 12, and a vacancy penalty of 4. Furthermore, the percentage of identity between two or more amino acid sequences can be determined using the Needleman and Wunsch algorithm in the GAP program, which has been incorporated into the GCG software package, with a Blossum 62 matrix or a PAM250 matrix, and vacancy weights of 16, 14, 12, 10, 8, 6 or 4, and length weights of 1, 2, 3, 4, 5 or 6.

[0080] The three-dimensional structure of any polypeptide or amino acid sequence can be obtained experimentally, for example by X-ray crystallography or using computational methods such as AlphaFold3. Structural similarity between three-dimensional structures can be determined by a TM score, calculated using a formula described by Zhang et al. (Zhang, Y. and Skolnick, J. Scoring Function for Automated Assessment of Protein Structure Template Quality. Proteins: Structure, Function, and Bioinformatics, 2004, 57, 702-710.), which is incorporated herein by reference. The maximum TM score is 1, for example, 1.0, corresponding to identical three-dimensional structures.

[0081] As used herein, “variants” refers to amino acid sequences that have similar binding function to the anchoring domains containing the amino acid sequences shown in SEQ ID Nos. 1-4, and that contain substitutions, insertions (including extensions), and / or deletions (e.g., truncations) at one or more positions of the amino acid sequences shown in SEQ ID Nos. 1-4. Substitution refers to replacing an amino acid occupying a position with a different amino acid; deletion refers to deleting an amino acid occupying a position; insertion refers to adding 1 to 10 amino acids (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acids) adjacent to and immediately following the amino acid occupying a position.

[0082] As used herein, “binding” refers to a specific interaction between proteins or peptides or between a protein and a ligand. Protein or peptide binding is primarily contributed by a variety of intermolecular forces, including electrostatic interactions, van der Waals forces, hydrogen bonds, and hydrophobic interactions. The synergistic effect of these forces determines the binding pattern and binding energy between proteins or peptides or between a protein and a ligand. In some embodiments, the binding force between the N-terminus of the second receptor-binding protein and the anchoring domain of the first receptor protein is mainly charge complementarity and hydrophobic interaction, as described by Plattner M et al. (Plattner M, et al., Structure and Function of the BranchedReceptor-Binding Complex of Bacteriophage CBA120. J Mol Biol. 2019 Sep 6;431(19):3718-3739), which is incorporated herein by reference.

[0083] The term "personalized" or "individualized" as used in this article refers to a medical model that utilizes individual patient information to select or optimize diagnostic, preventative, or treatment plans for that patient. The method for constructing plug-and-play engineered phages involved in this invention can be used in personalized phage therapy to screen or customize individualized phages.

[0084] This invention focuses on phages of the genus *Przondovirus* within the family Bacteriophageaceae of *Klebsiella pneumoniae*. It confirms that the anchoring domain of receptor-binding proteins, particularly the T4 gp10-like domain, is not only highly conserved evolutionarily but also widely distributed across phages of this genus, serving as a universal "molecular interface." Utilizing this structural feature, this invention establishes a modular assembly platform for receptor-binding proteins (RBPs). By freely switching and combining RBPs among phages of the same genus, precise customization of the phage host range is successfully achieved.

[0085] To address the challenges of construction complexity and safety associated with traditional genetically engineered bacteriophages, this invention further designs an assembly strategy based on exogenous plasmids providing RBPs. The method of this invention rapidly yields engineered bacteriophage mixtures modified only at the protein level without any editing of the bacteriophage genome. This "phenotypic engineering" strategy has dual advantages: firstly, it effectively expands the host range of bacteriophages to combat multidrug-resistant bacteria; secondly, because its genome remains wild-type, this expansion of a specific host spectrum has only a "one-off" effect, thus strictly limiting the risk of spillover of engineered traits into the environment, providing a new solution for developing highly biosafe bacteriophage formulations.

[0086] The following embodiments and accompanying drawings are provided to aid in understanding the present invention. However, it should be understood that these embodiments and drawings are for illustrative purposes only and do not constitute any limitation. The actual scope of protection of the present invention is set forth in the claims. It should be understood that any modifications and changes can be made without departing from the spirit of the present invention.

[0087] Unless otherwise stated, the embodiments of this invention will utilize conventional techniques of biology, cell culture, molecular biology, etc., which are described in the literature or performed in accordance with product instructions. For example, see J. Sambrook's *Molecular Cloning: A Laboratory Manual* (4th edition, Science Press). Unless otherwise specified, the materials, reagents, or instruments used in the embodiments are all commercially available conventional products.

[0088] Materials and Methods Strains and Culture Conditions Unless otherwise specified, all bacterial strains, bacteriophages, and plasmids used in this invention are listed in Table 1 below. Klebsiella pneumoniae and Escherichia coli strains were routinely cultured on LB (Luria-Bertani) medium (Thermo Fisher Scientific, CM0996B), in both liquid broth and solid plates containing 15 g / L agar (AMEKO). Cultures were typically incubated at 37°C with shaking at 220 rpm; antibiotics were added as needed, with the following final concentrations: kanamycin 50 μg / mL and gentamicin 20 μg / mL.

[0089] Table 1 Phage propagation involves transferring overnight phage-specific host cultures to fresh medium and culturing them to mid-log phase (approximately 10^6 days). 8 CFU / mL); add phage stock solution (approximately 10 CFU / mL); 7 Incubate the solution at 37°C for 3-5 hours until obvious lysis is observed (PFU / mL). Centrifuge the lysate (10000×g, 15min), filter the supernatant through a 0.22μm filter membrane, and store at 4°C for later use.

[0090] Phage DNA extraction was performed by propagating phages in liquid culture medium to the corresponding host strain. After incubation, chloroform was added to lyse the bacterial cells. The lysate was filtered through a 0.22 μm filter to clarify, and then phage genomic DNA was extracted using a λ phage genomic DNA extraction kit (Zhuangmeng Biotechnology) according to the kit instructions.

[0091] Phage titer was determined using the double-layer agar plaque method. The procedure was as follows: 100 μL of serially diluted phage solution was mixed with 100 μL of mid-log host bacterial solution (OD2). 600 Mix thoroughly with 0.6% melted soft agar and 5 mL of 0.6% melted soft agar, invert and gently shake, then spread onto a solidified LB agar plate. Incubate overnight at 37°C, then count plaques. A plaque count within the range of 10–300 PFU is considered a valid result; otherwise, repeat the platening and testing with the same dilution. Three independent technical replicates are set up for each sample, and the average value is used as the final titer.

[0092] Phage annotation and tail filament protein (RBP) identification: To achieve standardized annotation of phage genomes and obtain genomic information from unannotated entries, the genome of all phage sequences retrieved from public databases was rearranged using a one-stop phage analysis workflow (https: / / nmdc.cn / phage / tools / phage); and genome annotation was completed according to existing methods. The steps are as follows: Using Prokka software (v1.14.6), the parameters --kingdomViruses and --metagenome were set to predict and annotate open reading frames (ORFs); the obtained protein sequences were further annotated using multiple methods: (1) submitted to the National Center for Biotechnology Information Batch Conserved Domain Search Platform (NCBI Batch CD-Search), specifying the Pfam and TIGR databases; (2) submitted to the KofamKOALA platform based on the Kyoto Encyclopedia of Genes and Genomes (KEGG) database; (3) BLASTp alignment was performed in the VOGDB database (https: / / vogdb.org / ) with a consistency >40% and coverage >40% as thresholds. For phages with annotated tail filament protein (RBP), the RBP sequence was directly extracted. For phages where the RBP could not be identified through annotation, genome sequence alignment was performed using Easyfig software to determine the RBP gene locus before extraction. All extracted RBP sequences were re-annotated using HHpred software (https: / / toolkit.tuebingen.mpg.de / tools / hhpred) to verify the reliability of the RBP identification results.

[0093] RBP Clustering and Phylogenetic Analysis: Given that even minor amino acid substitutions can affect the host specificity of RBPs, CD-HIT software was used to remove redundant sequences with a 100% sequence identity threshold. Multiple sequence alignment of RBP sequences was performed using MAFFT software. Subsequently, an interactive Tree of Life (iTOL) tool was used to construct and visualize a phylogenetic tree to analyze the evolutionary relationships among RBP sequences. Ultrafast bootstrapping (UFBoot, 1000 repetitions) was used to verify node stability, and bootstrap support values ​​were labeled next to the corresponding phylogenetic branches.

[0094] Structural alignment was performed using the full-length RBP protein sequence as input. AlphaFold3 software (https: / / alphafoldserver.com) was used to predict the protein structure of each RBP, and the results were visualized using PyMOL software (v2.5.8). Foldseek software (https: / / search.foldseek.com / ) was used for inter-RBP structural alignment, with the Multiple Sequence Alignment Local Distance Difference Test (MSALDDT) score as the output metric. For the structural alignment of the T4 gp10-like domain, the following amino acid fragments were selected: RCIP0109_RBP1 (WPJ56834.1) positions 188-256 (SEQ ID No: 1), RCIP0018_RBP1 (WPJ49360.1) positions 189-255 (SEQ ID No: 2), and Klebsiella pneumoniae phage P560_RBP1 (QOV05501.1) positions 189-255 (SEQ ID No: 3). The typical spatial structure of the T4 gp10-like domain is shown in Figure 8. It has a 3-layer β-sheet structure and can covalently or non-covalently bind to the N-terminus of the second receptor-binding protein.

[0095] Phage engineering was performed using existing methods. The specific steps are as follows: When constructing the donor plasmid, the full-length donor RBP sequence and approximately 200 bp homologous arm sequences upstream and downstream of the chassis phage RBP were amplified using primers. These overlapping fragments were then assembled into a linearized pCOLADRed vector using the Gibson homologous recombination method, with the homologous arm positions adjusted according to design requirements. The donor plasmid, verified by sequencing, was transformed into the Klebsiella pneumoniae Kp8-41 host strain for subsequent phage recombination experiments.

[0096] In the recombinant experiment, 1 mM arabinose was added to the culture of Kp8-41 strain containing the donor plasmid to induce the expression of the recombinase system. After the strain entered the logarithmic growth phase, wild-type phage RCIP0109 was added and co-cultured at 37°C for 30-60 min. Chloroform was added and the mixture was vigorously stirred to lyse the cells and release the phage. The phage lysate was plated with the corresponding host bacteria using a double-layer agar method, and successfully recombined phages were screened. Positive plaques were subjected to at least three rounds of infection and screening to remove wild-type phage background and stabilize the phage phenotype.

[0097] The construction steps of pseudo-phage are as follows: Amplify the exogenous RBP2 fragment using specific primers with overlapping sequences; linearize the pCOLADRed plasmid, and knock out its original λERD (Exo, Beta, Gam) region using primer design, allowing the exogenous RBP2 to insert into the same site and be induced and regulated by arabinose. Assemble the above fragment using Gibson homologous recombination, and transform the recombinant plasmid into *E. coli* Top10 competent cells; after sequencing verification, introduce the pColad-donor plasmid into the target host strain. Add 1 mM arabinose to the strain containing the donor plasmid to induce exogenous RBP2 expression; after the strain enters the logarithmic growth phase, it is infected with the phage, and both native phage progeny and pseudophages are simultaneously produced within the host cells.

[0098] The host range of bacteriophages was determined using the spot method to ascertain the host range of wild-type and engineered bacteriophages. Brief procedure: 200 μL of mid-log Klebsiella pneumoniae bacterial suspension was mixed with 5 mL of 0.6% melted soft agar and spread onto LB agar plates to form a bacterial colony. 5 μL of serially diluted bacteriophage solution was spotted onto the surface of the bacterial colony, and the plates were incubated overnight at 37°C. Plaque formation was observed the following day. All experiments were performed in triplicate to ensure reproducibility.

[0099] Example 1: Broad Abundance Analysis of the T4 gp10-like Domain in Prz_RBP1 and Conservation of the N-terminal Domain in Prz_RBP2. Among the reported Klebsiella pneumoniae phages, the genus *Przondovirus* is the most abundant. As of March 2025, 234 strains of this genus have been identified from the 1662 phage genomes indexed in the Genebank Database. Current research confirms that the receptor-binding protein (RBP) of *Przondovirus* phages can functionally target 14 different capsular serotypes (confirmed by heterologous expression and activity verification experiments), exhibiting extremely strong broad-spectrum host capsule recognition. This unique combination of high abundance and broad-spectrum receptor recognition makes *Przondovirus* an ideal research subject for in-depth exploration of the molecular mechanisms by which phages recognize and bind to host bacteria.

[0100] In this embodiment, all receptor-binding protein sequences were extracted from the genome of Przondovirus phages, and protein structure prediction was performed using the AlphaFold3 tool. Based on structural characteristics and functional domain composition, these receptor-binding proteins can be divided into two categories, named Prz_RBP1 and Prz_RBP2 respectively (Figure 1). As shown in Figure 1, the Prz_RBP1 protein typically contains four functional domains: (1) a T7gp17-like N-terminal domain, which is responsible for anchoring the protein to the phage substrate; (2) a flexible T4 gp10-like domain, which serves as the binding anchoring site for the Prz_RBP2 protein; (3) a catalytic domain, which can also be considered as a receptor-binding protein in a narrow sense, with a characteristic right-handed parallel β-helix structure, which can act as a depolymerase to recognize and degrade bacterial capsular polysaccharide (CPS); and (4) a C-terminal domain, which participates in maintaining the stability of the protein trimer conformation. In contrast, the Prz_RBP2 protein contains only three domains (N-terminal domain, receptor-binding domain, and C-terminal domain) and lacks the conserved T7 gp17-like N-terminal domain responsible for binding to the substrate. To further analyze the functions of Prz_RBP1 and Prz_RBP2, some receptor-binding proteins were excluded from subsequent studies due to the lack of a catalytic domain. Ultimately, 280 non-redundant receptor-binding protein sequences and structures were screened for downstream analysis.

[0101] The Prz_RBP1 protein contains a T4 gp10-like domain, which is presumed to provide an anchoring platform for the Prz_RBP2 protein. Furthermore, the Prz_RBP2 protein has a short, conserved amino acid motif at its N-terminus, which is thought to mediate the interaction between Prz_RBP2 and Prz_RBP1 proteins. Based on this structural feature, the inventors hypothesized that the T4 gp10-like domain of Prz_RBP1 and the conserved N-terminal motif of Prz_RBP2 together constitute a modular interaction interface, which can enable the combinatorial assembly of receptor-binding proteins (RBPs) from different bacteriophage sources. To verify this hypothesis, the distribution of the T4 gp10-like domain in 183 Prz_RBP1 sequences was first examined, and the results showed that 84 sequences (with sequence identity of 41.03%–66.67%) contained this domain. These Prz_RBP1 proteins containing the T4 gp10-like domain are distributed across 28 of the 40 identified Prz_RBP1 protein clusters, and cover 19 of the 22 KL serotypes targeted by Prz_RBP1 protein (KL3, KL8, KL11, KL20, KL20 / KL112 / KL132, KL25, KL30 / KL69, KL43, KL47, KL57, KL64, KL71, KL101, KL103, KL104, KL107, KL110, KL111, and KL169), indicating that the T4 gp10-like domain is widely conserved (Figure 2).

[0102] Subsequently, this embodiment analyzed the N-terminal region of 97 Prz_RBP2 proteins, and a conserved motif of about 30 amino acids was identified in all sequences (sequence identity 37.50%–84.38%, Figure 3).

[0103] The above findings collectively confirm that these two complementary structural elements are widely present in the receptor-binding protein family of Przondovirus phages.

[0104] Example 2: RBP switching based on the T4 gp10-like domain of Prz_RBP1 and the N-terminus of Prz_RBP2. In this example, the representative strain RCIP0109 of the genus Przondovirus was selected as the chassis phage. The full-length genome of this phage is 41152 bp, with a GC content of 53.05%, and it does not carry sequences encoding transfer RNA, antibiotic resistance, toxins, virulence factors, or lysogenic gene clusters. RCIP0109 encodes two receptor-binding proteins, named RCIP0109_RBP1 (SEQ ID No: 7) and RCIP0109_RBP2 (SEQ ID No: 8, whose N-terminal amino acid sequence is shown in SEQ ID No: 5), respectively. Both possess a depolymerase functional domain and can target Klebsiella pneumoniae of types KL2 and KL111, respectively.

[0105] In this embodiment, the native receptor-binding protein RCIP0109_RBP1 of the chassis phage RCIP0109 was first replaced with the RCIP0018_RBP1 (SEQ ID No: 9) protein targeting the KL20 / KL112 serotype, while retaining its native Prz_RBP2 protein (Figure 4a). Sequence alignment showed that the T4 gp10-like domain sequence of RCIP0018_RBP1 and RCIP0109_RBP1 was 71.01% identical, and their tertiary structures were highly similar (Figure 5, left). The recombinant phage RCIP0109 obtained after the modification was... R5 The phage lost its ability to infect KL111 serotype strains but gained lytic activity against KL20 / KL112 serotype strains, while retaining its infectivity against KL2 serotype strains (Figure 4c). Next, the native Prz_RBP2 protein of RCIP0109 was replaced with the P560_RBP2 protein (SEQ ID No: 11, its N-terminal amino acid sequence is shown in SEQ ID No: 6) targeting KL47 serotype, while retaining its native RCIP0109_RBP1 protein (Figure 4b). Although the T4 gp10-like domain sequence identity between the RBP1 (SEQ ID No: 10) of P560 phage and RCIP0109_RBP1 was only 53.62%, their spatial structures were still highly similar (Figure 5, right panel). The obtained engineered phage RCIP0109 R6 It lost its lytic activity against KL2 serotype strains, but gained the ability to infect KL47 serotype strains, while retaining its infectivity against KL111 serotype strains (Figure 4c).

[0106] To verify the modular compatibility of receptor-binding proteins from different phage sources, this embodiment combines the RBP1 protein of BUCT-3589 phage (BUCT-3589_RBP1, SEQ ID No: 12) targeting the KL25 serotype with the RBP2 protein of P560 phage (P560_RBP2) targeting the KL47 serotype. The resulting engineered phage RCIP0109 R7 It completely lost its ability to infect the original hosts (KL2 and KL111 serotypes), but at the same time gained lytic activity against KL25 and KL47 serotypes (Figure 6).

[0107] The above data confirm that the Prz_RBP1 protein containing the T4 gp10-like domain and the Prz_RBP2 protein containing the N-terminal conserved motif can serve as combinable functional modules; through their free pairing, predictable and non-redundant customization of phage host tropism can be achieved.

[0108] Example 3: Using the T4 gp10-like domain of Prz_RBP1 and the N-terminus of Prz_RBP2 to achieve RBP switching, enabling "plug-and-play" engineered phage construction. Phages are highly modular nanoscale molecular machines, whose tail structures can typically assemble independently. Given that the combination of Prz_RBP1 and Prz_RBP2 modules demonstrated in Example 2 enables programmable control of phage host tropism, the inventors hypothesized that providing multiple Prz_RBP2 variants within bacteria could induce co-assembly with Prz_RBP1, thereby generating phage populations carrying diverse Prz_RBP1-RBP2 combinations. To verify this hypothesis, this example utilizes a plasmid within the host cell to express exogenous Prz_RBP2, enabling the phage to simultaneously integrate endogenous and exogenous RBP2 during infection and assembly. This strategy is expected to yield progeny phages with a significantly broadened host spectrum (Figure 7a).

[0109] First, an arabinose-inducible plasmid, pColad-donor1, was constructed, carrying an RCIP0109_RBP2 module targeting the KL2 capsular serotype. This plasmid was then transformed into host 161 (KL20 capsular serotype). When engineered phage RCIP0109... R1 Following infection of host 161 carrying the pColad-donor1 plasmid with KL20 / KL112, the host spectrum of the progeny phages was significantly broadened (Figure 7b). As shown in Figure 7b, the progeny phages could form phage plaques on KL20 strain plates, indicating that RCIP0109... R1The infection process can be completed normally. High-density spot experiments on KL2 host bacteria plates showed that the progeny bacteriophages exhibited a typical lysis-outside (LO) phenotype—causing only local cell death but failing to form well-defined plaques.

[0110] The above results indicate that the progeny phage population contains two types: one is the parental phage RCIP0109. R1 Secondly, there is a variant that has undergone engineering modifications (named camouflage type RCIP0109). R1 Abbreviated as PESEU-RCIP0109 R1 The latter retains RCIP0109. R1 Simultaneously with genome sequencing, RBP1 and exogenous RBP2 were successfully assembled. Furthermore, after passage of these progeny phages in the plasmid-carrying KL2 receptor strain 8-41, the engineered variant pseudo-RCIP0109 was found to be effective. R1 It can proliferate stably in this host context.

[0111] Subsequently, in this embodiment, the naturally occurring phage RCIP0109 carrying Prz_RBP1 and Prz_RBP2 was used to infect strain 17u111 carrying the plasmid pColad-donor2; this plasmid encodes P560_RBP2, which targets the KL47 serotype. The resulting progeny phages could simultaneously form plaques on both KL111 and KL2 strain plates. Notably, high-density spot experiments on KL47 strain plates showed that the progeny phages exhibited an exolytic lysis phenotype (Figure 7c). These progeny phages were able to stably proliferate in the plasmid-carrying KL47 recipient strain 334847, with titers comparable to those of the synthetic phage RCIP0109 targeting the KL47 serotype. R7 Quite similar (Figure 7d).

[0112] The results of this embodiment confirm that the highly flexible assembly architecture between the T4 gp10-like domain of Prz_RBP1 and the N-terminus of Prz_RBP2 allows these RBP modules to be delivered via extrachromosomal plasmids and exhibits excellent compatibility. This establishes a scalable, data-driven technical framework capable of transforming large-scale sequence libraries into easily accessible, targeted, and programmable therapeutic phages, achieving "plug-and-play" engineered phage construction and thus solving a core bottleneck problem in this field.

[0113] The RBP amino acid sequences involved in the examples are shown in Table 2 below.

[0114] Table 2 In the field of bacteriophage engineering, traditional strategies for expanding or altering the host range of bacteriophages primarily rely on direct modification of receptor-binding proteins (RBPs), including full-length replacement (switching to the complete RBP gene through gene editing) and chimeric modification (retaining the conserved N-terminal anchoring domain and replacing the host-specific C-terminal domain). However, current research lacks structural understanding of RBPs, such as the lack of coverage of the connection domains between RBPs; secondly, existing modification operations are cumbersome, genome editing is time-consuming and labor-intensive, and screening is difficult; furthermore, there are biosafety risks, as gene-edited engineered bacteriophages are transgenic organisms, and once released into the environment, the changes in their genetic material are permanent, posing an uncontrollable risk of biospillover.

[0115] Based on the above problems, this invention creatively proposes an innovative approach to construct a universal connectivity platform using anchoring domains, particularly the T4 gp10-like domain. The inventive aspects of this invention are: 1. Bioinformatics mining: In-depth pan-genome analysis of the genus Przondovirus (a representative species of short-tailed bacteriophages).

[0116] 2. Key structure identification: A highly conserved T4 gp10-like domain was found on the first receptor-binding protein (RBP1) of the vast majority of bacteriophages.

[0117] 3. Interaction pattern confirmed: Further analysis revealed that the N-terminal sequence (approximately the first 30 amino acids) of the second receptor-binding protein (RBP2) that it pairs with exhibits remarkable conservation among different bacteriophages.

[0118] 4. Theoretical Assumptions: Based on the "socket" characteristics of the T4 gp10-type structural domain and the "plug" characteristics of the N-terminal of RBP2, it is hypothesized that the T4 gp10-type structural domain is a natural and universal modular connection platform. This means that as long as the conservative sequence of the N-terminal of RBP2 is retained, a C-terminal structural domain with arbitrary functions can be mounted.

[0119] 5. Proof of Concept: Modular Assembly at the Genome Level To verify the above hypothesis, the inventors first conducted experiments at the genome level: Example 1 and Example 2 attempted to replace RBP1 (changing the connection platform) or RBP2 (changing the branching fibers), respectively, as well as simultaneously replacing both RBP1 and RBP2. The results successfully yielded chimeric phages with the expected host spectrum. This demonstrates that the T4 gp10-like domain of RBP1 and the N-terminus of RBP2 do indeed constitute a detachable and recombinant dual RBP functional module, validating the engineering potential of this domain as a universal platform.

[0120] 6. Technological Iteration and Breakthroughs (From "Genetic Engineering" to "Phenological Assembly") Despite successful validation at the gene level, the issues of cumbersome operation and biosafety remained unresolved. The inventors delved deeper into the assembly mechanism: phage tail assembly occurs in the cytoplasm through protein self-assembly, a process independent of genome replication. Could phages directly utilize "components" provided within the cell? This invention further developed a non-gene-editing, rapid approach: instead of modifying the chassis phage genome, the gene encoding exogenous RBP2 is constructed onto an expression plasmid and transformed into a host bacterium. When the chassis phage infects a host containing this plasmid, during the assembly phase, the T4 gp10-like domain on RBP1 randomly captures either the original RBP2 within the cell or the exogenous RBP2 expressed by the plasmid. The released progeny phage population contains engineered particles assembled with exogenous RBP2. Although the phenotype of these progeny phages changes (they are able to infect new hosts), their genotype remains wild-type. This means that they cannot pass on "engineered traits" to the next generation, thus perfectly solving the problem of biosafety spillover.

[0121] In summary, this invention utilizes a highly specific and conserved supramolecular interlocking mechanism between the T4 gp10-like domain and the N-terminus (~30aa) of the RBP to create a pioneering technical platform for expanding the host range of bacteriophages.

[0122] The core advantages of the technical platform of this invention are: 1. Versatility: It establishes the T4 gp10-like structural domain as a universal branching and connection platform, enabling free combination of different RBPs. It also allows for customization of the phage host range.

[0123] 2. Convenience: It eliminates the need for complex genome recombination engineering and only requires the construction of plasmids to achieve the customization of host profiles.

[0124] 3. Safety: By adopting a "phenotypic modification" strategy, the engineered bacteriophages will not cause genetic pollution after application, which greatly reduces the regulatory risks of environmental release.

[0125] The technical solutions of the present invention are not limited to the specific embodiments described above. Any technical modifications made in accordance with the technical solutions of the present invention fall within the protection scope of the present invention.

Claims

1. An engineered bacteriophage, characterized in that, The engineered phage comprises: a first receptor-binding protein, the N-terminus of which includes an anchoring domain; and optionally a second receptor-binding protein, the N-terminus of which binds to the first receptor-binding protein via the anchoring domain; wherein the C-terminus of the first receptor-binding protein is the same as or different from the C-terminus of the second receptor-binding protein, and part or all of the first receptor-binding protein and / or part or all of the second receptor-binding protein are engineered to specifically target a selected host bacterium.

2. The engineered bacteriophage according to claim 1, characterized in that, The anchoring structural domain is a T4 gp10 sample structural domain.

3. The engineered bacteriophage according to claim 1 or 2, characterized in that, The engineered bacteriophage is selected from the Myotail Phage Family, Longtail Phage Family, or Shorttail Phage Family, and preferably the engineered bacteriophage is from the Shorttail Phage Family.

4. The engineered bacteriophage according to any one of claims 1-3, characterized in that, The selected host bacterium is Klebsiella pneumoniae of the selected capsular serotype (KL).

5. The engineered bacteriophage according to any one of claims 1-4, characterized in that, The anchoring structural domain comprises a three-layer β-fold structure.

6. The engineered bacteriophage according to any one of claims 1-5, characterized in that, The anchoring domain comprises the amino acid sequence shown in SEQ ID No: 1-4 or a variant thereof; wherein the variant comprises the same three-layer β-sheet structure as the anchoring domain, and / or the variant comprises an amino acid sequence having a spatial structural similarity TM score of at least 0.70, at least 0.75, at least 0.80, at least 0.85, at least 0.90, at least 0.95, at least 0.96, at least 0.97, at least 0.98 or at least 0.99 with respect to the amino acid sequences shown in SEQ ID No: 1-4.

7. A method for constructing engineered bacteriophages, characterized in that, The method includes: a) providing an initial phage comprising an initial first receptor-binding protein, wherein the N-terminus of the initial first receptor-binding protein includes an anchoring domain; and b) engineering part or all of the initial first receptor-binding protein to specifically target a selected host bacterium.

8. The method according to claim 7, characterized in that, The method includes: the initial phage in step a) further includes an initial second receptor-binding protein, the N-terminus of which binds to the initial first receptor-binding protein via the anchoring domain; and in step b), partially or completely of the initial first receptor-binding protein and / or partially or completely of the initial second receptor-binding protein are engineered to specifically target a selected host bacterium.

9. The method according to claim 7 or 8, characterized in that, The anchoring structural domain is a T4 gp10 sample structural domain.

10. The method according to any one of claims 7-9, characterized in that, The selected host bacterium is Klebsiella pneumoniae of the selected capsular serotype (KL).

11. A method for constructing plug-and-play engineered bacteriophages, characterized in that, The method includes: (a) providing a plasmid containing an exogenous second receptor-binding protein and a receptor bacterium; (b) transforming the receptor bacterium with the plasmid to obtain transformed receptor bacteria; and (c) infecting the transformed receptor bacteria with an initial phage containing a first receptor-binding protein to obtain an engineered phage containing the first receptor-binding protein and the exogenous second receptor-binding protein to specifically target a selected host bacterium, wherein the receptor bacterium and the host bacterium targeted by the exogenous second receptor-binding protein are different, the N-terminus of the first receptor-binding protein includes an anchoring domain, and the N-terminus of the exogenous second receptor-binding protein binds to the first receptor-binding protein through the anchoring domain.

12. The use of the engineered phage according to any one of claims 1-6 or the engineered phage constructed using the method according to claims 7-11 in the preparation of Klebsiella pneumoniae inhibitors, drugs for the diagnosis, treatment or prevention of Klebsiella pneumoniae infection, antibiotic alternatives or disinfectants.

13. The method of claim 11 is used for screening or customizing individualized phages that target selected capsular serotypes of Klebsiella pneumoniae.

Images