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When proteins evolve new activity, a concomitant decrease in stability is often observed because the mutations that confer new activity can destabilize the native fold. In the conventional model of protein evolution, reduced stability is considered a purely deleterious cost of molecular innovation because unstable proteins are prone to aggregation and are sensitive to environmental stressors. However, recent work has revealed that nonnative, often unstable protein conformations play an important role in mediating evolutionary transitions, raising the question of whether instability can itself potentiate the evolution of new activity. We explored this question in a bacteriophage receptor-binding protein during host-range evolution. We studied the properties of the receptor-binding protein of bacteriophage λ before and after host-range evolution and demonstrated that the evolved protein is relatively unstable and may exist in multiple conformations with unique receptor preferences. Through a combination of structural modeling and in vitro oligomeric state analysis, we found that the instability arises from mutations that interfere with trimer formation. This study raises the intriguing possibility that protein instability might play a previously unrecognized role in mediating host-range expansions in viruses.

Background: Phage tail-like bacteriocins, or tailocins, provide a competitive advantage to producer cells by killing closely related bacteria. Morphologically similar to headless phages, their narrow target specificity is determined by receptor-binding proteins (RBPs). While RBP engineering has been used to alter the target range of a selected R2 tailocin from Pseudomonas aeruginosa, the process is labor-intensive, limiting broader application. Methods: We introduce a VersaTile-driven R2 tailocin engineering and screening platform to scale up RBP grafting. Results: This platform achieved three key milestones: (I) engineering R2 tailocins specific to Escherichia coli serogroups O26, O103, O104, O111, O145, O146, and O157; (II) reprogramming R2 tailocins to target, for the first time, the capsule and a new species, specifically the capsular serotype K1 of E. coli and K11 and K63 of Klebsiella pneumoniae; (III) creating the first bivalent tailocin with a branched RBP and cross-species activity, effective against both E. coli K1 and K. pneumoniae K11. Over 90% of engineered tailocins were effective, with clear pathways for further optimization identified. Conclusions: This work lays the groundwork for a scalable platform for the development of engineered tailocins, marking an important step towards making R2 tailocins a practical therapeutic tool for targeted bacterial infections. [ABSTRACT FROM AUTHOR]

Pyocins are high molecular weight bacteriocins produced by Pseudomonas aeruginosa that can be retargeted to new bacterial species by exchanging the pyocin tail fibers with bacteriophage receptor binding proteins (RBPs). Here, we develop retargeted pyocins called campycins as new antibacterials to precisely and effectively kill the major foodborne pathogen Campylobacter jejuni. We used two diverse RBPs (H-fibers) encoded by CJIE1 prophages found in the genomes of C. jejuni strains CAMSA2147 and RM1221 to construct campycin 1 and campycin 2, respectively. Campycins 1 and 2 could target all C. jejuni strains tested due to complementary antibacterial spectra. In addition, both campycins led to more than 3 log reductions in C. jejuni counts under microaerobic conditions at 42 °C, whereas the killing efficiency was less efficient under anaerobic conditions at 5 °C. Furthermore, we discovered that both H-fibers used to construct the campycins bind to the essential major outer membrane protein (MOMP) present in all C. jejuni in a strain-specific manner. Protein sequence alignment and structural modeling suggest that the highly variable extracellular loops of MOMP form the binding sites of the diverse H-fibers. Further in silico analyses of 5000 MOMP sequences indicated that the protein falls into three major clades predicted to be targeted by either campycin 1 or campycin 2. Thus, campycins are promising antibacterials against C. jejuni and are expected to broadly target numerous strains of this human pathogen in nature and agriculture. Key points: • Campycins are engineered R-type pyocins containing H-fibers from C. jejuni prophages • Campycins reduce C. jejuni counts by >3 logs at conditions promoting growth • Campycins bind to the essential outer membrane protein MOMP in a strain-dependent way [ABSTRACT FROM AUTHOR]

In tailed phages, the baseplate is the macromolecular structure located at the tail distal part, which is directly implicated in host recognition and cell wall penetration. In myophages (i.e., with contractile tails), the baseplate is complex and comprises a central puncturing device and baseplate wedges connecting the hub to the receptor-binding proteins (RBPs). In this work, we investigated the structures and functions of adsorption-associated tail proteins of Deep-Blue and Vp4, two Herelleviridae phages infecting members of the Bacillus cereus group. Their interest resides in their different host spectrum despite a high degree of similarity. Analysis of their tail module revealed that the gene order is similar to that of the Listeria phage A511. Among their tail proteins, Gp185 (Deep-Blue) and Gp112 (Vp4) had no structural homolog, but the C-terminal variable parts of these proteins were able to bind B. cereus strains, confirming their implication in the phage adsorption. Interestingly, Vp4 and Deep-Blue adsorption to their hosts was also shown to require polysaccharides, which are likely to be bound by the arsenal of carbohydrate-binding modules (CBMs) of these phages’ baseplates, suggesting that the adsorption does not rely solely on the RBPs. In particular, the BW Gp119 (Vp4), harboring a CBM fold, was shown to effectively bind to bacterial cells. Finally, we also showed that the putative baseplate hub proteins (i.e., Deep-Blue Gp189 and Vp4 Gp110) have a bacteriolytic activity against B. cereus strains, which supports their role as ectolysins locally degrading the peptidoglycan to facilitate genome injection. IMPORTANCE The Bacillus cereus group comprises closely related species, including some with pathogenic potential (e.g., Bacillus anthracis and Bacillus cytotoxicus). Their toxins represent the most frequently reported cause of food poisoning outbreaks at the European level. Bacteriophage research is undergoing a remarkable renaissance for its potential in the biocontrol and detection of such pathogens. As the primary site of phage-bacteria interactions and a prerequisite for successful phage infection, adsorption is a crucial process that needs further investigation. The current knowledge about B. cereus phage adsorption is currently limited to siphoviruses and tectiviruses. Here, we present the first insights into the adsorption process of Herelleviridae Vp4 and Deep-Blue myophages preying on B. cereus hosts, highlighting the importance of polysaccharide moieties in this process and confirming the binding to the host surface of Deep-Blue Gp185 and Vp4 Gp112 receptor-binding proteins and Gp119 baseplate wedge. [ABSTRACT FROM AUTHOR]

ABSTRACT A novel Hendra virus (HeV) genotype (HeV genotype 2 [HeV-g2]) was recently isolated from a deceased horse, revealing high-sequence conservation and antigenic similarities with the prototypic strain, HeV-g1. As the receptor-binding (G) and fusion (F) glycoproteins of HeV are essential for mediating viral entry, functional characterization of emerging HeV genotypic variants is key to understanding viral entry mechanisms and broader virus-host co-evolution. We first confirmed that HeV-g2 and HeV-g1 glycoproteins share a close phylogenetic relationship, underscoring HeV-g2’s relevance to global health. Our in vitro data showed that HeV-g2 glycoproteins induced cell-cell fusion in human cells, shared receptor tropism with HeV-g1, and cross-reacted with antibodies raised against HeV-g1. Despite these similarities, HeV-g2 glycoproteins yielded reduced syncytia formation compared to HeV-g1. By expressing heterotypic combinations of HeV-g2, HeV-g1, and Nipah virus (NiV) glycoproteins, we found that while HeV-g2 G had strong fusion-promoting abilities, HeV-g2 F consistently displayed hypofusogenic properties. These fusion phenotypes were more closely associated with those observed in the related NiV. Further investigation using HeV-g1 and HeV-g2 glycoprotein chimeras revealed that multiple domains may play roles in modulating these fusion phenotypes. Altogether, our findings may establish intrinsic fusogenic capacities of viral glycoproteins as a potential driver behind the emergence of new henipaviral variants.IMPORTANCEHeV is a zoonotic pathogen that causes severe disease across various mammalian hosts, including horses and humans. The identification of unrecognized HeV variants, such as HeV-g2, highlights the need to investigate mechanisms that may drive their evolution, transmission, and pathogenicity. Our study reveals that HeV-g2 and HeV-g1 glycoproteins are highly conserved in identity, function, and receptor tropism, yet they differ in their abilities to induce the formation of multinucleated cells (syncytia), which is a potential marker of viral pathogenesis. By using heterotypic combinations of HeV-g2 with either HeV-g1 or NiV glycoproteins, as well as chimeric HeV-g1/HeV-g2 glycoproteins, we demonstrate that the differences in syncytial formation can be attributed to the intrinsic fusogenic capacities of each glycoprotein. Our data indicate that HeV-g2 glycoproteins have fusion phenotypes closely related to those of NiV and that fusion promotion may be a crucial factor driving the emergence of new henipaviral variants.

ABSTRACT To date, complete genome sequences of 14 double-stranded RNA (dsRNA) phages are available, and studies have shown that the host range of dsRNA phages is limited. The hosts of most dsRNA phages belong to the genus Pseudomonas. However, the dsRNA phage phiNY, which has a non-lytic life cycle, was isolated from Microvirgula aerodenitrificans. Currently, the interaction between dsRNA phage phiNY and its host bacteria is unclear, which is not beneficial to a comprehensive understanding of dsRNA phage biology and the exploitation of dsRNA phage with non-lytic life cycle for biomedical applications and others. Phage adsorption is a crucial step through the interactions between receptor-binding protein (RBP) of the phage and its receptors to initiate the infection process, which dictates host range specificity. Thus, we identified the RBP and host receptor of phiNY. Through homology alignment, amino acid sequence similarity analysis, and the phylogenetic tree analysis, orf11, located in the M-segment of dsRNA phage phiNY, encodes a putative RBP. We further performed the whole-cell enzyme-linked immunosorbent assay (ELISA), western blotting assay, and indirect immunofluorescence assay and demonstrated that this orf11 is an RBP. Finally, using affinity chromatography, ELISA, and dynamic light scattering, we identified lipopolysaccharides (LPSs) on the surface of the host M. aerodenitrificans strain LH9 as host receptors involved in the adsorption of the dsRNA bacteriophage phiNY and observed the state of phiNY RBP after combining with LPS by atomic force microscopy. These results will guide future studies on phage-host interaction in a dsRNA phage with a non-lytic life cycle.IMPORTANCEThe interactions between the lytic dsRNA phages and their host receptors have been clarified in previous studies. However, the interaction between the dsRNA phage phiNY (which has a non-lytic life cycle) and its host receptors during the dsRNA phage adsorption process was unknown. Here, we found that phiNY uses the orf11 protein as a receptor-binding protein (RBP). In addition, we found that this orf11 recognizes lipopolysaccharide from the host bacterium Microvirgula aerodenitrificans strain LH9 as a specific receptor. These results suggest that phiNY, like lytic dsRNA phages, uses an RBP to bind to a similar host receptor (i.e., lipopolysaccharide). Determining the interaction between the dsRNA phage phiNY and its host receptors will help to elucidate the mechanisms underlying the phiNY non-lytic life cycle and enhance our understanding of its infection mechanism.

Background: Phage tail-like bacteriocins, or tailocins, provide a competitive advantage to producer cells by killing closely related bacteria. Morphologically similar to headless phages, their narrow target specificity is determined by receptor-binding proteins (RBPs). While RBP engineering has been used to alter the target range of a selected R2 tailocin from Pseudomonas aeruginosa, the process is labor-intensive, limiting broader application. Methods: We introduce a VersaTile-driven R2 tailocin engineering and screening platform to scale up RBP grafting. Results: This platform achieved three key milestones: (I) engineering R2 tailocins specific to Escherichia coli serogroups O26, O103, O104, O111, O145, O146, and O157; (II) reprogramming R2 tailocins to target, for the first time, the capsule and a new species, specifically the capsular serotype K1 of E. coli and K11 and K63 of Klebsiella pneumoniae; (III) creating the first bivalent tailocin with a branched RBP and cross-species activity, effective against both E. coli K1 and K. pneumoniae K11. Over 90% of engineered tailocins were effective, with clear pathways for further optimization identified. Conclusions: This work lays the groundwork for a scalable platform for the development of engineered tailocins, marking an important step towards making R2 tailocins a practical therapeutic tool for targeted bacterial infections.

The genus Acinetobacter comprises both environmental and clinically relevant species associated with hospital-acquired infections. Among them, Acinetobacter baumannii is a critical priority bacterial pathogen, for which the research and development of new strategies for antimicrobial treatment are urgently needed. Acinetobacter spp. produce a variety of structurally diverse capsular polysaccharides (CPSs), which surround the bacterial cells with a thick protective layer. These surface structures are primary receptors for capsule-specific bacteriophages, that is, phages carrying tailspikes with CPS-depolymerizing/modifying activities. Phage tailspike proteins (TSPs) exhibit hydrolase, lyase, or esterase activities toward the corresponding CPSs of a certain structure. In this study, the data on all lytic capsule-specific phages infecting Acinetobacter spp. with genomes deposited in the NCBI GenBank database by January 2024 were summarized. Among the 149 identified TSPs encoded in the genomes of 143 phages, the capsular specificity (K specificity) of 46 proteins has been experimentally determined or predicted previously. The specificity of 63 TSPs toward CPSs, produced by various Acinetobacter K types, was predicted in this study using a bioinformatic analysis. A comprehensive phylogenetic analysis confirmed the prediction and revealed the possibility of the genetic exchange of gene regions corresponding to the CPS-recognizing/degrading parts of different TSPs between morphologically and taxonomically distant groups of capsule-specific Acinetobacter phages. [ABSTRACT FROM AUTHOR]

Nipah virus (NiV) is a highly lethal, zoonotic Henipavirus (HNV) that causes respiratory and neurological signs and symptoms in humans. Similar to other paramyxoviruses, HNVs mediate entry into host cells through the concerted actions of two surface glycoproteins: a receptor-binding protein (RBP) that mediates attachment and a fusion glycoprotein (F) that triggers fusion in an RBP-dependent manner. NiV uses ephrin-B2 (EFNB2) and ephrin-B3 (EFNB3) as entry receptors. Ghana virus (GhV), a novel HNV identified in a Ghanaian bat, uses EFNB2 but not EFNB3. In this study, we employ a structure-informed approach to identify receptor-interfacing residues and systematically introduce GhV-RBP residues into a NiV-RBP backbone to uncover the molecular determinants of EFNB3 usage. We reveal two regions that severely impair EFNB3 binding by NiV-RBP and EFNB3-mediated entry by NiV pseudotyped viral particles. Further analyses uncovered two-point mutations (NiVN557SGhV and NiVY581TGhV) pivotal for this phenotype. Moreover, we identify NiV interaction with Y120 of EFNB3 as important for the usage of this receptor. Beyond these EFNB3-related findings, we reveal two domains that restrict GhV binding of EFNB2, confirm the HNV-head as an immunodominant target for polyclonal and monoclonal antibodies, and describe putative epitopes for GhV- and NiV-specific monoclonal antibodies. Cumulatively, the work presented here generates useful reagents and tools that shed insight to residues important for NiV usage of EFNB3, reveal regions critical for GhV binding of EFNB2, and describe putative HNV antibody-binding epitopes. [ABSTRACT FROM AUTHOR]

Stenotrophomonas maltophilia was discovered as a soil bacterium associated with the rhizosphere. Later, S. maltophilia was found to be a multidrug-resistant hospital-associated pathogen. Lytic bacteriophages are prospective antimicrobials; therefore, there is a need for the isolation and characterization of new Stenotrophomonas phages. The phage StenM_174 was isolated from litter at a poultry farm using a clinical strain of S. maltophilia as the host. StenM_174 reproduced in a wide range of clinical and environmental strains of Stenotrophomonas, mainly S. maltophilia, and it had a podovirus morphotype. The length of the genomic sequence of StenM_174 was 42,956 bp, and it contained 52 putative genes. All genes were unidirectional, and 31 of them encoded proteins with predicted functions, while the remaining 21 were identified as hypothetical ones. Two tail spike proteins of StenM_174 were predicted using AlphaFold2 structural modeling. A comparative analysis of the genome shows that the Stenotrophomonas phage StenM_174, along with the phages Ponderosa, Pepon, Ptah, and TS-10, can be members of the new putative genus Ponderosavirus in the Autographiviridae family. In addition, the analyzed data suggest a new subfamily within this family. [ABSTRACT FROM AUTHOR]

Diversity-generating retroelements (DGRs) are prokaryotic systems providing rapid modification and adaptation of target proteins. In phages, the main targets of DGRs are receptor-binding proteins that are usually parts of tail structures and the variability of such host-recognizing structures enables phage adaptation to changes on the bacterial host surface. Sometimes, more than one target gene containing a hypermutated variable repeat (VR) can be found in phage DGRs. The role of mutagenesis of two functionally different genes is unclear. In this study, several phage genomes that contain DGRs with two target genes were found in the gut virome of healthy volunteers. Bioinformatics analysis of these genes indicated that they encode proteins with different topology; however, both proteins contain the C-type lectin (C-lec) domain with a hypermutated beta-hairpin on its surface. One of the target proteins belongs to a new family of proteins with a specific topology: N-terminal C-lec domain followed by one or more immunoglobulin domains. Proteins from the new family were named tentaclins after TENTACLe + proteIN. The genes encoding such proteins were found in the genomes of prophages and phages from the gut metagenomes. We hypothesized that tentaclins are involved in binding either to bacterial receptors or intestinal/immune cells. [ABSTRACT FROM AUTHOR]

Identification of the interaction between the host membrane receptor and viral receptor-binding domain (RBD) represents a crucial step for understanding viral pathophysiology and for developing drugs against pathogenic viruses. While all membrane receptors and carbohydrate chains could potentially be used as receptors for viruses, prioritized searches focus typically on membrane receptors that are known to have been used by the relatives of the pathogenic virus, e.g., ACE2 used as a receptor for SARS-CoV is a prioritized candidate receptor for SARS-CoV-2. An ideal receptor protein from a viral perspective is one that is highly expressed in epithelial cell surface of mammalian respiratory or digestive tracts, strongly conserved in evolution so many mammalian species can serve as potential hosts, and functionally important so that its expression cannot be readily downregulated by the host in response to the infection. Experimental confirmation of host receptors includes (1) infection studies with cell cultures/tissues/organs with or without candidate receptor expression, (2) experimental determination of protein structure of the complex between the putative viral RDB and the candidate host receptor, and (3) experiments with mutant candidate receptor or homologues of the candidate receptor in other species. Successful identification of the host receptor opens the door for mechanism-based development of candidate drugs and vaccines and facilitates the inference of what other animal species are vulnerable to the viral pathogen. I illustrate these approaches with research on identification of the receptor and co-factors for SARS-CoV-2. [ABSTRACT FROM AUTHOR]

ABSTRACT The adsorption process is the first step in the lifecycle of phages and plays a decisive role in the entire infection process. Identifying the adsorption mechanism of phages not only makes phage therapy more precise and efficient but also enables the exploration of other potential applications and modifications of phages. Phage LP31 can lyse multiple Salmonella serotypes, efficiently clearing biofilms formed by Salmonella enterica serovar Enteritidis (S. Enteritidis) and significantly reducing the concentration of S. Enteritidis in chicken feces. Therefore, LP31 has great potential for many practical applications. In this study, we established an efficient screening method for phage infection-related genes and identified a total of 10 genes related to the adsorption process of phage LP31. After the construction of strain C50041ΔrfaL 58−358, it was found that the knockout strain had a rough phenotype as an O-antigen-deficient strain. Adsorption rate and transmission electron microscopy experiments showed that the receptor for phage LP31 was the O9 antigen of S. Enteritidis. Homology comparison and adsorption experiments confirmed that the tail fiber protein Lp35 of phage LP31 participated in the adsorption process as a receptor-binding protein. IMPORTANCE A full understanding of the interaction between phages and their receptors can help with the development of phage-related products. Phages like LP31 with the tail fiber protein Lp35, or a closely related protein, have been reported to effectively recognize and infect multiple Salmonella serotypes. However, the role of these proteins in phage infection has not been previously described. In this study, we established an efficient screening method to detect phage adsorption to host receptors. We found that phage LP31 can utilize its tail fiber protein Lp35 to adsorb to the O9 antigen of S. Enteritidis, initiating the infection process. This study provides a great model system for further studies of how a phage-encoded receptor-binding protein (RBP) interacts with its host's RBP binding target, and this new model offers opportunities for further theoretical and experimental studies to understand the infection mechanism of phages.

With the emergence and rapid spread of“super drug-resistant”bacteria, phages have become a research hot spot for antibiotic alternatives and a new way to solve the problem of antibiotic resistance and promote the healthy development of the farming industry. The key to the therapeutic role of phages is their ability of specifically lysing their host bacteria, while the specificity of phage lysis of bacteria depends on the recognition and adsorption of phage receptor-binding proteins to the receptor. Tailed bacteriophages use a broad range of receptor-binding proteins, such as tail fiber, tail spikes and the central tail spike, to target their cognate bacterial cell surface receptors lipopolysaccharide, outer membrane protein, capsule, flagella and pili, etc.，and finally the bacteria are lysed. In the present review, we systematically summarized the research advances in the types and structures of tailed bacteriophage and its receptors. We also discussed the selection strategies of phage therapeutic agents based on the research foundation of phage-host interaction mechanism. It is aimed to provide a solid theoretical foundation for the further study of the mechanism of interaction between phages and their host bacteria, modification of phages and creation of phage biocidal agents. [ABSTRACT FROM AUTHOR]

Although more than 12,000 bacteriophages infecting mycobacteria (mycobacteriophages) have been isolated so far, there is a knowledge gap on their structure-function relationships. Here, we have explored the architecture of host-binding machineries from seven representative mycobacteriophages of the Siphoviridae family infecting Mycobacterium smegmatis, Mycobacterium abscessus, and Mycobacterium tuberculosis, using AlphaFold2 (AF2). AF2 enables confident structural analyses of large and flexible biological assemblies resistant to experimental methods, thereby opening new avenues to shed light on phage structure and function. Our results highlight the modularity and structural diversity of siphophage host-binding machineries that recognize host-specific receptors at the onset of viral infection. Interestingly, the studied mycobacteriophages' host-binding machineries present unique features compared with those of phages infecting other Gram-positive actinobacteria. Although they all assemble the classical Dit (distal tail), Tal (tail-associated lysin), and receptor-binding proteins, five of them contain two potential additional adhesion proteins. Moreover, we have identified brush-like domains formed of multiple polyglycine helices which expose hydrophobic residues as potential receptor-binding domains. These polyglycine-rich domains, which have been observed in only five native proteins, may be a hallmark of mycobacteriophages' host-binding machineries, and they may be more common in nature than expected. Altogether, the unique composition of mycobacteriophages' host-binding machineries indicate they might have evolved to bind to the peculiar mycobacterial cell envelope, which is rich in polysaccharides and mycolic acids. This work provides a rational framework to efficiently produce recombinant proteins or protein domains and test their host-binding function and, hence, to shed light on molecular mechanisms used by mycobacteriophages to infect their host. IMPORTANCE Mycobacteria include both saprophytes, such as the model system Mycobacterium smegmatis, and pathogens, such as Mycobacterium tuberculosis and Mycobacterium abscessus, that are poorly responsive to antibiotic treatments and pose a global public health problem. Mycobacteriophages have been collected at a very large scale over the last decade, and they have proven to be valuable tools for mycobacteria genetic manipulation, rapid diagnostics, and infection treatment. Yet, molecular mechanisms used by mycobacteriophages to infect their host remain poorly understood. Therefore, exploring the structural diversity of mycobacteriophages' host-binding machineries is important not only to better understand viral diversity and bacteriophage-host interactions, but also to rationally develop biotechnological tools. With the powerful protein structure prediction software AlphaFold2, which was publicly released a year ago, it is now possible to gain structural and functional insights on such challenging assemblies. [ABSTRACT FROM AUTHOR]

Host range is a major determinant in the industrial utility of a bacteriophage. A model host range permits broad recognition across serovars of a target bacterium while avoiding cross-reactivity with commensal microbiota. Searching for a naturally occurring bacteriophage with ideal host ranges is challenging, time-consuming, and restrictive. To address this, SPTD1.NL, a previously published luciferase reporter bacteriophage for Salmonella, was used to investigate manipulation of host range through receptor-binding protein engineering. Similar to related members of the Ackermannviridae bacteriophage family, SPTD1.NL possessed a receptor-binding protein gene cluster encoding four tailspike proteins, TSP1-4. Investigation of the native gene cluster through chimeric proteins identified TSP3 as the tailspike protein responsible for Salmonella detection. Further analysis of chimeric phages revealed that TSP2 contributed off-target Citrobacter recognition, whereas TSP1 and TSP4 were not essential for activity against any known host. To improve the host range of SPTD1.NL, TSP1 and TSP2 were sequentially replaced with chimeric receptor-binding proteins targeting Salmonella. This engineered construct, called RBP-SPTD1-3, was a superior diagnostic reporter, sensitively detecting additional Salmonella serovars while also demonstrating improved specificity. For industrial applications, bacteriophages of the Ackermannviridae family are thus uniquely versatile and may be engineered with multiple chimeric receptor-binding proteins to achieve a custom-tailored host range. [ABSTRACT FROM AUTHOR]

Middle East respiratory syndrome coronavirus (MERS-CoV) causes viral pneumonia disease in humans. The close contact with camels and drinking milk may cause MERS-CoV transfer to humans. This study was designed to detect the existence of MERS-CoV in camel milk samples collected from healthy animals of various barns located around Saudi Arabia. Camel milk samples were examined for MERS-CoV RNA by real time-quantitative polymerase chain reaction, also enzyme-linked immunosorbent assay was performed to detect IgG antibodies directed against Middle East respiratory syndrome receptor-binding domain. Among 83 camel milk samples tested, the result showed that seven samples (8.4%) were positive for MERS-CoV RNA, whereas 40.9% of camel milk samples had antibodies directed against MERS-CoV. The findings indicate that some regions (East and South part) are characterized by a high incidence of viral antibodies. The Southwestern region displayed the lowest infection rates. Among the camel breeds, the lowest positivity for detection of MERS-CoV RNA and IgG antibodies was found in Sahilia. This could be related to a higher resistance to viral infection of the breed Sahilia and/or to the geographical origin of the camels sampled in the study. This needs to be more explored to reduce spread of infection and also to understand the underlying reasons. The presence of viral RNA in camel milk samples warrants for measures to prevent possible foodborne transmission of MERS-CoV through milk consumption. [ABSTRACT FROM AUTHOR]

Phages demonstrate remarkable promise as antimicrobial agents against antibiotic-resistant bacteria. However, the emergence of phage-resistant strains poses challenges to their effective application. In this paper, we presented the isolation of a phage adaptive mutant that demonstrated enhanced and sustained antibacterial efficacy through the co-evolution of Klebsiella pneumoniae ( K. pneumoniae ) 111-2 and phage ZX1Δint in vitro . Our experiments revealed that phage ZX1Δint successfully completed the adsorption phase by binding to the host surface, specifically targeting the capsular polysaccharide (CPS) receptor via the primary receptor-binding protein (RBP) ORF60 and the auxiliary RBP ORF59. Upon exposure to phage predation, mutations in genes wbaP , wbaZ or wzc , which encode the synthesis of the CPS, conferred resistance by reducing phage adsorption. In response to these host defense mechanisms, the adaptive mutant phages have evolved to utilize an alternative binding site located on an O-antigen site of lipopolysaccharide (LPS) through a mutation in the backup RBP ORF59. This evolutionary change enabled the phages to reinfect previously phage-resistant strains. Notably, the adaptive mutant phage PR2 carrying the ORF59 mutation Q777R, demonstrated the capacity to infect both wild-type and resistant strains, exhibiting prolonged antimicrobial activity against the wild strains. In conclusion, our findings elucidated a complex phage-host adsorption-antagonism mechanism characterized by mutation-driven alterations in phage receptor recognition. This work contributes to a deeper understanding of phage adaptability and highlights the potential for phages to combat phage-resistant bacteria through an in vitro evolutionary approach.

Receptor-binding proteins (RBPs) are located at the viral tail and mediate the initial recognition of phage to a specific bacterial host. Phage RBPs have co-evolved with numerous types of host receptors resulting in the formation of a diverse assortment of cognate pairs of RBP-receptors that function during the phage attachment step. Although several Clostridioides difficile bacteriophages have been discovered, their RBPs are poorly described. Using homology analysis, putative prophage-tail structure (pts) genes were identified from the prophage genome of the C. difficile HN10 strain. Competition and enzyme-linked immunosorbent assays, using recombinant PtsHN10M, demonstrated the interaction of this Pts to C. difficile cells, suggesting a role as a phage RBP. Gel filtration and cross-linking assay revealed the native form of this protein as a homotrimer. Moreover, truncated variants indicated that the C-terminal domain of PtsHN10M was important for binding to C. difficile cells. Interaction of PtsHN10M was also observed to the low-molecular weight subunit of surface-layer protein A (SlpA), located at the outermost surface of C. difficile cells. Altogether, our study highlights the function of PtsHN10M as an RBP and potentially paves the way toward phage engineering and phage therapy against C. difficile infection.

Receptor-binding proteins (RBPs) of bacteriophages initiate the infection of their corresponding bacterial host and act as the primary determinant for host specificity. The ever-increasing amount of sequence data enables the development of predictive models for the automated identification of RBP sequences. However, the development of such models is challenged by the inconsistent or missing annotation of many phage proteins. Recently developed tools have started to bridge this gap but are not specifically focused on RBP sequences, for which many different annotations are available. We have developed two parallel approaches to alleviate the complex identification of RBP sequences in phage genomic data. The first combines known RBP-related hidden Markov models (HMMs) from the Pfam database with custom-built HMMs to identify phage RBPs based on protein domains. The second approach consists of training an extreme gradient boosting classifier that can accurately discriminate between RBPs and other phage proteins. We explained how these complementary approaches can reinforce each other in identifying RBP sequences. In addition, we benchmarked our methods against the recently developed PhANNs tool. Our best performing model reached a precision-recall area-under-the-curve of 93.8% and outperformed PhANNs on an independent test set, reaching an F1-score of 84.0% compared to 69.8%. [ABSTRACT FROM AUTHOR]

Successful bacteriophage infection starts with specific recognition and adhesion to the host cell surface. Adhesion devices of siphophages infecting Gram-positive bacteria are very diverse and remain, for the majority, poorly understood. These assemblies often comprise long, flexible, and multi-domain proteins, which limits their structural analyses by experimental approaches such as X-ray crystallography and electron microscopy. However, the protein structure prediction program AlphaFold2 is exquisitely adapted to unveil structural and functional details of such molecular machineries. Here, we present structure predictions of whole adhesion devices of five representative siphophages infecting Streptococcus thermophilus, one of the main lactic acid bacteria used in dairy fermentations. The predictions highlight the mosaic nature of these devices that share functional domains for which active sites and residues could be unambiguously identified. Such AlphaFold2 analyses of phage-encoded host adhesion devices should become a standard method to characterize phage-host interaction machineries and to reliably annotate phage genomes.

Furin cleavage site (FCS) of the SARS-CoV-2 S protein, which connects the S1/S2 junction, is essential for facilitating fusion with the host cells. Wild-type (Wt) SARS-CoV-2 S protein, PDB ID: 6yvb, lacks a sequence of amino acid residues, including the FCS that links the S1/S2 junction. For the first time, we demonstrated that a stretch of 14 amino acid residues (677QTNSPRRARSVASQ689) forms an antiparallel β-sheet comprising of PRRAR sequence in the FCS within a short loop. Upon comparing the loop content of the S1/S2 junction with that of Wt SARS-CoV-2 containing PRRAR in the FCS, we observed a decrease in antiparallel β-sheet content and an increase in loop content in the B.1.1.7 variant with HRRAR in the FCS. This short loop within antiparallel β-sheet can serve as a docking site for various proteases, including TMPRSS2 and α1AT. We performed a 300-ns simulation of the SARS-CoV-2 receptor binding domain (RBD) using several antibacterial and antiviral ligands commonly used to treat various infections. Our findings indicate that the receptor binding domain (RBD) comprising the receptor binding motif (RBM) utilizes β6 and a significant portion of the loop to bind with ligands, suggesting its potential for treating SARS-CoV-2 infections., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 The Authors. Published by Elsevier B.V. All rights reserved.)

Klebsiella pneumoniae is one of the most threatening multi-drug-resistant pathogens today, with phage therapy being a promising alternative for personalized treatments. However, the intrinsic capsule diversity in Klebsiella spp. poses a substantial barrier to the phage host range, complicating the development of broad-spectrum phage-based treatments. Here, we have isolated and genomically characterized phages capable of infecting each of the acquired 77 reference serotypes of Klebsiella spp. , including capsular types widespread among high-risk K. pneumoniae clones causing nosocomial infections. We demonstrated the possibility of isolating phages for all capsular types in the collection, revealing high capsular specificity among taxonomically related phages, in contrast to a few phages that exhibited broad-spectrum infection capabilities. To decipher the determinants of the specificity of these phages, we focused on their receptor-binding proteins, with particular attention to depolymerases. We also explored the possibility of designing a broad-spectrum phage cocktail based on phages isolated in reference capsular-type strains and determining the ability to lyse relevant clinical isolates. A combination of 12 phages capable of infecting 55% of the reference Klebsiella spp. serotypes was tested on a panel of carbapenem-resistant K. pneumoniae clinical isolates. Thirty-one percent of isolates were susceptible to the phage cocktail. However, our results suggest that in a highly variable encapsulated bacterial host, phage hunting must be directed to the specific Klebsiella isolates. This work is a step forward in the understanding of the complexity of phage-host interactions and highlights the importance of implementing precise and phage-specific strategies to treat K. pneumoniae infections worldwide.IMPORTANCEThe emergence of resistant bacteria is a serious global health problem. In the absence of effective treatments, phages are a personalized and effective therapeutic alternative. However, little is still known about phage-host interactions, which are key to implementing effective strategies. Here, we focus on the study of Klebsiella pneumoniae, a highly pathogenic encapsulated bacterium. The complexity and variability of the capsule, where in most cases phage receptors are found, make it difficult for phage-based treatments. Here, we isolated a large collection of Klebsiella phages against all the reference strains and in a cohort of clinical isolates. Our results suggest that clinical isolates represent a challenge, especially high-risk clones. Thus, we propose targeted phage hunting as an effective strategy to implement phage-derived therapies. Our results are a step forward for new phage-based strategies to control K. pneumoniae infections, highlighting the importance of understanding phage-host interactions to design personalized treatments against Klebsiella spp., Competing Interests: P.D.-C. is a co-founder of Evolving Therapeutics SL and a member of its scientific advisory board.

Pseudomonas aeruginosa is a Gram-negative bacterium associated with life-threatening healthcare-associated infections (HAIs), including burn wound infections, pneumonia and sepsis. Moreover, P. aeruginosa has been considered a pathogen of global concern due to its rising antibiotic resistance. Efficient identification of P. aeruginosa would significantly benefit the containment of bacterial infections, prevent pathogen transmission, and provide orientated treatment options. The accuracy and specificity of bacterial detection are primarily dictated by the biorecognition molecules employed. Lytic bacteriophages (or phages) could specifically attach to and lyse host bacterial cells. Phages' host specificity is typically determined by their receptor-binding proteins (RBPs), which recognize and adsorb phages to particular bacterial host receptors. This makes RBPs promising biorecognition molecules in bacterial detection. This study identified a novel RBP (Gp130) from the P. aeruginosa phage Henu5. A modified enzyme-linked phage receptor-binding protein assay (ELPRA) was developed for P. aeruginosa detection employing Gp130 as biorecognition molecules. Optimized conditions provided a calibration curve for P. aeruginosa with a range from 1.0 × 103 to 1.0 × 107 CFU/mL, with a limit of detection as low as 10 CFU/mL in phosphate-buffered saline (PBS). With VITEKⓇ 2 Compact system identification (40 positives and 21 negatives) as the gold standard, the sensitivity of ELPRA was 0.950 (0.818–0.991), and the specificity was 0.905 (0.682–0.983) within a 95 %confidence interval. Moreover, the recovery test in spiked mouse serum showed recovery rates ranging from 82.79 %to 98.17%, demonstrating the prospect of the proposed ELPRA for detecting P. aeruginosa in biological samples. • A novel receptor-binding protein Gp130 was identified from P. aeruginosa phage Henu5. • Gp130 had a more broad-spectrum binding ability for P. aeruginosa strains than Henu5. • A modified ELPRA based on Gp130 has been developed for P. aeruginosa detection. • ELPRA presented high specificity and sensitivity to recognize P. aeruginosa strains. [ABSTRACT FROM AUTHOR]

Summary: SARS-CoV-2, the virus responsible for COVID-19, is causing a devastating worldwide pandemic, and there is a pressing need to understand the development, specificity, and neutralizing potency of humoral immune responses during acute infection. We report a cross-sectional study of antibody responses to the receptor-binding domain (RBD) of the spike protein and virus neutralization activity in a cohort of 44 hospitalized COVID-19 patients. RBD-specific IgG responses are detectable in all patients 6 days after PCR confirmation. Isotype switching to IgG occurs rapidly, primarily to IgG1 and IgG3. Using a clinical SARS-CoV-2 isolate, neutralizing antibody titers are detectable in all patients by 6 days after PCR confirmation and correlate with RBD-specific binding IgG titers. The RBD-specific binding data were further validated in a clinical setting with 231 PCR-confirmed COVID-19 patient samples. These findings have implications for understanding protective immunity against SARS-CoV-2, therapeutic use of immune plasma, and development of much-needed vaccines.

The Gram-positive pathogen Listeria monocytogenes can be subdivided into at least 12 different serovars, based on differential expression of a set of somatic and flagellar antigens. Of note, strains belonging to serovars 1/2a, 1/2b and 4b cause the vast majority of foodborne listeriosis cases and outbreaks. The standard protocol for serovar determination involves an agglutination method using a set of sera containing cell surface-recognizing antibodies. However, this procedure is imperfect in both precision and practicality, due to discrepancies resulting from subjective interpretation. Further, the exact antigenic epitopes remain unclear, due to the preparation of the absorbed sera and the complex nature of polyvalent antibody binding. Here, we present a novel method for quantitative somatic antigen differentiation using a set of recombinant affinity proteins (cell-wall-binding domains and receptor-binding proteins) derived from a collection of Listeria bacteriophages. These proteins enable rapid, objective and precise identification of the different teichoic acid glycopolymer structures, which represent the O-antigens, and allow a near-complete differentiation. This glycotyping approach confirmed serovar designations of over 60 previously characterized Listeria strains. Using select phage receptor-binding proteins coupled to paramagnetic beads, we also demonstrate the ability to specifically isolate serovar 1/2 or 4b cells from a mixed culture. In addition, glycotyping led to the discovery that strains designated as SV 4e actually possess an intermediate 4b-4d teichoic acid glycosylation pattern, underpinning the high discerning power and precision of this novel technique. [ABSTRACT FROM AUTHOR]

Bacteriophage has become an attractive alternative for the treatment of antibiotic-resistant Staphylococcus aureus. For the success of phage therapy, phage host range is an important criterion when considering a candidate phage. Most reviews of S. aureus (SA) phages have focused on their impact on host evolution, especially their contribution to the spread of virulence genes and pathogenesis factors. The potential therapeutic use of SA phages, especially detailed characterizations of host recognition mechanisms, has not been extensively reviewed so far. In this report, we provide updates on the study of SA phages, focusing on host recognition mechanisms with the recent discovery of phage receptor-binding proteins (RBPs) and the possible applications of SA phages in phage therapy. [ABSTRACT FROM AUTHOR]

To provide insights into phage-host interactions during winemaking, we assessed whether phenolic compounds modulate the phage predation of Oenococcus oeni. Centrifugal partition chromatography was used to fractionate the phenolic compounds of a model red wine. The ability of lytic oenophage OE33PA to kill its host was reduced in the presence of two collected fractions in which we identified five compounds. Three, namely, quercetin, myricetin and p-coumaric acid, significantly reduced the phage predation of O. oeni when provided as individual pure molecules, as also did other structurally related compounds such as cinnamic acid. Their presence was correlated with a reduced adsorption rate of phage OE33PA on its host. Strikingly, none of the identified compounds affected the killing activity of the distantly related lytic phage Vinitor162. OE33PA and Vinitor162 were shown to exhibit different entry mechanisms to penetrate into bacterial cells. We propose that ligand-receptor interactions that mediate phage adsorption to the cell surface are diverse in O. oeni and are subject to differential interference by phenolic compounds. Their presence did not induce any modifications in the cell surface as visualized by TEM. Interestingly, docking analyses suggest that quercetin and cinnamic acid may interact with the tail of OE33PA and compete with host recognition.

Bacteriophages can play beneficial roles in phage therapy and destruction of food pathogens. Conversely, they play negative roles as they infect bacteria involved in fermentation, resulting in serious industrial losses. Siphoviridae phages possess a long non-contractile tail and use a mechanism of infection whose first step is host recognition and binding. They have evolved adhesion devices at their tails’ distal end, tuned to recognize specific proteinaceous or saccharidic receptors on the host’s surface that span a large spectrum of shapes. In this review, we aimed to identify common patterns beyond this apparent diversity. To this end, we analyzed siphophage tail tips or baseplates, evaluating their known structures, where available, and uncovering patterns with bioinformatics tools when they were not. It was thereby identified that a triad formed by three proteins in complex, i.e., the tape measure protein (TMP), the distal tail protein (Dit), and the tail-associated lysozyme (Tal), is conserved in all phages. This common scaffold may harbor various functional extensions internally while it also serves as a platform for plug-in ancillary or receptor-binding proteins (RBPs). Finally, a group of siphophage baseplates involved in saccharidic receptor recognition exhibits an activation mechanism reminiscent of that observed in Myoviridae.

With the availability of an increasing number of 3D structures of bacteriophage components, combined with powerful in silico predictive tools, it has become possible to decipher the structural assembly and functionality of phage adhesion devices. In the current study, we examined 113 members of the 936 group of lactococcal siphophages, and identified a number of Carbohydrate Binding Modules (CBMs) in the neck passage structure and major tail protein, on top of evolved Dit proteins, as recently reported by us. The binding ability of such CBM-containing proteins was assessed through the construction of green fluorescent protein fusion proteins and subsequent binding assays. Two CBMs, one from the phage tail and another from the neck, demonstrated definite binding to their phage-specific host. Bioinformatic analysis of the structural proteins of 936 phages reveals that they incorporate binding modules which exhibit structural homology to those found in other lactococcal phage groups and beyond, indicating that phages utilize common structural “bricks” to enhance host binding capabilities. The omnipresence of CBMs in Siphophages supports their beneficial role in the infection process, as they can be combined in various ways to form appendages with different shapes and functionalities, ensuring their success in host detection in their respective ecological niches.

The R-type pyocins are high-molecular weight bacteriocins produced by some strains of Pseudomonas aeruginosa to specifically kill other strains of the same species. Structurally, the R-type pyocins are similar to “simple” contractile tails, such as those of phage P2 and Mu. The pyocin recognizes and binds to its target with the help of fibers that emanate from the baseplate structure at one end of the particle. Subsequently, the pyocin contracts its sheath and drives the rigid tube through the host cell envelope. This causes depolarization of the cytoplasmic membrane and cell death. The host cell surface-binding fiber is ~340 Å-long and is attached to the baseplate with its N-terminal domain. Here, we report the crystal structures of C-terminal fragments of the R1 and R2 pyocin fibers that comprise the distal, receptor-binding part of the protein. Both proteins are ~240 Å-long homotrimers in which slender rod-like domains are interspersed with more globular domains—two tandem knob domains in the N-terminal part of the fragment and a lectin-like domain at its C-terminus. The putative substrate binding sites are separated by about 100 Å, suggesting that binding of the fiber to the cell surface causes the fiber to adopt a certain orientation relative to the baseplate and this then triggers sheath contraction.

Background: Despite continuous research efforts, bacterio(phages) infecting Lactococcus lactis starter strains persist as a major threat to dairy fermentations. The lactococcal P335 phages, which are currently classified into four subgroups (I-IV), are the second most frequently isolated phage group in an industrial dairy context. Results: The current work describes the isolation and comparative genomic analysis of 17 novel P335 group phages. Detailed analysis of the genomic region of P335 phages encoding the so-called "baseplate", which includes the receptor binding protein (RBP) was combined with a functional characterization of the RBP of sub-group III and IV phages. Additionally, calcium-dependence assays revealed a specific requirement for calcium by sub-group IV phages while host range analysis highlighted a higher number of strains with CWPS type A (11 of 39 strains) are infected by the P335 phages assessed in this study than those with a C (five strains), B (three of 39 strains) or unknown (one of 39 strains) CWPS type. Conclusions: These analyses revealed significant divergence among RBP sequences, apparently reflecting their unique interactions with the host and particularly for strains with a type A CWPS. The implications of the genomic architecture of lactococcal P335 phages on serving as a general model for Siphoviridae phages are discussed. [ABSTRACT FROM AUTHOR]

Objectives: To characterize the lytic coliphage vB_EcoM_JS09 (phage JS09) isolated from sewage samples of a swine farm in Jiangsu Province, China, which infects antibiotic-resistant avian pathogenic Escherichia coli (APEC) and enterotoxigenic E. coli (ETEC). Methods and Results: Transmission electron microscopy revealed that phage JS09 has an isometric icosahedral head (76 nm in diameter) and a long contractile tail (140 nm in length) and features a T-even morphology. Its latent period was 30 min and the average burst size was 79 phage particles per infected cell. It attached to the host cells within 9 min. JS09 could infect 16 clinically isolated APEC and ETEC strains and the laboratory-engineered E. coli K and B strains. Ten of the clinical isolates of E. coli were resistant to antibiotics. At a multiplicity of infection of 10, 3, 1, or 0.3, the phage caused rapid cell lysis within 2 h, resulting in 5- to 10-fold reductions in cell concentration. Sequencing of the JS09 genome revealed a 169.148-kb linear but circularly permuted and terminally redundant dsDNA with 37.98% G+C content. Two hundred seventy-three open reading frames were predicted to be coding sequences, 135 of which were functionally defined and organized in a modular format which includes modules for DNA replication, DNA packaging, structural proteins, and host cell lysis proteins. Phage JS09 is assigned to the Caudovirales order (Myoviridae phage family), and it is considered a T4-like phage based on its morphological, genomic, and growth characteristics. JS09 gp37, a receptor-binding protein (RBP) important for host cell infection, shares little homology with other RBP in the NCBI database, which suggests that the variable regions in gp37 determine the unique host range of phage JS09. Protein sequence comparisons cluster the putative 'RBP' of JS09 much more closely with those of Yersinia phage phiD1, phage TuIa, and phage TuIb. Conclusions: A novel lytic coliphage named JS09 was isolated from sewage samples of a swine farm in Jiangsu Province, China. It could infect antibiotic-resistant APEC and ETEC. The morphological, genomic, and growth characteristics of JS09 were studied, and this will be helpful for phage therapy in controlling diseases caused by APEC and ETEC. © 2015 S. Karger AG, Basel [ABSTRACT FROM AUTHOR]

Bacillus anthracis is a potent biowarfare agent, able to be highly lethal. The bacteria dwell in the soil of certain regions, as natural flora. Bacteriophages or their lytic enzymes, endolysins, may be an alternative for antibiotics and other antibacterials to fight this pathogen in infections and to minimize environmental contamination with anthrax endospores. Upon screening environmental samples from various regions in Poland, we isolated three new siphophages, J5a, F16Ba, and z1a, specific for B. anthracis. They represent new species related to historical anthrax phages Gamma, Cherry, and Fah, and to phage Wbeta of Wbetavirus genus. We show that the new phages and their closest relatives, phages Tavor_SA, Negev_SA, and Carmel_SA, form a separate clade of the Wbetavirus genus, designated as J5a clade. The most distinctive feature of J5a clade phages is their cell lysis module. While in the historical phages it encodes a canonical endolysin and a class III holin, in J5a clade phages it encodes an endolysin with a signal peptide and two putative holins. We present the basic characteristic of the isolated phages. Their comparative genomic analysis indicates that they encode two receptor-binding proteins, of which one may bind a sugar moiety of B. anthracis cell surface. [ABSTRACT FROM AUTHOR]

To provide insights into phage-host interactions during winemaking, we assessed whether phenolic compounds modulate the phage predation of Oenococcus oeni. Centrifugal partition chromatography was used to fractionate the phenolic compounds of a model red wine. The ability of lytic oenophage OE33PA to kill its host was reduced in the presence of two collected fractions in which we identified five compounds. Three, namely, quercetin, myricetin and p-coumaric acid, significantly reduced the phage predation of O. oeni when provided as individual pure molecules, as also did other structurally related compounds such as cinnamic acid. Their presence was correlated with a reduced adsorption rate of phage OE33PA on its host. Strikingly, none of the identified compounds affected the killing activity of the distantly related lytic phage Vinitor162. OE33PA and Vinitor162 were shown to exhibit different entry mechanisms to penetrate into bacterial cells. We propose that ligand-receptor interactions that mediate phage adsorption to the cell surface are diverse in O. oeni and are subject to differential interference by phenolic compounds. Their presence did not induce any modifications in the cell surface as visualized by TEM. Interestingly, docking analyses suggest that quercetin and cinnamic acid may interact with the tail of OE33PA and compete with host recognition. [ABSTRACT FROM AUTHOR]

The high specificity of bacteriophages is driven by their receptor-binding proteins (RBPs). Many Klebsiella bacteriophages target the capsular exopolysaccharide as the receptor and encode RBPs with depolymerase activity. The modular structure of these RBPs with an N-terminal structural module to attach the RBP to the phage tail, and a C-terminal specificity module for exopolysaccharide degradation, supports horizontal transfer as a major evolutionary driver for Klebsiella phage RBPs. We mimicked this natural evolutionary process by the construction of modular RBP chimeras, exchanging N-terminal structural modules and C-terminal specificity modules. All chimeras strictly follow the capsular serotype specificity of the C-terminal module. Transplanting chimeras with a K11 N-terminal structural RBP module in a Klebsiella phage K11 scaffold results in a capsular serotype switch and corresponding host range modification of the synthetic phages, demonstrating that horizontal transfer of C-terminal specificity modules offers Klebsiella phages an evolutionary highway for rapid adaptation to new capsular serotypes. IMPORTANCE The antimicrobial resistance crisis has rekindled interest in bacteriophage therapy. Phages have been studied over a century as therapeutics to treat bacterial infections, but one of the biggest challenges for the use of phages in therapeutic interventions remains their high specificity. In particular, many Klebsiella phages have a narrow spectrum constrained by the high diversity of exopolysaccharide capsules that shield access to the cells. In this work, we have elaborated how Klebsiella phages deal with this high diversity by exchanging building blocks of their receptor-binding proteins., (Copyright © 2021 Latka et al.)