Bacteriophages are viruses that specifically infect bacteria and are found in nearly all microbial ecosystems. Estimates suggest their global numbers reach 10²⁹ -10³⁰ particles, making them one of the most abundant biological entities on Earth. These viruses are environmentally stable and, once inside a susceptible host, replicate efficiently.¹ Since their discovery over a century ago, phages have attracted considerable attention for their practical applications. They have been explored as alternatives to antibiotics, as tools for bacterial biocontrol, and as vectors in gene delivery studies. Structurally simple and genetically compact, phages are obligate parasites that rely entirely on the replication and translation machinery of their bacterial hosts to produce progeny. Most phages are highly specific, infecting only one bacterial species or a narrow range of strains.² Beyond their therapeutic potential, phages play an important ecological role. By infecting and lysing bacteria, they influence host population size, species composition, and metabolic activity, thereby shaping the dynamics of microbial communities. This capacity to modulate bacterial diversity and function makes them key contributors to ecosystem balance and a useful model for studying the molecular basis of host-microbe interactions.³

Most bacteriophages belong to the order Caudovirales and are characterized as tailed viruses with a double-stranded DNA genome. This predominance has been documented in large-scale surveys of the virion.⁴ Phage replication can proceed through several infection strategies. In the lysogenic pathway, for example, the phage genome, known as a prophage, becomes stably maintained within the bacterial cell. It may integrate into the host chromosome or persist as a plasmid-like element. In either case, the prophage is replicated along with the host genome and can remain dormant for extended periods, while expressing specific regulatory proteins that control both host and phage gene activity. Under stressful environmental conditions, prophages may exit their dormant state, transitioning into the lytic cycle, which culminates in the lysis of the host cell and the release of new virions. Alternatively, pseudo-lysogeny represents a non-canonical phage life strategy wherein the phage neither lyses the host nor integrates into its genome, avoiding stable long-term residence.^5,6 A critical and universal step in all phage life cycles is adsorption, which hinges upon the precise recognition of specific host surface proteins. Independent of the subsequent replication strategy, the initial phase of infection invariably involves the identification and attachment of the phage particle to the bacterial cell surface. This adsorption process is a fundamental mechanism shared among viruses infecting organisms across the three domains of life.^7,8

Although phages are small and present at low density on the bacterial surface, they usually succeed in attaching to their receptors after very few encounters. The speed at which a bacterial population is cleared depends largely on how quickly virions adsorb to their hosts. In simplified experimental systems, this adsorption rate can be estimated from basic factors such as phage diffusion, bacterial cell size, and the likelihood that a collision results in a stable attachment. When bacterial numbers are high, free phages disappear from the environment more quickly because adsorption events are more frequent. Conversely, when phage concentrations rise, bacteria are infected at a faster rate.⁹

In the past decade, interest in bacteriophages has grown well beyond microbiology and now includes applications in biotechnology, medicine, agriculture, and even food safety.^10-12 For these applications to be both safe and effective, it is essential to understand in detail how phages interact with their bacterial hosts. This involves not only identifying the receptors that allow phage attachment but also clarifying the mechanisms of infection and ways bacteria evolve resistance. In this review, we highlight current knowledge on these processes, with particular emphasis on host cell recognition, receptor specificity, and the molecular strategies bacteria use to defend themselves.

Bacteriophage structure and classification
Bacteriophage morphology plays a central role in shaping their interactions with bacterial hosts. Variations in capsid architecture and tail structures influence how effectively a phage can recognize surface receptors, attach to the cell, and initiate infection. Phages that have been described to date display considerable diversity in their host specificity and infection strategies, ranging from narrow to broad host ranges and from strictly lytic to more complex lifestyles. As a result, members of different taxonomic groups often affect microbial communities in distinct ways, reflecting their structural and functional adaptations to particular ecological environments. The morphology of bacteriophages fundamentally governs their interactions with bacterial hosts. Structural complexities, including the organization of the capsid and tail apparatus, critically determine their capacity to recognize, adhere to, and penetrate bacterial cells. Currently characterized bacteriophages exhibit remarkably diverse life strategies, particularly in terms of host range specificity and lytic capabilities. Consequently, phages belonging to distinct taxonomic groups may exert unique influences on the microbial dynamics within specific ecological niches.¹³

Phages possess a relatively simple architecture, consisting of a proteinaceous capsid that encases their nucleic acid, which may be either DNA or RNA. Based on their structural morphology, they are broadly categorized into tailed, non-tailed, and filamentous types.¹⁴ Among these, tailed phages are the most extensively studied. They typically contain three major parts: a capsid that contains the genome, a tail that acts as a conduit for DNA delivery into the host cell, and a specialized tail tip structure that is responsible for recognizing and binding to bacterial receptors. The assembly of double-stranded DNA (dsDNA) phages occurs in a stepwise manner. First, capsid proteins form a precursor “prohead” around a portal complex. The viral genome is then actively pumped into this prohead by an ATP-driven motor. Once the DNA is fully packaged, the head undergoes maturation to stabilize the enclosed genome. Finally, tail and neck components are attached, giving rise to a complete and infectious viral particle.¹⁵

Bacteriophages were initially categorized into four primary morphological groups: Tailed phages (Caudoviridae), Icosahedral or polyhedral form (Microviridae), filamentous phages (Inoviridae), and pleomorphic types (Plasmaviridae)

Among bacteriophages, the tailed types grouped under the order Caudovirales are by far the most widespread. It is estimated that they account for close to 96% of all known phages. These viruses typically carry double-stranded DNA, which is packaged inside an icosahedral head and linked to a tail structure. The tail is more than a passive appendage; it acts as a nanomachine that mediates host recognition, binding, and the transfer of genetic material. Tail morphology has been used as a taxonomic marker, leading to the division of Caudovirales into three families: Myoviridae, with their contractile tails; Siphoviridae, which have long but non-contractile and flexible tails; and Podoviridae, characterized by their short, stubby tails.¹⁶ More recently, however, the International Committee on Taxonomy of Viruses (ICTV) revised this system, bringing these families under a broader class, Caudoviricetes, to better reflect genome-based and evolutionary relationships.¹⁷ Members of this class are not just abundant but also ecologically significant. Through both lytic and lysogenic interactions, they help regulate microbial populations. Beyond ecology, they are attracting interest in applied fields ranging from therapeutic development to biotechnology and bacterial genetics.

The family Microviridae consists of small, non-enveloped bacteriophages that carry single-stranded DNA within an icosahedral capsid. Within this group, three main lineages have been described. The genus Microvirus infects free-living bacteria such as enterobacteria. In contrast, members of the subfamily Gokushovirinae specialize in intracellular parasitic bacteria, including Chlamydia and Spiroplasma. A third lineage, the Alpavirinae, has so far been identified only in the form of prophages. Once these viruses enter a bacterial cell, they produce a complementary minus-strand of DNA. This strand provides the template needed for transcription of viral mRNAs and subsequent gene expression.^18,19

The Inoviridae family comprises non-enveloped, flexible, filamentous bacteriophages that primarily infect Gram-negative bacteria by adsorbing to bacterial pili. Their circular, positive-sense single-stranded genomes range from 5.5 to 10.6 kb, encoding 4-15 proteins. These phages replicate via a rolling circle mechanism and release progeny through extrusion without lysing the host. The family includes 43 species across two genera, with genomes that may remain episomal or integrate with the bacterial chromosome.²⁰

The Plasmaviridae family comprises quasi-spherical, membrane-enveloped bacteriophages that infect cell-wall-less bacteria, specifically Acholeplasma species. These phages possess a circular, supercoiled double-stranded DNA genome approximately 12 kb in length, containing 14 open reading frames encoding at least 15 proteins. With a diameter ranging from 50-125 nm, they replicate within the host cytoplasm. The family has no assigned order and includes a single genus and species (Acholeplasma virus L2).²¹

Strategies underpinning the phage-host binding and invasion
Unlike typical predator-prey relationships, phage-host interactions exhibit remarkable specificity, with phage replication intricately dependent on the metabolic and replicative systems of the bacterial hosts. Bacteriophage infection begins with adsorption, a critical step involving specific interactions between phage binding proteins and bacterial surface receptors. Attachment of the bacteriophage to the host cell may result from Brownian motion, passive diffusion, or directional fluid-mediated transport.²² There are two types of phage adsorption to the host cell. Reversible attachment enables phage particles to detach and subsequently infect other cells of the same bacterial strain within the culture. Upon successful engagement with primary receptors, this transient interaction transitions into irreversible anchoring, triggering the activation of the phage’s injection apparatus. Adsorption not only initiates infection but also defines the host range by determining which bacterial strains can be targeted. Consequently, it serves as the primary determinant of phage-host specificity.⁷

The process is initiated by a reversible interaction between the phage and the bacterial cell surface, facilitated by the binding of phage tail fibres or specialised appendages to primary receptors located on the bacterial outer envelope- typically lipopolysaccharides (LPS), outer membrane proteins, teichoic acids, or surface structures such as flagella and pili. This transient association serves as a preliminary host recognition step, enabling the phage to assess the compatibility of the potential host before committing to infection.^23,24 The vast majority of bacteriophages are classified under the order Caudovirales and are distinguished by their tail structures, which play a pivotal role in host recognition and the delivery of phage genetic material into the bacterial cell.²⁵

A critical initial phase in the life cycle of most bacteriophages entails the attachment of the virion to a specific recognition site on the host cell membrane, subsequently facilitating the injection of phage genomic DNA into the bacterial cell. A given bacteriophage typically exhibits a narrow host range, often targeting a specific microbial strain. This high degree of host specificity is primarily governed by the adsorption process, which is dictated by the structural and molecular characteristics of receptor molecules present on the surface of the bacterial cell against phage receptor-binding proteins (RBPs).²²

Receptor-binding proteins are essential components of bacteriophages as they determine the recognition and attachment to the host. In general, RBPs contain three regions: a conserved N-terminal domain, a hinge-like flexible segment, and a C-terminal domain, which often participates in receptor recognition and, in some cases, enzymatic degradation of host surface proteins.²⁶ The exact architecture of RBPs differs across phages, reflecting variations in phage morphology and strategies of host interaction. For example, in the Podovirus Sf6, which infects Shigella flexneri, short tail fibres (tail spike proteins) first engage with the lipopolysaccharide (LPS) layer, allowing a reversible binding step.²⁷ In contrast, in the well-studied Escherichia coli phage T4, the gp12 protein located at the short tail fibres mediates irreversible binding to the LPS core region.²⁸ A different arrangement is seen in Siphoviridae, such as Lactococcus lactis phage TP901-1, where RBPs form part of a larger structural baseplate complex that coordinates attachment.²⁹

The bacterial cell envelope
As illustrated in Figure 1, Bacterial surfaces provide a variety of molecular targets that bacteriophages can exploit as receptors. The type of receptor available depends on the bacterium-phage pairing and can range from short peptide sequences to more complex polysaccharide structures.

The architecture of the cell envelope differs markedly between Gram-positive and Gram-negative organisms, and these differences shape both bacterial physiology and their interactions with phages. Gram-positive bacteria contain a thick peptidoglycan layer with embedded teichoic acids but lack an outer membrane. By contrast, Gram-negative bacteria have a much thinner peptidoglycan layer situated between the cytoplasmic membrane and an outer membrane rich in lipopolysaccharides (LPS). Such variations not only contribute to differences in antibiotic sensitivity but also strongly influence the way bacteriophages recognize and adsorb to their hosts.³⁰

Surface-localized receptors in Gram-positive bacterial cell wall
Gram-positive bacteria are characterized by a relatively thick cell wall that contains a large amount of peptidoglycan proteins together with teichoic acid (TA). In some species, Teichoic acid may represent more than half of the total cell wall mass. These polymers occur in two major forms: lipoteichoic acids (LTA), which are linked to the cytoplasmic membrane, and wall teichoic acids (WTA), which are covalently associated with the peptidoglycan layer.³¹ The peptidoglycan itself forms the main structural framework of the wall and lies just outside the cytoplasmic membrane, a feature that is conserved across most bacterial groups. While its primary role is to preserve cell shape and structural integrity, it also provides anchoring sites for various proteins and lipopolysaccharides. Structurally, peptidoglycan comprises linear glycan chains composed of alternating N-acetylglucosamine and N-acetylmuramic acid residues, linked via 1-4 glycosidic bonds, which are further crosslinked by short peptide bridges to form a robust, mesh-like matrix.³² Bam35, a temperate double-stranded DNA bacteriophage that infects Gram-positive Bacillus thuringiensis, uses a sequential entry mechanism involving three distinct stages: rapid receptor binding, peptidoglycan penetration, and interaction with the plasma membrane. Binding to the bacterial surface is dependent on N-acetyl-muramic acid, while peptidoglycan degradation is mediated by a virion-associated hydrolytic activity, and both of these steps occur independently of divalent cations. In contrast, successful penetration through the plasma membrane requires the presence of divalent cations, highlighting a unique ion-dependence at the final entry step.³³

In Listeria Phage A500, host recognition occurs via protein-glycopolymer interaction involving the endolysin Ply500, which specifically targets Listeria serovars 4, 5, and 6 through its l-alanoyl-d-glutamate endopeptidase activity. The cell wall binding domain (CBD500) of Ply500 specifically binds 3’-O-acetylated N-acetylglucosamine (GlcNAc) residues in the wall teichoic acid (WTA) polymer. These GlcNAc moieties serve as critical epitopes recognised by a binding site within SH3b-like repeats of the viral domain, ensuring selective cell wall interaction.³⁴ In bacteriophage SPP1, irreversible adsorption to its host, Bacillus subtilis, is facilitated by the host cell membrane protein YueB. However, reversible attachment primarily targets the glucosylated poly (glycerophosphate) chains of the cell wall teichoic acid.³⁵ Similarly, in the broad host range, virulent Listeria phage A511, receptor binding protein Gp108 specifically recognizes N-acetylglucosamine and rhamnose substituents present on the wall teichoic acids of the bacterial cell wall. These examples highlight the pivotal role of teichoic acid modifications in phage host recognition and specificity.³⁶

Upon attachment of the phage to the bacterial surface, a mechanical or chemical signal is transmitted along the tail fiber to the capsid, where the viral genome is encapsulated. The contraction of the phage’s tail sheath triggers the precise penetration of the bacterial cell wall by the internal tail tube, facilitating the release of the viral genome through the portal pore located at a distant capsid vertex. The DNA is subsequently translocated into the host cytoplasm in a sequential manner, leaving the empty phage particle firmly anchored to the bacterial surface.³⁷

Surface-localized receptors in the Gram-negative bacterial cell wall
Bacteriophages targeting Gram-negative bacteria must first overcome the lipopolysaccharide (LPS) barrier, a structurally robust component of the outer membrane stabilized by strong noncovalent interactions. Unlike peptidoglycan, which is susceptible to enzymatic degradation, LPS poses a formidable challenge, prompting many phages to utilize specialized proteins to navigate through it and engage outer membrane protein (Omp) receptors a strategy observed across all tail morphotypes.³⁹

Bacteriophages that target the smooth (S) type of lipopolysaccharide (LPS) exhibit an exceptionally narrow host range due to the high structural variability of the O-antigen across different bacterial taxa. In contrast, phages that recognize rough (R) type LPS or vice versa tend to possess a broad host spectrum, as the conserved nature of the LPS core region is shared among various species and genera of Gram-negative bacteria.⁴⁰

Recent advancements in the characterization of novel phage isolates infecting Gram-negative bacteria have leveraged lipopolysaccharide profiling to elucidate phage infection strategies, particularly those targeting hosts with impaired or modified LPS biosynthesis.⁴¹

The host cell receptors interact with phage proteins either reversibly or irreversibly, and these distinct modes of adsorption are mediated by different sets of viral proteins and host receptors, highlighting a functional specialization in the phage infection process.⁴² The attachment to the host begins with a selective but reversible interaction with LPS on the bacterial surface. Tail-associated enzymes, particularly those embedded within spike structures, exhibit LPS-degrading activity by targeting and cleaving O-antigen repeats. By breaking down the LPS barrier, the phage gains access to deeper cell surface components, including secondary receptors or the outer membrane itself, where stable and irreversible binding is ultimately established. One of the previously explained examples, where T4 bacteriophage mediates the reversible attachment to the host E. coli surface, is by gp53 proteins located in the long tail fibres, whereas irreversible binding involves gp12 proteins in the short tail fibres, which specifically recognize the heptose region within the host’s lipopolysaccharide.²⁸

Peptidoglycan, although shielded by the outer membrane in Gram-negative bacteria, plays a crucial role during the later stages of phage entry by acting as a secondary barrier and target for enzymatic processing. Escherichia phage T7, a well-studied Podovirus infecting Escherichia coli, follows a multi-step infection pathway involving initial attachment to the outer membrane protein FhuA, followed by localized degradation of the peptidoglycan to enable DNA translocation.⁴³ This process is mediated by a virion-associated lysozyme domain within the tail machinery, which specifically cleaves the β-1,4 glycosidic bonds between N-acetylglucosamine and N-acetylmuramic acid residues. While peptidoglycan recognition in T7 is secondary to outer membrane binding, its efficient hydrolysis is essential for successful DNA delivery into the cytoplasm, highlighting a coordinated interaction between outer membrane receptor engagement and peptidoglycan penetration in Gram-negative hosts.⁴⁴

The flagella are the initial host contact targets on the bacterial cell, by which the phages enhance their chances of establishing attachment and achieving successful infection. The interaction between the phage head-associated filament and the bacterial flagellum represents a distinct infection strategy, wherein the filament transiently coils around the rotating flagellum, facilitating phage transport towards the cell pole and proximal host receptors.⁴⁵

In some instances, the phage adsorption process may involve multiple receptors on the bacterial surface, indicating a mechanism that requires sequential or simultaneous interaction with two distinct host receptors. For example, Flagellotropic phage 7-7-1, a member of Myoviridae, infects the soil bacterium Agrobacterium spp. strain H13-3 by targeting actively rotating bacterial flagella. The number, length, and rotational speed of the flagella influence the efficiency of infection. The phage initially binds to the flagellar filament and is subsequently translocated towards the bacterial surface via clockwise rotation. Upon reaching the cell surface, the phage engages with the lipopolysaccharide (LPS), enzymatically degrades it to access the outer membrane, and then injects its genomic DNA into the host cell through a contractile, syringe-like tail structure.⁴⁶

A distinct class of bacteriophages exhibits plasmid dependency, wherein infection is restricted to bacterial cells that harbor and actively express specific plasmid-encoded genes required for phage adsorption and entry. These typically exploit plasmid-derived surface structures – most notably conjugative pili -as receptors for attachment. A well-characterized example is bacteriophage MS2, which selectively infects Escherichia coli strains carrying the F (fertility) plasmid, which encodes the biosynthesis of F-pili functioning as primary receptors for MS2 binding and subsequent infection.⁴⁷ The phage maturation (A) protein specifically binds to the pilus shaft, and natural pilus retraction draws the virion towards the cell envelope, enabling direct translocation of the RNA genome into the cytoplasm. In the absence of these plasmid-encoded surface components, the bacterial host remains resistant to phage invasion. Recently, Parra et al. successfully isolated and characterized two novel dsDNA plasmid-dependent bacteriophages designated Lu221 and Hi226, which specifically target Salmonella enterica harbouring the IncN plasmid Pkm101.⁴⁸ These examples highlight how plasmid-encoded extracellular appendages can be co-opted by bacteriophages to mediate precise host recognition and efficient genome delivery.

Some bacteria develop a thick polysaccharide barrier, such as a capsule, enveloping the cell, which often acts as a key recognition site for bacteriophages. In many phage-host systems, phages employ capsule depolymerases located on tail fibres or the baseplate to degrade this layer, granting access to underlying receptors. For example, Klebsiella phage KP32 encodes two such enzymes, KP32gp37 and KP32gp38, which are highly specific for capsular serotypes K3 and K21, respectively. By targeting and breaking down these serotype-specific polysaccharides, these polymerases limit the narrow host range of phage KP32 to strains that express either the K3 or K21 capsule type.⁴⁹

Bacterial antiphage defence mechanisms
Bacteria and bacteriophages are locked in a perpetual evolutionary arms race, marked by dynamic cycles of coevolution and coexistence. Within this ongoing interaction, bacteria evolve phage resistance mechanisms to protect their populations, while phages simultaneously adapt through counter-resistance strategies that enable them to bypass these defences and maintain infectivity.⁵⁰

As shown in Figure 2, bacteria orchestrate intricate defense mechanisms that specifically target distinct phases of the bacteriophage life cycle. These defenses encompass the inhibition of phage adsorption, prevention of viral genome injection, and disruption of essential processes, including phage DNA replication, capsid formation, and progeny assembly. Moreover, components derived from these phage resistance systems, most notably the CRISPR-Cas machinery, have laid the foundation for the development of cutting-edge genome editing technologies.

Blocking Phage binding/Interference with phage adsorption
The initiation of bacteriophage infection is universally dependent on the successful adsorption of the virion to specific receptors on the bacterial cell surface. To counter this, certain bacterial strains have evolved sophisticated strategies to impede phage attachment. These adsorption-inhibiting mechanisms generally fall into three categories: direct masking or modification of surface receptors, secretion of extracellular matrix components that hinder access, and production of competitive inhibitors that obstruct phage binding.

Receptor blocking
OmpA is a major outer membrane protein in Escherichia coli that not only facilitates F-mediated conjugation but also serves as a receptor for various bacteriophages. Studies have indicated a potential interaction between the F-plasmid encoded lipoprotein TraT and OmpA of the bacteria, wherein TraT appears to interfere with the binding of OmpA-specific phages, likely through direct interaction. Additionally, notable sequence homology between TraT and phage protein 38-known to recognize bacterial outer membrane proteins-further supports its role in modulating phage-host interactions.⁵¹

Competitive inhibitor production
Outer membrane vesicles (OMVs) appear to serve as innate decoys, shielding bacteria from the attack of virulent bacteriophages. In Vibrio cholerae, secreted OMVs have been shown to function as a protective barrier against predation by three lytic phages-ICP1, ICP2, and ICP3. This OMV-mediated defense was demonstrated to be both dose-dependent and reliant on specific receptor interactions, thereby significantly limiting phage infectivity.⁵² Similarly, hyper-vesiculating mutants of Escherichia coli have been observed to produce OMVs that confer resistance not only against bacteriophage infection but also against antimicrobial peptides such as Polymyxin B and colistin. Notably, when T4 phages were co-incubated with OMVs, they exhibited rapid and irreversible binding, resulting in a marked decrease in phage infectivity due to the sequestration of virions by vesicular structures.⁵³

Extracellular matrix
In Escherichia coli K-12, a quorum-sensing-regulated antiphage defense mechanism has been observed, wherein AHL-mediated signalling in the presence of autoinducers modulates the bacterial response to reduce susceptibility to phage λ and the broad-host-range phage χ.⁵⁴ Furthermore, quorum-sensing peptides were found to induce abortive infection in E. coli by activating the mazEF toxin-antitoxin system, effectively restricting the propagation of phage P1.⁵⁵ These findings suggest that quorum-sensing-based regulation of phage sensitivity may represent a widespread strategy within microbial communities.

CRISPR-Cas mediated defense
Among the various strategies bacteria employ to defend against phage infection, the CRISPR-Cas system (Clustered Regularly Interspaced Short Palindromic Repeats and CRISPR-associated proteins) is unique as the only known adaptive and heritable immune mechanism.⁵⁶ It is prevalent in most archaea and nearly half of bacteria, which defends against lytic phages. This system incorporates short DNA fragments (26-72 bp) from invading phages as spacers, enabling the production of small RNAs that recognize and block future infections by matching viral sequences, thereby generating bacteriophage-insensitive mutants (BIMS).⁵⁷ In a comprehensive analysis, 44 lysogenic strains of Pseudomonas aeruginosa PA14 were evaluated for their susceptibility to infection by three distinct bacteriophages: JBD18, JBD25, and JBD67. These phages are efficiently neutralized by the native CRISPR-Cas immune system of P. aeruginosa PA14, which prevents successful phage replication. Notably, productive infection by these phages occurred only in mutant PA14 strains in which either the CRISPR loci or the associated cas genes had been deleted, underscoring the essential role of CRISPR-Cas in conferring phage resistance in this bacterial model.⁵⁸

Blocking phage genome entry
Superinfection exclusion (SIE) is a mechanism by which bacterial strains harbouring a temperate phage genome (prophage) within their chromosome gain resistance to subsequent infection by the same or closely related phages, thereby preventing viral coinfection. The principal strategies by which phages mediate SIE involve either the inactivation of host surface receptors essential for phage binding or the obstruction of phage DNA injection and intracellular translocation.⁵⁹ In the case of T-even coliphage, rapid superinfection exclusion is established through the action of two phage-encoded proteins: Imm, which blocks DNA translocation and partially restricts its release from superinfecting virions, and Sp protein, which inhibits murein degradation by the phage-associated lysozyme.⁶⁰

In Pseudomonas aeruginosa, a highly conserved Pf phage protein called PfsE (PA0721) confers resistance to superinfection by phages that utilize the type IV pilus as their receptor by interacting with PilC to block pilus assembly.⁶¹ However, the minor capsid protein pVII confers complete protection against pilus-dependent lytic phages by interacting with T4P components PilC and PilJ. In contrast, pIII offers partial superinfection exclusion by interacting with PilJ and TolR/TolA, providing limited defense against pilus-dependent phages.⁶²

Abortive infection
The toxin-antitoxin (TA) systems have emerged as a significant component of bacterial antiviral defense mechanisms by inducing cellular stress in infected bacteria, thereby disrupting the phage replication cycle and preventing the release of progeny virions. It has been increasingly recognized for its widespread presence across sequenced bacterial genomes – some of which encode dozens of distinct TA modules. These systems are generally arranged as two-gene operons comprising a toxin that disrupts essential cellular functions and a specific antitoxin that counteracts its activity under normal physiological conditions.⁶³ In Salmonella typhimurium, the retron Sen-2 element encodes the accessory toxin RcaT, which is normally inactivated by a complex formed between reverse transcriptase (RT) and multicopy single-stranded DNA (msDNA). Upon disruption of msDNA biosynthesis, RcaT is released from inhibition and becomes toxic. Phage infection can activate RcaT through direct modification of msDNA, leading to growth arrest of the host. Conversely, certain prophage-encoded proteins evade this defense by directly neutralizing RcaT activity.⁶⁴ Collectively, retron-based TA systems serve as abortive infection mechanisms that contribute to phage resistance in bacteria. Another example of DarTG, a recently identified toxin-antitoxin system with two subtypes, DarTG1 and DarTG2, that provide strong protection to Escherichia coli MG1655 against phages RB65 and T5, respectively. Upon infection, the DarT toxin – a DNA ADP-ribosyl transferase -is released and modifies phage DNA, blocking replication and preventing virion production. Some phages, particularly T-even types, have evolved resistance by acquiring mutations in their DNA polymerase or expressing the anti-DarT protein gp61.2, demonstrating phage adaptability against host defenses.⁶⁵ Another notable recent example is provided by the toxin-antitoxin immunity protein CapRel(SJ46) in E. coli, which, in contrast to the conventional one-to-one recognition paradigm, utilizes a flexible pseudo zinc finger domain to sense two structurally unrelated phage proteins the major phage capsid and small phage protein GP54Bas11. This dual recognition mechanism introduces a robust safeguard for bacteria, as phages must simultaneously mutate both triggers to fully evade detection, thereby limiting their evolutionary escape routes.⁶⁶

Nucleic acid interference
The restriction-modification (RM) system serves as a critical bacterial defense strategy against foreign genetic elements, such as bacteriophages. It consists of two coordinated enzymatic activities: a restriction endonuclease (REase) and a methyl transferase (MTase). The REase detects and cleaves specific short DNA sequences, typically 4-8 base pairs in length, if these sequences are unmethylated. To safeguard the host genome from inadvertent cleavage, the MTase selectively methylates these same recognition sites on the host DNA, thus protecting it from restriction activity. This system enables bacteria to discriminate between self and non-self-DNA. When the infecting phage DNA enters the cell and lacks the host-specific methylation, it is rapidly targeted and degraded by the REase. The level of immunity conferred by the RM system depends on the compatibility of the phage and the specificity of the RM components present in the host. Collectively, RM systems function as precise molecular sentinels, providing robust protection by promoting the selective destruction of exogenous DNA while maintaining the integrity of the host genome.^67,68

In Escherichia coli carrying a Type I RM system (e.g., EcoP1), the restriction endonuclease targets unmethylated DNA at specific recognition motifs. The complementary methyltransferase modifies the host’s DNA at these sites, thereby preventing self-restriction. Phage λ DNA grown in an RM-negative host lacks methylation and is therefore efficiently cleaved upon infection of an RM-positive strain.⁶⁹ In addition to methylation- based RM system, bacteria can modify their genomes through phosphorothioation, a process in which sulfur replaces a non-bridging oxygen in the DNA backbone and forms the basis of phosphorothioation-dependent RM systems. This chemical signature distinguishes self-DNA from non-self-DNA and directs restriction activity toward invading phage genomes that lack the appropriate modification.⁷⁰ The Dnd system exemplifies this strategy in E. coli and Salmonella enterica, where DndABCDE introduces phosphorothioate marks at specific motifs in the host chromosome, while the associated DndFGH restriction complex targets and cleaves unmodified phage DNA, conferring resistance to a broad range of bacteriophages.⁷¹

Phage counter-defense strategies against bacterial resistance
Phages are also capable of evolving and developing novel strategies to circumvent bacterial defenses, enabling them to sustain their predatory activity. A comprehensive investigation into both microbial defense systems and phage countermeasures is essential for advancing and refining phage therapy.

Phage infectivity is compromised when host receptors are either masked or no longer compatible with the phage’s receptor-binding proteins (RBP). As a result, alterations or loss of these receptors constitute a fundamental strategy by which bacteria develop resistance to phage infection. To overcome bacterial resistance arising from receptor modification or loss, bacteriophages can adapt by altering their receptor-binding proteins (RBPs), which are crucial for host recognition and attachment. Such modifications enable phages to recognize alternative or mutated bacterial surface structures, thereby restoring infectivity. Escherichia coli phage λ, which typically binds to the LamB porin on the bacterial outer membrane. When E. coli develops resistance by mutating or downregulating LamB, V can evolve variants with mutations in its tail fiber protein (encoded by gene J), allowing it to use alternative receptors such as OmpF.^72,73 This receptor-switching mechanism demonstrates the phage’s ability to circumvent bacterial resistance through targeted changes in RBPs, thus sustaining the infection cycle and evolutionary battle between phages and their hosts.

Another example is the Escherichia coli podophage K1-5, which encodes two distinct types of tail fiber proteins. This adaptation allows K1-5 to attach to and infect E. coli strains expressing different capsule antigens (K1 and K5), despite antigenic variation in the bacterial population. Thus, by molecular evolution of receptor-binding proteins, bacteriophages can adapt to changes in bacterial surface structures and continue their infection cycle despite bacterial attempts at evading phage adsorption.⁷⁴

Bacteriophages evade the bacterial CRISPR-Cas adaptive immune system by encoding anti-CRISPR (Acr) proteins within their genomes. These Acr proteins inhibit the assembly and the activity of CRISPR-Cas complexes, thereby blocking target recognition and cleavage, and ultimately allowing successful phage infection and replication within the bacterial host.⁷⁵ A characterized instance involves Type I-F CRISPR-Cas systems in Pseudomonas aeruginosa and their inhibition by several AcrIF proteins; AcrIF1 binds to the Cas7f backbone of the Csy surveillance complex, thereby blocking the hybridization between the CRISPR RNA (crRNA) and the target DNA. AcrIF2 interacts with the DNA-binding site, specifically blocking the recognition of the protospacer adjacent motif (PAM) on the phage DNA. AcrIF3 acts as a direct inhibitor of the Cas3 helicase-nuclease, an essential enzyme for DNA cleavage in the Type I-F system. Experimentally, engineered bacteriophages carrying these acrIF genes (termed EATPs) have demonstrated the successful suppression of CRISPR immunity in P. aeruginosa, permitting productive infection and replication even in multidrug-resistant (MDR) bacterial strains.⁷⁶ The inclusion of Acr proteins significantly enhances the ability of therapeutic phages to overcome bacterial immune defenses in both in vitro and in vivo infection models.

Phages can resist bacterial restriction-modification (RM) systems by interfering with the DNA methylation process that bacteria use to distinguish their DNA from foreign invaders. Bacteriophage T7, for example, encodes the OCR (Overcome Classical Restriction) protein that structurally mimics the phosphate backbone of B-form DNA, allowing it to inhibit host restriction enzymes competitively.⁷⁷ This mimicry not only shields phage DNA from cleavage but also disrupts host methylation by preventing methyltransferase access to DNA, thereby simultaneously blocking both restriction and methylation pathways to ensure efficient phage infection and replication.^78,79 Similarly, phages have evolved mechanisms to overcome alternative RM systems, such as those based on DNA phosphorothioation. Notably, phage JSS1 can bypass PT-mediated immunity, allowing it to successfully infect and replicate within PT-positive bacterial hosts.⁸⁰