Bacterial Immunity: Mechanisms Shielding Cells from Phages

Bacteria are constantly under attack from bacteriophages, viruses that infect and replicate within them. To survive, they have evolved a range of defense mechanisms to recognize, block, and destroy these invaders.

Understanding bacterial immunity provides insight into microbial survival strategies and has applications in medicine and biotechnology. Scientists study these systems for their role in bacterial resistance and their potential in gene editing and antibacterial therapies.

Physical Barriers

Bacteria possess structural defenses that serve as the first line of protection against bacteriophages. These barriers prevent viral attachment, impede entry, or create inhospitable conditions for infection. While not absolute defenses, they significantly reduce susceptibility.

Peptidoglycan

The bacterial cell wall, composed primarily of peptidoglycan, acts as a shield against external threats, including phages. This mesh-like polymer consists of alternating N-acetylglucosamine (NAG) and N-acetylmuramic acid (NAM) chains cross-linked by peptide bridges. In Gram-positive bacteria, the thick peptidoglycan layer, sometimes exceeding 40 nm, provides a formidable barrier. Some phages produce specialized enzymes, such as endolysins, to degrade peptidoglycan and access the cytoplasmic membrane. However, bacterial modifications, including altered cross-linking density and the incorporation of secondary cell wall polymers like teichoic acids, can hinder phage attachment or penetration. Research in Nature Reviews Microbiology (2021) highlights how structural alterations reduce susceptibility to infection.

Capsules

Many bacteria produce extracellular polysaccharide capsules that serve as an additional protective layer. These capsules, composed of long-chain carbohydrates, vary in composition and thickness. By masking receptor sites, they prevent phages from binding to their target structures. Encapsulated bacteria like Klebsiella pneumoniae and Streptococcus pneumoniae exhibit increased resistance due to dense capsule layers. A study in Cell Reports (2022) found that phages targeting Escherichia coli struggle to penetrate certain capsule types, reducing infection efficiency. Some phages counteract this defense with capsule-degrading enzymes, but constant variation in capsule composition presents a persistent challenge.

Biofilms

Bacteria often exist in biofilms—structured communities encased in a self-produced extracellular matrix—that provide collective resistance against phages. These biofilms, composed of polysaccharides, proteins, and extracellular DNA, create a dense physical barrier that limits phage diffusion. Within a biofilm, bacteria exhibit altered metabolic states, reducing phage susceptibility by slowing down viral replication. Research in Nature Microbiology (2023) demonstrated that phages struggle to penetrate mature biofilms, leading to incomplete eradication of bacterial populations. Additionally, biofilms promote genetic diversity through horizontal gene transfer, allowing bacteria to share resistance traits. Some phages have evolved enzymes to degrade biofilm components, but bacterial communities respond by increasing matrix production or altering biofilm architecture.

CRISPR-Cas

Bacteria have an adaptive immune system known as CRISPR-Cas, which enables them to recognize and neutralize bacteriophages based on previous encounters. This system relies on Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) and associated Cas proteins to store genetic fragments from past infections. When a bacterium survives a phage attack, it incorporates short sequences of viral DNA, known as spacers, into its CRISPR array. These spacers serve as a genetic record, allowing for precise targeting of viral genomes upon subsequent infections.

Once integrated, spacer sequences are transcribed into CRISPR RNA (crRNA), which guides Cas proteins to recognize and degrade matching viral DNA. The Cas proteins act as molecular scissors, scanning for sequences complementary to stored spacers. If a match is found, the Cas complex cleaves the viral genome, preventing replication. Different bacterial species employ variations of the CRISPR-Cas system, categorized into Class 1 and Class 2. Class 2 systems, particularly CRISPR-Cas9, have been extensively studied for their simplicity and efficiency in gene targeting, making them valuable tools in genetic engineering.

Phages have evolved countermeasures such as anti-CRISPR proteins that inhibit Cas activity or mutations that prevent recognition. This evolutionary struggle drives the diversification of CRISPR loci, with bacteria acquiring new spacers to keep pace with viral mutations. Research in Nature Microbiology (2022) found that bacterial populations exposed to high phage pressure rapidly expand their CRISPR repertoire. Some bacteria regulate CRISPR-Cas activity based on environmental conditions, balancing immunity with the need for horizontal gene transfer.

Restriction-Modification

Bacteria use enzymatic systems to differentiate their own genetic material from foreign DNA, allowing them to defend against bacteriophages. One of the most effective is restriction-modification (R-M), which relies on two enzymes: a restriction endonuclease that cleaves specific DNA sequences and a methyltransferase that modifies the host genome to prevent self-targeting. This ensures only unmodified foreign DNA is degraded while the bacterium’s genome remains intact.

Restriction enzymes recognize short palindromic sequences, typically 4 to 8 base pairs, making them highly efficient at detecting viral genomes. These systems are classified into four types—Type I, II, III, and IV—each differing in enzymatic complexity and cleavage mechanism. Type II systems, like EcoRI from Escherichia coli, are widely studied for their precise DNA cleavage, a feature that has also made them invaluable in molecular biology. Type IV systems, rather than targeting unmethylated DNA, recognize and degrade modified viral genomes, highlighting bacterial adaptability.

Phages have evolved strategies to bypass restriction-modification defenses, such as incorporating chemical modifications like glucosylation or hydroxymethylation to evade detection. Some encode their own methyltransferases to mimic bacterial DNA modifications, effectively disguising themselves. Others produce anti-restriction proteins that inhibit bacterial endonucleases, preventing DNA cleavage. These adaptations drive bacterial refinement of restriction-modification specificity and regulatory control.

Toxin-Antitoxin Systems

Bacteria have developed self-regulatory mechanisms to enhance survival under hostile conditions, including phage infection. Toxin-antitoxin (TA) modules consist of paired genes encoding a stable toxin and a more transient antitoxin. Under normal conditions, the antitoxin neutralizes the toxin’s effects. However, when a phage disrupts bacterial metabolism, the balance shifts, triggering a defensive response that inhibits viral replication.

These systems come in multiple forms, categorized into six types based on how the antitoxin neutralizes the toxin. Type II TA systems, the most extensively studied, involve protein-protein interactions where the antitoxin binds and inhibits the toxin. A well-characterized example is the MazEF system in Escherichia coli, where MazF cleaves mRNA at specific sequences, shutting down protein synthesis. This dormancy-like state can prevent phage replication since viruses rely on active bacterial machinery. Some TA systems even induce programmed cell death, sacrificing infected cells to protect the bacterial community.

Phage Evasion Strategies

While bacteria have developed a range of defenses, phages have evolved countermeasures to bypass these protections. By modifying their genetic material, altering infection mechanisms, or interfering with bacterial defense pathways, phages continuously adapt to evade destruction.

One effective strategy is the use of anti-CRISPR proteins, which inhibit bacterial CRISPR-Cas immunity by blocking Cas enzyme activity. Studies have identified multiple families of these proteins, each targeting different components of the CRISPR-Cas system. Phages also mutate their genomes to escape recognition by restriction-modification systems, altering or masking sequences targeted by bacterial restriction enzymes. Some incorporate host-derived methylation patterns to mimic bacterial DNA, further increasing their chances of avoiding cleavage.

Beyond molecular mimicry, phages modify their infection strategies to circumvent physical barriers. Some produce enzymes that degrade bacterial capsules and biofilm matrices, enabling them to penetrate protective layers. Others exhibit host range mutations that allow them to recognize alternative surface receptors when primary attachment sites are blocked or modified. This rapid adaptability ensures that even when bacterial populations develop resistance to specific phage strains, new viral variants emerge to maintain infectious potential.