Bacterial defense mechanisms against bacteriophages

Bacterial defense mechanisms against bacteriophages

B.G.E.T Jayashantha B.Sc (ug) Microbiology (Special), University of Kelaniya, Sri Lanka

Objective • To study the defense mechanisms associated with bacteria against the pathogenicity of bacteriophages and mechanisms which are associated with phages to overcome these bacterial defense mechanisms.

PIC Source :- http://www.microbiologyonline.org.uk/

Introduction • Bacteriophages infect bacteria. • Bacteria have several genetically regulated defense mechanisms to protect from the phages. • Different bacteria have different defense mechanisms. • Bacteria and Bacteriophages have been co-evolving during the evolutionary process. • Therefore, phage mechanisms are evolved to overcome these bacterial resistance. • This continuous evolutionary relationship between the pathogenicity of Bacteriophages and the resistance of Bacteria is described by Evolutionary arms race hypothesis

Anti-phage mechanisms of bacteria • Main mechanisms can be categorized under following topics 1. Preventing phage adsorption 2. Preventing phage DNA entry

3. Restriction modification systems 4. CRISPR-Cas system

5. Abortive infection systems

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1.) Preventing phage adsorption • Adsorption is the initial step of phage infection to a bacterium. • Phage adsorption to the bacterial cell surface occurs through recognition of a phage receptor on the surface • Adsorption-blocking mechanisms can be divided into mainly three categories i) Blocking of phage receptors ii) Production of extracellular matrix iii) Production of competitive inhibitors

Blocking of phage receptors 1. Bacteria can become resistant to phages by modifying cell surface receptors which recognize distinctive pages.

• Bacteriophages make resistance by Adapting to recognize these new receptors

How phages overcome this resistance

PIC Source :- Nature reviews | Microbiology

Blocking of phage receptors cond.. 2.) Bacteria can also produce proteins that mask the phage receptor

• Examples:  Staphylococcus aureus produces protein A, which reduces phage adsorption

Masking Protein

PIC Source :- Nature reviews | Microbiology

When bacteria produce low amount of Protein A , adsorption is improved

Blocking of phage receptors cond.. Examples: Escherichia coli produces a lipoprotein (Llp) that blocks its own receptor ferrichrome-iron receptor (FhuA). • Llp is expressed at the beginning of the infection of E.coli by T5 • It also prevents superinfection

 E. coli has outer-membrane receptor protein named as OmpA for many T-even-like E. coli phages

• It is masked and modified its conformation by protein TraT which is encoded by the F plasmid,

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Production of extracellular matrix 1.) Phage adsorption can also be blocked by the production of exopolysaccharide (EPS)

but phages overcome the EPS layer by producing a polysaccharide lyase or a polysaccharide hydrolase to cleave EPS

How phages overcome this resistance

PIC Source :- Nature reviews | Microbiology

Production of extracellular matrix • Examples: Pseudomonas spp. and Acetobactor spp. • phage F116, targets Pseudomonas, produces an alginate lyase, produce Alginates (a type of EPS ) which increase the resistance against phages • It facilitates the dispersion of the alginate matrix as well as reducing the viscosity of this matrix. How phages overcome this resistance

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PIC Source :- http://www.nature.com/

Production of extracellular matrix 2.) Phages have also evolved to specifically recognize polysaccharides such as LPS O antigens and K antigens. • Some bacteria modifies these LPS antigens . Therefore phage can not recognize and adsorb to the cell.

Examples: E.coli O157:H7 has O-antigen and Kantigen. • Which are responsible for specific adsorption of Phage ɸV10 • E.coli O157:H7 produces O-acetyltransferase that modifies the O157 antigen to block the adsorption of ɸV10 and similar phages39

PIC Source :- Nature reviews | Microbiology

Production of competitive inhibitors • Molecules that are naturally present in the bacterial environment can bind specifically to the phage receptors • Becoming these receptors are unavailable for phages

• Examples:  In E. coli ,FhuA is a receptor for adsorption of coliphages such as T1, T5 and ɸ80  Microcin J25 is an antimicrobial molecule which is synthesized by phylogenetically related strains for competition.  It also acts as a competitive inhibitor for FhuA receptor on E.coli.  Microcin J25 bound FhuA receptors are resist for adsorption of such coliphages

2.) Preventing phage DNA entry • Superinfection exclusion (Sie) systems responsible for blocking phage DNA entry in to host cells.

• They make immunity against specific phages. • Sie proteins are membrane anchored or associated with membrane components • Sie proteins encoding genes can be found in prophages • They are expressed in prophage stage. • They are responsible for phage-phage interactions rather than phage – host interactions.

Preventing phage DNA entry

contd…

• Normal phage T4 infection of an Escherichia coli cell. • The peptidoglycan layer is degraded • Inner-membrane protein is involved for the translocation of the DNA into the cytoplasm. How this DNA entry is blocked PIC Source :- Nature reviews | Microbiology

Preventing phage DNA entry

contd…

PIC Source :- Nature reviews | Microbiology

• Phage T4 encodes the protein Imm • It blocks the translocation of phage DNA into the cytoplasm, • thus preventing infection by other Teven-like phages.

• The protein Sp, also encoded by phage T4 • It blocks degradation of the peptidoglycan • It causes trapping the DNA between peptidoglycan layer and the outer membrane.

3.) Restriction modification systems • The restriction enzyme cleaves specific patterns in the incoming foreign bacteriophage DNA  Restriction • The host cell also protects its own genetic material from enzymatic degradation by modifying it  Modification • These systems are found in 90% of all sequenced prokaryotic genomes • Both activities are mediated by recognition of a short specific DNA sequence • Protection is normally conferred by usually methylation of specific bases in this recognition sequence in the host genome • All non-methylated DNA are recognized as foreign and they are cleaved • RM systems minimally contains methyltransferase (MTase) gene for protection of host DNA • Restriction endonuclease (REase) gene performs the foreign restriction activity

• Restriction Modification systems show arms race between bacteria and phages. • In bacteria, defense mechanisms are evolved to protect from pages, • Phages are evolved to overcome these bacterial defense mechanisms • It is a evolutionary continuous process • Evidence for co-evolution of bacteria and phages. • We can describe several RM systems by using E.coli and its infector T4 phage as models

EM of T4 phages infecting E.coli PIC Source :- http://www.hyglos.de/

EM of T4 phages infecting E.coli PIC Source :- http://www.thenakedscientists.com/

Phage T4 infecting a susceptible host (E.coli) cell. It causes the infection PIC Source :- Nature reviews | Microbiology

How this infection is prevented

How phage overcome the host’s resistance A classical R-M system PIC Source :- Nature reviews | Microbiology

• Phage T4 infecting a phage-resistant E. coli cell containing a R–M system. The phage genome is cut at specific sites by the restriction enzyme • The host cell contains methylase activity to prevent enzymatic degradation of its own DNA

• The genome of phage T4 contains hydroxymethylcytosine (HMC) and can also be methylated, • Thereby avoiding specific endonucleases. • Resist for host's endonucleases • Bacterial cell is infected

How phage overcome the host’s resistance

PIC Source :- Nature reviews | Microbiology

• Some bacteria have acquired ModificationDependent systems (MDS) that can exclusively cleave HMC-containing DNA • It prevents infection by HMC-containing phages.

• Phage T4 acquired a resistance to MDSs through the glucosylation of HMC residues (glu–HMC).

• Eg:DpnI from Streptococcus pneumoniae McrA, McrBC and Mrr from E. coli

• Bacterial cell is infected

• Host resistance has been overcame

How phage overcome the host’s resistance

PIC Source :- Nature reviews | Microbiology

• Some E. coli strains have acquired a Glucose-modified restriction (Gmr) system that targets and cleaves glu–HMC-modified phage T4 genomes • Block phage infection. • Two subunits (GmrS and GmrD) specifically cut glu–HMC DNA . • No effect on unglucosylated DNA. Therefore protects host DNA

• Some T4-like phages have a gene encoding internal protein I (IPI), a protein that hinders the Gmr • Phage successfully infect E. coli strains containing Gmr system.

How phage overcome the host’s resistance

PIC Source :- Nature reviews | Microbiology

• Some E. coli strains achieve a modified Gmr system • It consists translational fusion of GmrS and GmrD is produced, • Presence of such system causes IPI ineffective. • Host cell is not infected

• Phage T4 mutants can bypass the GmrS–GmrD fusion by a unknown mechanism,

• Leads to a successful infection of the host cell.

4.) CRISPR-Cas system

CRISPR = Clustered Regularly Interspaced Short Palindromic Repeat Cas = CRISPR associated systems

5.) Abortive infection systems (Abi) • Abi is a collective term describing host mechanisms that interrupt phage development at different stages such as; • Genomic replication • Transcription • Translation • Packaging of genome • Abi-mediated resistance leads to death (suicide) of the infected bacterial cell • Prevent further infection • In most Abi systems Phage is trapped inside the cell. • There are few Abi systems are found, mechanisms of some of them are not understood yet. • Rex system in E.coli is well studied and a typical example for a Abi systems

Rex Abortive infection system in E .coli • Rex is Two- compornent system

• RexA and RexB proteins are involved • RexA is an intracellular sensor that activates the membrane anchored RexB. • RexA is activated during phage infection by production of phage protein – DNA complex as an inter.mediate of replication and recombination. • At least two RexA proteins are needed to activate one RexB protein • Therefore Rex is a protein ratio mediated mechanism.

• RexB is an ion channel that reduces membrane potential • Causing reduction of cellular ATP levels • Decrease the synthesis of macromolecules and stopping cell multiplication • Phage infection will also abort, because it needs either ATP or ATP-dependent cellular components.

An inactive form of RexA, the sensor protein

RexA is activated by the phage protein–DNA complex that forms as a replication or recombination intermediate during the phage infection

• Two activated RexA proteins are needed to trigger the membrane-anchored protein RexB, which acts as an ion channel . • It allows the passage of monovalent cations through the bacterial inner membrane, • Cause for destroying the membrane potential and killing the cell.

Examples for Some of other Abi systems in bacteria 1. Lit Abi system in E.coli against phage T4 • Translation elongation factor - TU (EF-TU) is cleaved and abort the phage infection.

2. PrrC Abi system in E.coli against phage T4 • Cleaves tRNA(lysine) in the anticodon loop and stop the translation. Therefore protein synthesis is inhibited.

3. PifA Abi system in E.coli against phage T3 & T7 • Macromolecule synthesis is severely reduced, only half of the phage genome is injected into the cell , Limiting the late transcription, bacterial chromosomal DNA is degraded , membrane permeability is altered causing molecules leaking through the cell membrane

4. Toxin-Antitoxin (TA) Abi systems in Erwinia • Toxic molecule is formed by different regulatory interactions, including protein– protein, RNA–RNA or protein–RNA interactions. Expression of these toxic molecules are causing promoter repression, specific transcriptional termination. Toxin can be neutralized by antitoxin molecule.

Examples for Some of other Abi systems in bacteria contd.. 5. Lactococcal Abi systems ( eg: Lactobacillus lactis ) • Abi ( A,F,P,K,T )  Interfere with DNA replication • Abi ( B,G,U )

 RNA transcription

• Abi C

 Restricts production of major capsid protein

• Abi ( E,I,Q )

 Disrupt DNA packaging

• Ani 2

 Premature lysis of infected cell

References • Labrie, S. J., Samson, J. E., & Moineau, S. (2010). Bacteriophage resistance mechanisms. Nature Reviews Microbiology, 8(5), 317-327. • Abedon, S. T. (2012). Bacterial ‘immunity’against bacteriophages.Bacteriophage, 2(1), 50-54. • Stern, A., & Sorek, R. (2011). The phage‐host arms race: shaping the evolution of microbes. Bioessays, 33(1), 43-51. • https://microbewiki.kenyon.edu/index.php/Bacterial_phage_defense_mechani sms_with_applications

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