Bacteriophage Khansa BukhariMaryum Zeb

History and Definition• Frederick Twort (1915) and Felix d'Herelle (1917) were the first

to recognize bacteriophage.• In the 1930s and after words virologists utilized these viruses

as model systems to investigate many aspects of virology

Viruses which infect bacteria are called bacteriophages (eaters of bacteria).

Classification • Phages are classified by the International Committee on Taxonomy

of Viruses (ICTV) according to morphology and nucleic acid.

Bacteriophage M13• Belongs to family Inoviridae• Filamentous• Circular single-stranded DNA• 6400 base pairs long• It infects F+ E. coli• It just slows growth of host cell• Virions leak out from the cell. • The virus is not very lytic.

Genome• The genome codes for a total of 10 genes (named using

Roman numerals I - X)• Gene VIII codes for the major structural protein, forms a

tubular array of approx. 2,700 identical subunits surrounding the viral genome

• Gene III codes for the minor coat protein• Approximately five to eight copies of the gene III protein are

located at the ends of the filamentous phage• Allows binding to bacterial "sex" pilus• Pilus is a bacterial surface structure of E. coli which harbor the "F

factor" extrachromosomal element

REPLICATION• Single strand genome ( '+' strand) attached to pilus enters host cell• Major coat protein (gene VIII) stripped off• Minor coat protein (gene III) remains attached

• Host components convert single strand (+) genome to double stranded circular DNA (replicative form or "RF")

• Transcription takes place by host RNA polymerase• Viral g2p protein nicks RF DNA strand at the origin of replication.• (+)strand replication occurs by rolling circle.• New (+)ssDNA genomes are converted into new RF molecules, and further

transcription occurs.• When enough g5p protein is synthesized, conversion into RF dsDNA is

inhibited, as neo-synthesized genomic ssDNA is covered with g5p.• g5p are replaced by g8p proteins to assemble the viral capsid.• New virions bud out from host cell.• Infected cells continue to divide and produces virion indefinitely.

Bacteriophage lambda

Classification• Virus classification• Group: Group I (dsDNA)• Order: Caudovirales• Family: Siphoviridae• Genus: λ-like viruses• Species: λ Phage

Bacteriophage lambda• Bacteriophage lambda (λ) was discovered by

Joshua and Esther Lederberg. While mutagenizing strains E. coli using UV

• Genome is 48,502 bp dsDNA • Linear dsDNA circularizes due to annealing

between 3 sticky cos sites.′• The capsid has two parts:• Head, composed of B, C, Nu3, D, and E proteins.• Tail, composed of J and H proteins.

• It is a temperate phage: being either lytic or lysogenic.

• Chooses developmental pathway depending on nutrient availability

PHI-6 PHAGE

Classification• Group group III, dsRNA virus• Family Cystoviridae• Genus Cystovirus• Specie Pseudomonas phage phi6

Structure & Entry• Multi-segmented, Spherical enveloped virus of Pseudomonas

syringae• The nucleocapsid NC, consisting of the five proteins P1 , P2, P4, P7 and P8 • Entry begins by viral attachment, followed by fusion with the

host cell outer membrane • NC penetrates the cytoplasmic membrane via membrane

invagination with the aid of protein P8.• steps releases a transcriptionally active polymerase complex

called the core into the cytoplasm.

Genome• Genome consists of three segments: small (S), medium (M)

and large (L).• Core is encoded by L segment.• P1 is the major structural protein• P4 is identified as nucleoside triphosphotase, which is

necessary for +strand RNA packaging.• P7 stabilizes RNA packaging.• P2 is rdrp, carries out –strand synthesis and RNA transcription.

Replication• Pseudomenas synirgae pili brings the virion into contact with

the NC associated lytic enzyme locally digests the peptidoglycan.

• Upon entry, viral polymerase is activated to produce early transcripts.

• Translation of L transcripts produces the early proteins, which assemble to form PC.

• Packaging of +s transcripts and synthesis of –s transcripts inside the PC

• Transcription by these PC produces message for late gene synthesis

• The NC surface proteins assemble on the PC and inactivated transcription.

• The NC acquires membrane from the host plasma membrane.• The cell eventually lyses and liberates mature progeny

particles.

PHAGE RESISTANCE MECHANISMS

Preventing Phage Adsorption

Restriction Modification System

CRISPR Mode of Action

APPLICATIONS

PHAGE AS CLONING VECTOR

Phage Therapy• Use of phages to treat bacterial infections in animals and

humans.• treat bacterial dysentery, staphylococcal lung infections,

surgical wound infections,• staphylococcal septicemia • Easier to develop new phage than a new antibiotic.• Replicate at the site of infection and are thus available where

they are most needed • Phage-resistant bacteria remain susceptible to other phages

having a similar target range

Phage Lysins as Antimicrobials• Use of phage endolysins as potential therapeutics instead of

whole phage.• Phage endolysins, or lysins, are enzymes that damage the cell

walls' integrity• phage lysins composed of at least two distinctly separate

functional domains: • C-terminal cell-wall binding domain, which directs the enzyme

to its target• N-terminal catalytic domain

Phage Display• 1. Insert a diverse group of genes into the phage genome. Each

phage receives a different gene. • 2. Create a library of genetically modified phages which are all

related but each of which have a different gene.• 3. Isolate a disease causing molecule, such as a receptor or an

enzyme, and expose the phage library to the molecule. • 4. Some of the phages will bind to the disease causing molecule.

Wash the phage library. All of the phages with proteins that didn't bind to the disease causeing molecule will be removed.

• 5. Replicate the phages that remain so that they can be sequenced.

• 6. Since we know where the new gene was inserted we can determine the amino acid sequence of the protein that bound to the disease

Applications of Phage Display• To select proteins, peptides, or antibodies with affinity to a

molecule or protein of interest• To clone antibodies from unstable hybridoma.• To identify molecules that can be recognized and internalized

by eukaryotic cells.• To identify epitopes, functional and accessible sites from

antigens.• To study protein-protein interaction.• To design vaccines.

Use of Phage in Vaccination• Whole bacteriophage acts as efficient DNA vaccine delivery

vehicles.• Phage-display vaccination.• Phage DNA vaccination. • Hybrid phage vaccine.

The End