Bacteriophages Flashcards
what is one common bacteriophage found in 50% of human guts
crAssphage
what type of phages are most abundant
tailed phages
what are the components of tailed phages?
linear dsDNA, ssDNA, capsid, tail, adsorption apparatus (baseplate, tail fibres, tail spikes, tail tip)
what is the mechanism of adsorption
- first contact of the phage with receptor (reversible adsorption) (WEAK)
- phage walks on the cell surface to find an ideal place to adsorb irreversibly (STRONG)(irreversible adsorption)
- DNA injection into the cell
how is the moment of energy conserved
virus keeps protein upwards
what is the difference between the specialised and generalised phages
specialist- able to kill only one type of strain within one species
generalist- able to kill more than one strain over more than one species by being able to detect >1 receptor type
there is not a phage known that can bind to both ______ and ______
gram negative and gram positive bacteria
describe the viral contractile tail ejection system
binds to receptor, dock into position, tail tube contracts, DNA injected and then tail comes out. -degradation of the peptidoglycan layers makes injection possible
describe the viral long flexible tail ejection system (non contractile)
non contractile tail is like a screw and penetrates the membrane in this way
describe the short tail ejection system
same as long but the tail sheath goes all the way in
bacteriophages that only use the lyric cycle are called_________
virulent phages
what are the three potential lifecycles
lytic, pseudolysogenic, lysogenic
describe the pseudolysogenic life cycle
stage of stalled development. without multiplication of the phage genome or it’s replication synchronised with cell cycle. usually caused by unfavourable conditions. termination when growth conditions improve
what is the lysogenic life cycle characterised by
integration of bacteriophage nucleic acid into gosh bacterium’s genome or formation of a circular replicon in the bacterial cytoplasm
describe the mechanism of the final stage of the lyric cycle - cell lysis
- lytic proteins accumulate in the cell wall (lysins and holins)
- inner cell membrane disruption - at critical concentration holins oligomerise to form nonspecific holes. these pores result in membrane depolarisation
- peptidoglycan disruption - degradation of cell wall by endolysins SAR lysins bound to periplasmic region of cell membrane and degrade peptidoglycan layer. release occurs through membrane depolarisation via pinholes
- outer membrane fusion w inner membrane - spanins span both membranes. after cell wall degradation spanin complexes are free to diffuse and aggregate. lytic blowout occurs when sufficient spanin complexes are liberated within the degraded area. they mediate fusion of the inner and outer membrane.
what is the latency period
after initial infection, proliferation of virus particles ceases (the viral genome is not eradicated)
how can temperate phages survive in extreme environments
they can integrate into the chromosome and stay there in a dormant state
what is the best way to survive when environmental conditions are not favourable
lysogenic life cycle
what small molecule do phages use to communicate
Arbitrium
what is transduction
the process by which foreign DNA is introduced into the cell by a virus or viral vector
which transduction process causes anti microbial resistance
transfer by generalised transduction
what are viral quasispecies
a well defined distribution of mutants that is generated by mutation selection process. selection does not act on a single mutant but on the quasispecies as a whole
describe generalised transduction
after adsorption, upon degradation of host genome, by mistake phage packages prices of bacterial DNA into its capsid
describe specialised transduction
integration of temperate phage in lysogenic life cycle, every time it excises itself from the chromosomes it will take info with it
what is the eclipse period
the time between infection by a bacteriophage or other virus and the appearance of the mature virus within the cell (viral infectivity cannot be recovered)
how does phage DNA packaging occur
by DNA translocation in a prohead
what protects nucleic acid before capsid matures
pro capsid
what are terminases
enzymes which initiate DNA packaging by cutting the DNA concatemer
what is the range of burst size per infection
100-150 particles
how does the phage recognise receptors
via its adsorption apparatus
give some examples of frequently used enzymes that come from phages
T4 DNA ligase, polynucleotide kinase, DNA polymerase, RNA ligase, RNA polymerase. AMB reverse transcriptase. phi29 DNA polymerase
what are 5 viral resistance mechanisms
preventing virus adsorption, preventing virus DNA entry, cutting virus nucleic acids, abortive infection, CRISPR-Cas
how do bacteria prevent virus adsorption
blocking virus receptors. producing extra cellular matrix
how do bacteria prevent virus DNA entry
superinfection exclusion (if i am infected there’s no room for you). expression of two proteins to prevent entry of DNA (Sp blocks degradation of peptidoglycan layer (t4 lysozyme) and Imm redirects DNA)
how do bacteria protect themselves by cutting viral nucleic acid
restriction/ modification system. viruses modify nucleotides then host modify nuclease (host dna modified and protected viral dna not)
give two examples of abortive infection (host defence)
membrane puncturing
self toxification
how do bacteria protect themselves by membrane puncturing
phage DNA replication detected by RexA activating it to bind RexB on inner membrane to form complex. causes escape of protons and loss of membrane potential (that helps membrane degradation) leads to death
how are bacteria protected by self toxification
virus infection alters antitoxin-toxin balance (by shutting down host transcription), now more toxin that antitoxin, so self toxification
why is abortive infection useful for the bacterial population
individual cells infected with phage use it to prevent spreading to other cells. sacrifice for the survival of the overall population
describe broadly the three steps of the lysogenic cycle
- virus attaches and injects DNA - incorporation into host dna = prophage
- prophage dna passed on to daughter cells
- dna can excise and enter lytic cycle
what are the advantages of phage therapy compared to antibiotics
very specific, replication at the site of infection, no side effect, developmental resistance, finding new phage is fast
what is phage therapy
a therapy that uses bacteriophages to treat bacterial infections.
(because they only attack bacteria; phages are harmless to people, animals, and plants)
what is required for phages used in therapy
Uses strictly virulent phages to avoid potential transfer of virulence genes through transduction
Only use phages that go through the lytic cycle
Requires active immune system for phages to work as a cure
what are the limitations of using phage therapy
narrow host range (limits resumptive treatment have to identify the bacteria)
inflammatory responses due to bacterial toxin release after lysis
resistance may occur
isolation dependent on the culturability of the bacteria
difficult to obtain clear intellectual property rights
undefined regulatory frame for phage product approval
what are the advantages of using phage therapy
high specificity causes minimal disruption of the normal flora
replication at the site of infection
low inherent toxicity
low potential for inducing resistance
phage resistant bacteria remain susceptible to other phages having similar target range)
lack of cross resistance with antibiotics
active against antibiotic-resistant bacteria
rapid discovery
what are the features of a good phage candidate for phage therapy
- Infects starain of interest
- Infects efficiently
- Does not intergrate the bacterial chromosome
- Reduced development of resistance
how do you screen the phage collection for good phage candidates
add bacteria to 33 wells and incubate at 37degrees celcius (Add 180 μl LB and 10 μl bacteria)
add 10 μl each phage to 3 wells (10 phages + control)
measure optical densitiy every 10 mins for 12hrs
plot as graphics and compare bacterial growth with and without phage
- the good phages are ones who show increasing phage density and a drop in bacterial density
what does a good phage cocktail need to be
need to infect efficiently, have reduced development of resistance, work well together and use different receptors
how do you test for the best combination of phages
Efficacy of plating assay
individual phages and combination of phages at 10^10 PFU/ml
90 μl buffer in all wells + 10 μl phages - test different concentrations
calculate EOP
what equation allows you to calculate the efficacy of plating EOP (individual phages used)
PFU/ml divided by
Concentration of phages when plated on host strain (10^10 PFU/mL)
how do you work out the plaque-forming units per millilitre (individual phages used)
(No. phage plaques x 10^dilution) divided by Volume plated (0.01 mL)
how do you calculate the efficacy of plating of phage cocktails
Concentration of phage, divided by
Concentration of phage if plated on its host strain (1010 PFU/mL)
how do you work out the percentage bacteriophage insensitive mutants
%BIM =
Number of colonies resistant to the phage, divided by
Total number of colonies evaluated
times by 100
how do you test the development of resistance in phages
add bacteria and phages together, incubate at 37 degrees and centrifuge at 170rpm
dilute and plate
isolate specific colonies and streak plate - repeat many times
put a phage on top
- see what die out what dont and calculate BIM%
how do you calculate colony forming units per mililitre
CFUs/mL =
(Number of colonies X dilution factor) divided by
Volume plated (0.01 mL)
how to test the synergistic effects of phage and antibiotic combined therapy
add bacteria to multiple tubes, one is control, add antibiotics on their own, add phages on their own then add a combination
incubate at 37 degrees, centrifuge for 34hr at 170rpm, dilute and plate
then calculate colony forming units
