Toxin-Antitoxin (TA) Systems and Bacteria Persistence (MCB3026F) notes
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Course
Molecular Genetics and Genomics (MCB3026F)
Institution
University Of Cape Town (UCT)
Comprehensive lecture notes for the TA systems and bacterial persistence notes module covered in MCB3026F. These notes cover all content taught in lectures as well as additional materials (powerpoints, textbooks) required to succeed. These notes were created by a student who achieved a distinction ...
Toxin-Antitoxin (TA) Systems and Bacteria Persistence
Introduction
- Bactericidal antibiotics quickly kill most of a bacterial population however, a small fraction of cells typically survives
through entering the so-called persister state
- persister cells are increasingly being viewed as a major cause of the recurrence of chronic infectious disease and is
considered a key factor in the emergence of multi-drug antibiotic resistance
Lecture 1: The importance of antimicrobial resistance
Antimicrobial resistance (AMR)
- the antibiotic crisis is the most pressing concern of the 21 st centaury and is caused by two main factors:
1) effective antibiotics are becoming increasingly scarce, or even non-existent for some pathogens (as pathogens are
becoming increasingly more resistant to the common antibiotics)
2) the increasing occurrence of bacterial pathogens in clinical settings (superbugs) that can overcome antibiotic killing
- superbugs are bacteria that are resistant to a wide variety of antibiotics and becomes very difficult to treat (such as TB)
- it is expected that there will be 10 million deaths in 2050 due to antimicrobial resistant infections
- there is a decline in new antibiotics each year yet there is a rise in antibiotic resistance each year
- generating new antibiotics is a huge bottleneck as need to test all candidate antibiotics, test if it is effective, whether there
are any detrimental effects on clinical patients
- Africa in particular is under a large threat with antimicrobial resistance
What is antimicrobial resistance
- antimicrobial resistance covers three distinct phenomena:
1) Bacteria Antibiotic Resistance
2) Bacterial Antibiotic Tolerance
3) Bacteria Persistence
Bacterial Antibiotic Resistance
- inherited trait that allows a resistant bacterial s train to grow in the presence of a specific antibiotic above the minimal
inhibitory concentration (MIC) compared to a susceptible strain
- the genetic trait is often carried on mobile genetic elements (MGEs) such as transposons, conjugative or mobilizable
plasmids (plasmid that can be transferred from one bacterial species to another) and integrated elements in the bacterial
chromosome (genomic islands)
- mobile genetic elements allow for DNA shuffling of antibiotic resistance genes via horizontal gene transfer among
different bacterial species of clinical importance
- bacterial antibiotic resistance due to MGEs often allows for the emergence of multidrug resistant bacteria (superbugs)
Bacterial Antibiotic Tolerance
- bacterial tolerance is homogenously displayed by the whole population and allows the population to transiently survive
antibiotic exposure
- the entire bacterial population is slow growing due to the environmental limitations of a genetic alteration (auxotroph’s
tend to grow very slowly or not at all depending on their environmental conditions and nutrient status) which causes the
population to become tolerant to a specific class of antibiotic that requires actively growing cells
- antibiotics kill actively dividing cells therefore the cells need to be actively growing, antibiotics are not effective against
slow growing cells and will need a much longer antibiotic duration time to have the “normal effect”
- the tolerant bacteria require a longer exposure to the antibiotic to observe the same level of killing as observed with a
susceptible strain, minimal duration of killing (MDK)
- MDK99 is the minimum duration of antibiotic treatment required to kill 99% of the bacterial population
Bacterial Persistence
- bacterial persistence is found only in a fraction (1%) of the bacterial population and are generally indistinguishable from
sensitive cells within the population
- interestingly, are usually persistent to a number of drugs
- transiently survive antibiotic treatment and will not influence the MIC or MDK
- bacterial persistence can be ‘spontaneous/stochastic persistence’ or ‘triggered/induced persistence’ which allow the
persistent bacteria to survive antibiotic exposure
- in triggered/induced persistence, conditions such as starvation, oxidative stress, carbon source transition, DNA damage or
antibiotics increases the frequency of persister cells in the population
,Formation of Persister cells
- Definition of Stochastic: having a random
probability distribution or pattern that may be
analyzed statistically but may not be predicted
precisely
- responsive diversification: within a population,
have persister cells and if the environmental
conditions change, these cells are already adapted
to tolerance and resist that change
- induced: adding an external stress/challenge induces the persistent state
- the treatment of a population with an antibiotic results in cell death,
leaving only persister cells or resistant mutants alive
- when regular (sensitive) cells are treated with antibiotics, they cells
will be killed
- if you have resistance cells, cell growth will continue and will not
be affected by the addition of the antibiotic
- with persister cells, they are part of the initial normal population so
these cells will always be growing in the background (flat baseline)
Difference between Bacterial Tolerance and Persistence
a) tolerant cells are slow growing due to conditions
or because they are an auxotroph
- with a susceptible population, when an antibiotic
is added, expect that population to be killed and
will observe a dramatic drop in cell numbers
- with tolerant bacteria, a decrease in numbers will
be observed but the time taken for this decrease
will be much longer
- the MDK of the sensitive bacteria will be different
(smaller) to the MDK of the tolerant bacteria as the
tolerant bacteria take much longer to be affected by
the antibiotic
b) when the antibiotic is added, there will be a decrease in the number of cells of the persistent and susceptible bacteria at
the same rate, but at a certain point, the bacteria will not be affected by the antibiotic due to the presence of the persister
cells where only the persisters will remain in that population as all the susceptible bacteria are killed
Bacterial persistence
- persister cell does not undergo a genetic change to escape antibiotic treatment and survives the antibiotic treatment as a
result of phenotypic bistability, persisters are not resistant but considered phenotypic variants that are tolerant to antibiotics
i.e. they are genetically the same but phenotypically different
- the survival of persister cells is explained by their transition into a “dormant” state hallmarked by a reversible and
substantial reduction of growth rate and metabolism that protects the cellular processes
- most antibiotics and environmental threats effect only growing cells, hence persisters are more protected from killing
- persister cells can revert or ‘awake’ from the persister state stochastically or through environmental conditions, and upon
exit from antibiotic-tolerant state, a new population can be formed when antibiotics are removed from the environment and
can recolonize the environment
Persister cells in biofilms
- a biofilm is a microbial population growing on a surface where the cells are enclosed by an extracellular matrix (consists
of glycoproteins, eDNA, polysaccharides) and exhibit multicellular-like behavior
- biofilms are involved in 80% of human bacterial chronic inflammatory and infectious diseases and are multidrug tolerant
as well as resistant to host immune system
- biofilms tend to contain increased prevalence of persister cells which appear to be responsible for the cause and
recalcitrance of chronic infections
- biofilm environments tend to have an increased number of persister cells in a community
- biofilms are protective habitats for persisters against antibiotics and the immune system and persisters remain viable and
repopulate biofilms when the level of antibiotics drops and can recolonize the host after antibiotic removal
- bacterial cells attach to a surface, the cells then produce EPS (extracellular polymeric substances) and other cells then
other bacterial cells bind to the EPS (may include different bacterial species)
, - formation of pores for nutrients to go through the biofilm, there is cell to cell
commutation between the bacteria, it will grow and can have different layers of
communities (such as anaerobic bacteria on the bottom and aerobic bacteria on the
top), there is exchange of nutrients, so it is a very complex dynamic environment
- it grows, and eventually starts shedding bacterial cells which can spread to new
locations or communities
- on the surface, such as the surface of the lungs, will have the mucosal surface and on
this will have a biofilm and within the biofilm, will have bacteria and in that bacterial
population, will have the persister cells
- the EPS protects it from the immune system as well as antibiotic treatment
- if antibiotics are added, the antibiotic diffuses within the EPS, minimizing the impact of
the antibiotic
- most bacteria will be killed but persister cells will remain in the biofilm and are in a
dormant state to protect itself from the antibiotic
- once antibiotic treatment is discontinued, the persister cells will repopulate the
biofilm environment, regrow and the host will have a re-infection
Persister cells
- why does a bacterial population have persister cells?
- theory is it considered a bet-hedging strategy in which a subpopulation of clonal cells sacrifices fast proliferation to ensure
survival of the population in adverse conditions
- in the long term, the persisters will benefit the entire population as they have the ability to reestablish the population after
the majority has been killed off from an external pressure/environmental change
Periodic antibiotic treatment selects for increased Persistence
- periodic doses of lethal antibiotic concentrations select for high-persistent mutants rather than antibiotic resistance
- has two setups: first had an environment where they had a subinhibitory concentration of antibiotics and application was
constant, selected for the antibiotic resistant bacteria
- applied the stress over time and expected to obtain the antibiotic resistant mutants as those with antibiotic resistance would
thrive in this environment
- continually select for antibiotic resistant mutants with a higher and higher MIC
- second treatment was they treated the bacterial population with a lethal dosage of antibiotic well above the MIC
- after application, found that it killed the bacteria, but the persistent bacteria remained
- they allowed them to regrow and then retreated the population with an antibiotic
- this was repeated several times, and this resulted in an increase in the number of persistence in that population
- this is much worse than when selecting for the antibiotic-resistant mutants because with the persisters, they are able to
tolerate the antibiotic and allow the entire population to regrow again with more persisters present
Lecture 2: Toxin-antitoxin (TA) systems concept and classification
Basic concept of toxin-antitoxin systems
- requires a stable toxin produced in low amounts and an unstable antitoxin produced in large amounts in the cell
- antitoxin is unstable due to being the target of host RNAses or proteases and is produced in excess compared to antitoxin
- toxin must attach specific host target to cause cell death
- toxin is neutralized by antitoxin and cell protected against action of toxin
- the cognate antitoxin is either a protein or a small RNA molecule
- antitoxin counteracts the toxin by acting as a direct inhibitor or by controlling toxin production
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