Staphylococcus aureus: A super bug Flashcards
Staphyloccus aureus
Its a classical organism first identified by Robert Koch.
Its gram positive so has a thick cell wall around the outside. It means a bunch of grapes - forming clusters which is important for its pathogenicity. Its golden because of a pigment found in its membrane, giving it its colour.
It has the capacity to divide in 3 different planes and for the daughter cell remain stuck together- becomes important in pathogenesis.
S. aureus Lifecycle
Its an opportunist pathogen. It will try and inhbait anywhere and will cause disease
In doing so, it causes a wide range of different infections. For example nosocomial infections (that you pick up in hospitals). It forms biofilms because the cells stick together and can stick to surfaces, therefore its quite difficult to eradicate. Its also antibiotic resistant
What diseases can it cause
Many are initiated via wounds
- Furnacles (boils): superficial, self limiting infections- they then have the capacity to spread around your own body of someone else
-Pyomyositis: This is when an abscess forms underneath the skin - easiest way to treat this is to be an incision and release the bacteria. In itself isn’t life threatening but the bacteria can spread to cause other things - Endocarditis: This is on the heart valve- it degrades the heart valve.
- Indwelling devices: Many S. aureus infections you pick up in hospital, because the bacteria are there and because you might in a situation where your skin might be being breached.
- Toxic shock syndrome: S. aureus can also cause TSS. This is mostly prevalent in younger children
All of these can spread, leading to septicemia in the blood
How can one organism cause so many diseases?
- Highly adaptable- its an opportunist pathogen. It finds itself in a particular pathogen, it will try and survive and grow- it responds to environment conditions
- It has multiple virulence determinants
Infection-associated dynamics
- Interaction with specific target tissue: needs to recognise that environment.
- Then needs to proliferate and it needs to avoid our host defences - our primary line of defence against it- avoid innate
- It needs to cause local damage
- It then needs to disseminate of product or pathogen which can lead to systemic disease
Importance of the immune system
We can survive against S. aureus because of our innate immune system. Initial abscess leads to acute inflammatory reactions. Without these, we would be dead
Neutrophils are produced 10 to the 9/ day/ kg in a human - they are the gatekeepers for infection.
What is chronic granulatomous disease
Its a fatal hereditary defect - neutrophils fail to make H2O2 so no oxidative killing. These individuals have frequent serious S. aureus infections - primary defence.
Neutrophils
Neutrophils are important in the ability to control S. aureus
They have to be recruited via signals such as IL8, C5A, LTA and formyl peptides.
Neutrophils then need to kill off the bacteria they find e.g. phagocytosis (for example, opsonisation involves complement and antibodies)
Killing: reactive oxygen dependent (HOCL) and oxygen independent (cathespin, antimicrobial peptides etc.)
Neutrophil recruitment
TLR-2 recognises lipoproteins - s. aureus component that binds to it and inhbits it.
PSGL1 in migration of neutrophils
CD47 is involved in the migration of neutrophils
C5AR is the complement receptor involved in the opsonisatoon receptor
FPR1 is involved in recognising formyl peptides
CXCR2 is involved in binding chemokines and migration.
Phagocytosis
Complement activation leads to assembly of C3 convertase on the outside of the cell. The C3a cleaves bits to it. C3b is the marker (becomes covalently bound to bacterial surface for opsonisation)
This process has a lot of S. aureus components to prevent opsonisation.
Virulence factors that are needed to interact with environment
Cell wall anchored (sortase)- specific signal that sortase regocnises and covalently binds them.
ISD proteins- Iron acquisiation and innate defences resistance.
Protein A - (Spa) binds IgG prevents opsonisation- because opsonisation if its binding immunoglobulins - spa binds them in a way where they’re non functional
ClfA, FnBP etc- adhesions (bind host ligands).
Toxins
Hemolysins (over 3 types)
Alpha hemolysin- A heptameric pore forming toxin
As its becomes more and more prevalent - it has more than one function.
- It lyses red blood cells
- On the epithelial cells Hla interacts with ADAM10 (protease) and enhances its activity. It cleaves a protein called E-cadherin and destroys adherens junctions. It disrupts the epithelial barrier (important for getting in).
TSST- super antigen
This antigen is worse. It leads to IL-2 and other proinflammatory cytokines, fever and shock (death)
Children are suseptible to reoccuring bouts of it
MHC binding site, forms a bridge between the two. Leading to lesions leading to shock and death
Exfoliative toxins A and B. They are proteases and lead to the sloughing of the skin (scalded skin syndrome- mostly prevalent in children)
Leukocidin
Damages white blood cells.
There are a couple of classic toxins e.g. phanton valentine leukocidin.
Another leukocidin known as LukED
Phenol soluble modulins
They are associated with community acquried MRSA. - hospital acquried MRSA
Community acquried MRSA became prevelent - its not a fitness cost. - if someone gets those antibiotics, they might not work
Small peptides- PSM
Coagulase
It can clot your plasma
Used as a test for S. aureus. Coagulase reactions clots plasma - makes a fibrin clot. Fibrin is produced in order to clot wounds. Can tell the difference between S. aureus etc.
They convert Prothrombrin to Staphylothrombin
Staphylothrombin converts fibrinogen to fibrin (insoluble matrix).
This prevents neutrophil access and protects the bacteria in abscess.
Regulation of virulence determinant production
Organism has to be highly adaptable in order to produce these virulence determinant’s at appropriate amounts and modulate their expression in different scenarios.
- In response to environmental stimuli that it gets from that environment
- Specific virulence determinants expressed - doesn’t make them all at the same time. Not only does it control the virulence determinants individually etc.
- It allows adaptation to that specific niche
Growth phase in exponential phase into stationary phase
you get production of Surface Proteins (+++), Exoproteins (-) and as they go into stationary phase, they switch: They stop making Surface Proteins- adhesens and invasins (-), and they start to make toxins- Exoproteins (+++)
Surface proteins: Spa, Fnbp etc
Exoproteins: Hla, TSST etc
How many DNA binding proteins are there
There are 194 putative DNA binding proteins in S. aureus which bring about this regulation. All coordinate the production of virulence determinants
What is agr
accessory gene regulator- virulence determinants are accessory genes (not required for the life of the organism, they’re required for virulence) - are required to cause disease
key pivotal regulator- switch that leads from making immuno invasins during the exponential phase to switch to making toxins. Important regulator. Agr is a positive regulator of toxin production as they enter post exponential growth. Its a negative regulator of surface proteins
Agr kicks in in the interface between exponential phase and post exponential phase
What is sar
staphylococcal accessory regulator-
Sar: upregulates agr expression- regulatory protein
SarA: DNA binding protein- binds to agr promotor region (positively regulates agr) - modulates agr.
The agr locus
All of the processes that are required for this switch in hundreds of genes is controlled by this divergon
There are two operons which are divergently transcribed going from p2 leftwards and p3 rightwards.
Its called AgrBCDA. There are 4 genes in the leftward facing operon and they’re all transcribed as a result of the p2 promoter.
agrBDCA- AgrD
Encodes a prepeptide of extracellular density dependent signalling molecule.
AgrD makes a larger version of something which is then chewed up which is a signalling molecule that brings around a change
AgrD is a 45 a.a peptide. Out of it is clipped an 8 a.a. peptide which is modified (this is the extracellular signalling molecule) AKA autoinducing peptide (AIP)
AgrB
Is the AgrD processing enzyme so AgrB is the first gene in that leftward facing operon.
AgrD encodes the prepeptide of this extra cellular density signalling molecule.
This needs to be processed by AgrB (product of second gene in that operon).
Density dependent signalling process
- Effective concentration of AIP rising in that environment. During the growth within that culture, you’re getting more bacteria and because they’re all producing AIP at this constant rate, effective concentration of AIP rises in this environment
AgrC
Is the third gene in that operon. AgrC is a sensor protein (transmembrane) sensor protein which is membrane associated monitoring the external environment
AgrC senses the concentration of AIP in the environment. It needs a cognate regulator in order to bring about change
AgrA
Cognate regulator. Is the 4th gene in the operon. Its a transcriptional activator and will cause changes in gene expression
What does activated agrA do
It activates expression from p2 and p3. P3 is the promotor upstream of that rightward facing operon.
Activated AgrA upregulates expression from P2 and P3 (promotor upstream of rightward facing operon)
P3 promoter è RNAIII transcript
Turns on expression of itself via P2 but activated agrA also turns on the rightward facing operon controlled by p3. P3 controls expression of that RNA 3 transcript- you end up with a big burst of expression from RNA 3.
What does RNA III encode
RNAIII encodes d-hemolysin (pehnol soluble modulents). Thats the only translated product from RNA3.
You can mutate the ribosome binding site of the delta hemolysin so get no delta hemolysin production but still get the switch. The switch is not due to the production of delta hemolysin. The switch is due to the RNA molecule itself.
BUT regulatory effector is RNAIII itself
RNAIII acts at level of:
1.Transcription: mechanism unknown.
(possibly intermediate proteins)
2.Translation (works on a large number of differnent transcripts) eg. spa (surface protien- turned off), hla (alpha heamolysin- turned off)
Negative regulation of spa translation:
RNA 3 is an anti sense RNA molecule (has a sequence that is anti sense to this upstream region - binds in an anti sense fashion - now have a double stranded RNA molecule- it masks the ribosome binding site.
When RNA 3 is produced it binds to spa transcript and stops translation.
When RNA 3 is bound to the spa transcript - it allows bases down stream to form a stem loop structure (substrate for RNAse). It sets up the transcript to be degraded
RNA 3 can control the expression of spa and surface proteins in a negative fasion - less spa
Positive regulation of hla translation
No RNAIII: 5’ region hla transcript complementary bases form stem loop. These cover the ribosome binding site and prevents translation and sets transcript up to be degraded.
Positive regulation of hla translation
Presence of RNA 3 allows RNA binding site to be exposed. The presence of RNA 3 allows the turn off of translation of surface proteins but turn on translation
SarA
Repressor of proteases
Acts via and independently of Agr
Proteases control virulence determinant stability
Allows swift adaptation by turnover of adhesins etc.
Control of virulence determinants at the level of:
Transcription/Translation/Stability
What is MRSA and what is it resistant to
MRSA - methicillin resistant S. aureus
Resistant to all b-lactams (penicillins, cephalosporins, carbapenems, penems etc.)
Mechanism of methicillin resistance
PBP2a (MecA): low B-lactam affinity, altered site. PBP2A is a transpeptidase (cross links the peptidoglycan
MecA hasn’t evolved from s. aureus but from HGT from another source, potentially from S. sciuri
MecA is a 30-50kb mec element flanked by insertion sequences (mec element)
How is MecA regulated
Only want to express this in the antibiotic
MecR is a membrane associated protein when there’s no beta lactam, it’s inactive.
When beta lactam binds to a mecR motif (similar to PBP), it allows the depression of mecA
MecR undergoes a confirmational change and chews up MecI allowing MecA expression
New antibiotics that have soon become ineffective
fluoroquinolones (synthetic)- probably isn’t resistant as it hasn’t encountered the drug
Ciprofloxacin(most famous drug of this class). MRSA MIC <2gml-1- means theryre clinically effective.
Resistance went from 5% to 80% strains in 1 year- resistance appears multiple times - driven by a point mutation.
Resistance due to altered codon 84 or 85 in DNA gyrase (changed target)
Now use mupirocin (resistance appeared)
VISA where was it found
Vancomycin Insensitive S. aureus
Vancomycin was the only effective drug inhibiting the peptidoglycan.
Insensitivity appeared in 1996 in Japan
1997 in USA
1998 in France
1999 in Scotland
The insensitive cells had a thicker cell wall which stops the vancomycin getting in
VRSA
Vancomycin Resistant S. aureus
In 2002, the first case of VRSA in the USA was found
These strains are completely resistant, they no longer have the target in which vancomycin binds.
Acquired resistance mechanism from VanA from Enterococcus. VanA encodes for D-Ala D-Lac ligase
This was because someone had an enterococcus and S. aureus infection at the same time and VanA was found on a plasmid
VRSA is rare
New treatment/prophylaxis
Vaccine/antibody therapy:
Capsule (CP5, CP8)- creates a problem clinically.
Extracellular polysaccharide
Surface proteins (IsdB)
Three Phase III clinical trial failures- because s. aureus is a human organism
Drug Discovery and methods
All major antibiotics discovered before 1980
Economics: $1 billion for 1 drug to sale
10-12 years
Cost effectiveness?
- Resistance
- Broad spectrum
- Use in developing world
Methods:
Empirical - screening
Rational - existing targets and change the drug
Novel targets- end up with novel chemistry
Existing targets
1.Analogues of existing drugs
2.Understand molecular basis of resistance
Knowing all the resistance mechanisms, you can design novel compounds which can bypass those resistance mechanisms.
- Drug analogues stable to degradation e.g. amikacin (kanamycin analogue)
- Inhibit inactivating enzymes e.g.
- Analogues not effluxed e.g. glycylcyclines (tetracycline analogues)
- Analogues binding to modified target e.g. carbapenem derivatives bind to MecA
Novel Drugs
New classes of chemistry which have appeared in the last 40 years:
Daptomycin (Cubicin)- different structure and works in a different way. Acts at the membrane of s. aureus. It oligermizes at the membrane - forms a patch in which potassium ions are lost from the cell. It inserts into the membrane and is effective at killing cells. Its a cyclic lipopeptide and membrane active
Linezolid (Zyvox)- target for lots of drugs. Its a Oxazolidinone that binds ribosome 50s. It prevents initiation complex formation- it stops the assembly initiation stage
There’s Clinical resistance to both of them
Beta lactam derivatives
Ceftobiprole: PBP2a is a membrane associated protein, it assembles the bit of the peptidlocycan on the outer membrane. The muropeptide substrates come as this lipid linked molecule known as lipid 2. Lipid 2 is flipped across the membrane and then the penicillin binding proteins and others take it and use transglycolyase activity and transpeptidase activity knitted into its existing wall for it to get bigger. There’s a membrane spanning domain at the bottom and a stalk as as they they poke out the membrane.
Ceftaroline (teflaro): Its a cephalosporin it binds to the active site and it also binds the allosteric site. Ceftaroline binds to the pbp2a allosteric site - opens up and gets it to insert into the active site- well thought out mechanism to combat this PBP2A.
Teixobactin discovery
IChip:
got some mud, shook it up and diluted it down so each well in a microtiter plate, you would have one organism. Then you put a bit of permeable membrane on top and the bottom of the wells also had a permeable membrane and you stuck it in the soil/ mud. The cells in the wells can interact with other soil organisms through the semi permeable membranes - allows those organisms in the membranes to grow, you seed it with S. aureus on top and look for where the S. aureus is growing because that organism in that well has produced an organism that will inhibit the S. aureus on top.
Where does teixobactin come from
Eleptheria terrae and its a gram negative proteobacterium- this could produce teixobactin
Structure and Biosynthesis of Teixobactin
Complicated molecule and is made as a product of two genes
1,242 Molecular mass- big as vancomycin
Depsipeptide
(enduracididine, methylphenylalanine and 4 D-amino acids- residues in it)
Non-ribosomally encoded- has two enzymes that make it. Have different activities that stick the different residues together to make the large molecule
Peptide synthetase proteins
Teixobactin activity
Selection of resistant mutants - did this experiment by using a drug that they knew they could get resistance to as a control but they didn’t get any resistance at all.
Mode of Action of Teixobactin
Resistance is trickier because it has two activities. Its the first class of antibiotic that has more than one target - makes resistance more difficult to evolve
It inhibits cell wall biosynthesis in two ways
it binds to the lipid 2 molecule and prevents it being used as a substrate for peptidoglycan biosynthesis
It binds to a molecule required for teichoacid biosynthesis- lipid 3.
