Staph. aureus

Question 1

What are the characteristics of Staph. aureus?

Answer 1

Gram positive and coccus in shape. Can divide into three planes with daughter cells remaining stuck together. Opportunistic pathogen and causes nosocomial infections. Biofilm formation is key for infection due to the sticking together and so hard to eradicate due to cell clumping. This confers antibiotic resistance.
Very good at abiotic survival, also stress resistant to salt and heat.

Question 2

Why can Staph. aureus cause so many infections?

Answer 2

It is highly adaptable. Responds to different environmental conditions and also has multiple virulence determinants. Within an individual infection, the environment changes and so must adapt to these changes to.

Question 3

What are the infection associated dynamics?

Answer 3

1. Interaction with specific target tissue
2. Proliferate (by avoiding host defences)
3. Local damage
4. Dissemination of pathogen or products (systemic disease)

Question 4

What is the main killing mechanism targeting staph. aureus?

Answer 4

Phagocytes. Importance of this is shown in chronic granulatomous disease. It is a fatal hereditary defect where neutrophils fail to make H2O2 and so no oxidative killings.

Question 5

What are neutrophil characteristics with Staph. aureus?

Answer 5

Important in ability to control Staph infections. Recruited through chemotactic signals such as IL8, C5a, LTA. LTA and formyl peptides are part of bacteria recognised by neutrophils. Recruitment leads to phagocytosis using opsoniation from complement and antibodies. Oxygen dependent such as HOCl or oxygen independent killing occurs through cathepsin.

Question 6

How does immune evasion occur?

Answer 6

Bacterial components bind to TLRs to inhibit innate immunity. TLR-2 recognises lipoproteins and is inhibited by SSL3. PSGL1 is associated with neutrophil migration and is inhibited by SSL5 and SelX. CD47 is involved in neutrophil migration and is inhibited by SSL6. C5aR is complement receptor inhibited by CHIPS. FPR1 recognises formyl peptides and is inhibited by CHIPS and FLIPr.

Question 7

How does staph. aureus evade the immune system?

Answer 7

Staph can inhibit the ability to opsonise the bacteria. Also inhibits neutrophil killing by inhibiting NADPH oxidase by producing SodM and SodA. Cathepsin is inhibited by AhpCF. Lysozyme is inhibited through OatA which oacetylates the bacterial wall.

Question 8

What are the main virulence factors in Staph. aureus?

Answer 8

1. Cell wall proteins - Anchored to cell wall by sortase. Isd proteins are involved in Fe acquisition and innate defence resistance. Protein A binds IgG preventing opsonisaiton. ClfA and FnBP are adhesins that bind host ligands.
2. Toxins - Hemolysins to bind RBC. Alpha hemolysin is a heptameric pore forming in RBC to lyse them. Interacts with ADAM10 on epithelial cells to enhance the activity to cleave E-cadherin and destroy adherens junctions between epithelial cells. This disrupts the epithelial barrier to allow Staph in. Enterotoxins are super-antigens. They undergo indiscriminate binding to APC (antigen-presenting cell). Causes an excess production of IL-2 which stimulates the production of TNF alpha and other cytokines causing cytokine shock. IL-2 is what causes the nausea and vomiting.

Question 9

What is toxic shock syndrome?

Answer 9

Toxic shock syndrome toxin is worse than the enterotoxin. Leads to IL-2 and then fever, shock and death. Most mortalities come from children and can get recurring syndromes. Has exfoliative toxins A and B. These are proteases that lead to sloughing of the skin leading to scalding skin syndrome. Also prevalent in children.

Question 10

What is leukocidin?

Answer 10

Leukocidin damages WBC and has a well-known strain called Panton Valentine Leukocidin and LukED. Phenol soluble modulins are also leukocidins is community acquired MRSA. This makes small peptides and took so long to find as the genome is ridiculously small and so weren’t even sure they were encoding something.

Question 11

What are coagulases?

Answer 11

Coagulase can clot the plasma – Coa and vWbp. Cause the conversion of prothrombin to staphylothrombin. Staphylothrombin is a protease that converts fibrinogen to fibrin and causes the clots. This prevents neutrophil access and protects bacteria in abscesses.

Question 12

What regulates virulence determinant production?

Answer 12

1. In response to environmental stimuli
2. Specific virulence determinants expressed
3. Allows adaptation to specific niches
   Growth phase dependent virulence determinant production is a key part of the ability to coordinate regulation.

Question 13

What happens to Staph. aureus in exponential phase?

Answer 13

Surface proteins are adhesins and so establish the infection meaning needed in exponential phase. When nutrients become limiting, toxins allow dispersal through tissue damage. There are 194 putative binding proteins that bring about regulation, done through positive or negative regulation. The organism is responding to the environment all the time.

Question 14

What are regulators of staph. aureus?

Answer 14

1. Agr: accessory gene regulator
2. Sar: staphylococcal accessory regulator
   Sar upregulates agr expression with agr being a pivotal regulator. Agr regulates the switch between immune evasions to toxin production. Agr is a positive regulator of toxin production and a negative regulator of surface proteins.

Question 15

What is the structure of Staph. aureus regulators?

Answer 15

SarA is a DNA binding protein that binds to agr promoter region. It positively regulates agr. Agr is a complex divergon (meaning two divergently transcribed operons with linked function).

Question 16

What is AgrD?

Answer 16

pre-peptide of extracellular density dependant signalling molecule. Bacterial pheromone is an auto-inducing peptide (AIP). AIP is an octapeptide which allows bacteria to signal to each other. AgrB is an AgrD processing enzyme.

Question 17

What is promoter P2?

Answer 17

low level expression during exponential phase. There is a slow build-up of AIP in the medium until it reaches a threshold level. This is density dependant signalling. Bacteria are using this as a proxy for the density.

Question 18

What senses AIP?

Answer 18

AgrC which is a transmembrane sensor protein. AgrC is a member of family of two component sensor regulator pairs. Very common regulator mechanism. AgrA is a regulator protein (transcriptional activator).

Question 19

What is activated AgrA?

Answer 19

A transcriptional activator that upregulates expression from P2 and P3. There is an auto-regulatory circuit with density dependent signalling. Once AIP threshold is met, enhancement of signal switches to turn on P3.
P3 promoter -> RNAIII transcript. RNAIII encodes -hemolysin. This is a phenyl soluble lysin and lyses RBC. The switch is NOT due to delta hemolysin it is because of the P3 itself. RNA3 contains the regulatory effector within itself - it is a regulatory RNA molecule.

Question 20

Where does RNA III act at the level of?

Answer 20

1. Transcription = mechanism is unknown possible using intermediate proteins
2. Translation = spa, hla “Antisense RNA.” Works at transcript level.
   There is negative regulation of spa translation.
   Masks the shine delgardo sequence and masks RBS. Stops translation of spa transcript by forming stem loop structure which is RNase substrate. SO prevents it by masking RBS and setting transcript up to be degraded.

Question 21

What is positive regulation of hla translation?

Answer 21

No RNAIII: 5’ region hla transcript complementary bases form stem loop. This covers the ribosome binding site and prevents translation. With RNAIII: RNAIII binds antisense to hla transcript. This stops the stem loop from forming so with the RBS being free, allows translation.

Question 22

What is SarA?

Answer 22

Repressor of proteases. Acts via and independently of Agr. Proteases control virulence determinant stability which affect surface proteins on the bacteria itself. Allows swift adaptation by turnover of adhesins etc. Control of virulence determinants at the level of transcription, translation and stability in response to the environment. Allows adaptation to inhabit a wide range of niches and is a versatile pathogen.

Question 23

What is bacterial interference?

Answer 23

Novel mode of competition between related strains, mediated by AgrD peptide. This inhibits toxin production in other groups and gives a competitive advantage in mixed infections.

Question 24

What are beta lactams?

Answer 24

Beta lactams bind to penicillin binding proteins which are involved in final stage synthesis of peptidoglycan for cell wall. As it is on outside of the cell, has to be assembled on outside of the cell. Therefore PBPs are also on outside of the cell meaning it is easy to get compounds to them.

Question 25

What is PBP2a?

Answer 25

PBP2a (MecA) has a low affinity for beta lactams meaning it has an altered site where high affinity drugs bind to is different. This means they can’t bind at an effective concentration and so the drugs are ineffective. PBP2a is a transpeptidase and has come from an exogenous source. It is only present in methicillin resistant isolates through horizontal transfer. The 30-50kb mec element is flanked by insertion sequences, meaning if there is a transposase, it can jump in and out of the chromosome. MecA encodes PBP with a low affinity for antibiotics.

Question 26

What affects MecA transcription?

Answer 26

MecI is a dimer that represses mecA transcription. MecR is a membrane associated metalloprotease and responds to beta lactams, degrading MecI allowing MecA transcription.

Question 27

What is the only effective drug against MRSA?

Answer 27

Vancomycin. This is a glycano peptide which inhibits peptidoglycan biosynthesis. However VISA (Vancomycin Insensistive S.Aureus) now has a change in permeability with a thicker cell wall.

Question 28

What is VRSA?

Answer 28

Vancomycin Resistant Staph. aureus. First reported in 2002 and was acquired from Enterococcus.

Question 29

What are new therapies for MRSA?

Answer 29

New therapies include vaccine / antibody therapy such as capsule (CP5/CP8), extracellular polysaccharides and surface proteins (IsdB). There have since been three Phase 3 clinical trial failures.

Question 30

How did existing drugs offer new drugs to be made?

Answer 30

1. Analogues of existing drugs
2. Understand molecular basis of resistance
   a. Drug analogues stable to degradation
   b. Inhibit inactivating enzymes
   c. Analogues not effluxed
   d. Analogues binding to modified target

Question 31

What are novel drugs for MRSA?

Answer 31

• Daptomycin (Cubicin) which is a cyclic lipopeptide and membrane active
• Linezolid (Zyvox) which is an oxazolidinone and binds ribosome 50S preventing initiation complex formation.
• Teixobactin which is a depsipeptide and non ribosomally encoded producing peptide synthetase proteins. Binds to Lipid II (peptidoglycan biosynthesis) and Lipid III (teichoic acid biosynthesis).